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The epileptic brain is the result of a sequence of events transforming normal neuronal populations into hyperexcitable networks supporting recurrent seizure generation. These modifications are known to induce fundamental alterations of circuit function and, ultimately, of behavior. However, how hyperexcitability affects information processing in cortical sensory circuits is not yet fully understood. Here, we investigated interlaminar alterations in sensory processing of the visual cortex in a mouse model of focal epilepsy. We found three main circuit dynamics alterations in epileptic mice: (i) a spreading of visual contrast-driven gamma modulation across layers, (ii) an increase in firing rate that is layer-unspecific for excitatory units and localized in infragranular layers for inhibitory neurons, and (iii) a strong and contrast-dependent locking of firing units to network activity. Altogether, our data show that epileptic circuits display a functional disruption of layer-specific organization of visual sensory processing, which could account for visual dysfunction observed in epileptic subjects. Understanding these mechanisms paves the way to circuital therapeutic interventions for epilepsy.

Melanoma is a malignant and metastatic form of skin cancer, which is not diagnosed in early stages of the disease. Nowadays, immunotherapy is changing the treatment landscape for metastatic melanoma. Placenta-specific1 (PLAC1) is a cancer-testis-placenta (CTP) antigen with differential expression in melanoma tissues. Here, we evaluated the potential of plac1 to induce anti-cancer immune responses as well as to prevent cancer development in a mouse model of melanoma.Two proteins containing full extracellular domain (ED) of mouse plac1+KDEL3 and full ED of mouse plac1+ tetanus toxin P2 and P30+ pan DR epitope (PADRE) ​+ ​KDEL3 were produced and injected in mice to evaluate their capacity to induce anti-cancer immune responses as well as their potential to prevent melanoma development. Induction of plac1-specific humoral and cellular responses as well as tumor-associated parameters were tested in a series of 36 mice.Sera of mice immunized with ED ​+ ​P2P30+PADRE ​+ ​KDEL3 contained antibodies able to react with surface plac1 in B16F10 ​cells. Both proteins induced proliferative cellular immune responses against B16F10 ​cells and plac1-specific cytotoxic T cells (CTL) and CD107a ​+ ​CTL responses, which was higher in mice immunized with ED ​+ ​P2P30+PADRE ​+ ​KDEL3. Splenocytes of mice vaccinated with ED ​+ ​P2P30+PADRE ​+ ​KDEL3 exerted a significant cytotoxicity against B16F10 ​cells. Vaccination with ED ​+ ​P2P30+PADRE ​+ ​KDEL3 significantly delayed B16F10-induced tumor onset, reduced tumor growth, and increased survival. Tumors induced by B16F10 expressed plac1 in vivo.Our results pave the way for development of effective melanoma preventive vaccine in humans, although further studies are needed.

Emerging evidence shows that spontaneous synaptic transmission plays crucial roles on neuronal functions through presynaptic molecular mechanisms distinct from that of action potential (AP)-evoked transmission. However, whether the synaptic vesicle (SV) population undergoing the two forms of transmission is segregated remains controversial due in part to the conflicting results observed in cultured neurons. Here we address this issue in intact neuromuscular synapses using transgenic zebrafish larvae expressing two different indicators targeted in the SVs: a pH-sensitive fluorescent protein, pHluorin, and a tag protein, HaloTag. By establishing a quantitative measure of recycled SV fractions, we found that ∼85% of SVs were mobilized by high-frequency AP firings. In contrast, spontaneously recycling SVs were mobilized only from SIGNIFICANCE STATEMENTSynaptic vesicles (SVs) are divided into functionally distinct pools depending on how they respond to action potential (AP) firing. The origin of SVs used for spontaneous fusion remains enigmatic despite intensive studies in cultured preparations. We addressed this question in intact neuromuscular synapses and provided two findings. First, prolonged AP inhibition activated a distinct population of fusion, which needs to be distinguished from genuine spontaneous fusion arising from a highly limited fraction. Second, the limited fraction observed early in the AP inhibition period exhibited the characteristics of readily releasable pool in the subsequent round of stimulation. Our study revealed that the origin of spontaneous SV fusion is restricted to the readily releasable pool among the SV pools involved in AP-evoked fusion.

The recombinant carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts neuroprotective and neurorestorative effects on the dopaminergic system of animal models of Parkinson's disease (PD). The present study aimed to determine the effect of the Hc-TeTx fragment on the markers of oxidative stress and nitrosative stress generated by the acute toxicity of 1-methyl-4-phenylpyridinium (MPP+). For this purpose, the Hc-TeTx fragment was administered once a day in three 20 μg/kg consecutive injections into the grastrocnemius muscle of the rats, with an intra-striatal unilateral injection of 1 μL of MPP+ [10 μg/mL] then administered in order to cause a dopaminergic lesion. The results obtained show that the rats treated with Hc-TeTx plus MPP+ presented an increase in the expression of tyrosine hydroxylase (TH), a significantly greater decrease in the levels of the markers of oxidative stress, nitrosative stress, and neurodegeneration than that observed for the group injured with only MPP+. Moreover, it was observed that total superoxide dismutase (SOD) and copper/zinc SOD activity increased with the administration of Hc-TeTx. Finally, immunoreactivity levels were observed to decrease for the levels of 3-nitrotyrosine and the glial fibrillary acidic protein in the ipsilateral striatum of the rats treated with Hc-TeTx plus MPP+, in contrast with those lesioned with MPP+ alone. Our results demonstrate that the recombinant Hc-TeTx fragment may be a potent antioxidant and, therefore, could be suggested as a therapeutic tool against the dopaminergic neuronal impairment observed in the early stages of PD.

RESUMEN INTRODUCCIÓN: El tétanos es una enfermedad inmunoprevenible, ocasionada por la bacteria Clostridium tetani, desencadenando una enfermedad caracterizada por espasmos musculares, insuficiencia respiratoria y disautonomías, potencialmente mortal. MATERIALES Y MÉTODOS: Presentamos una serie de tres pacientes que consultaron al servicio de urgencias por presentar trismus, rigidez muscular generalizada y dificultad respiratoria, requiriendo manejo en la unidad de cuidados intensivos, con relajación muscular y administración intramuscular e intratecal de inmunoglobulina antitetánica, con evolución satisfactoria en todos los casos. DISCUSIÓN: Su tratamiento está divido en dos grandes secciones; la primera parte, el control de la infección y eliminación del agente causal, con lavado y desbridamiento de heridas, administración de antibióticos y neutralización de la neurotoxina. La segunda parte del tratamiento está en el soporte vital en la unidad de cuidados intensivos, con la administración de sedación, relajación muscular, control de disautonomías y manejo de complicaciones. CONCLUSIONES: El tétanos a pesar de los avances en vacunación aún es una enfermedad presente, cuyo diagnóstico y tratamiento rápido y adecuado, permite sobrevivir a los pacientes, como en los casos aquí reportados. ABSTRACT INTRODUCTION: Tetanus is an immuno-preventable disease, produced by the bacterium Clostridium tetani, that causes a disease characterized by muscle spasms, respiratory insufficiency and life-threatening dysautonomia. MATERIALS AND METHODS: We present a series of three patients who consulted for trismus, muscle stiffness and respiratory failure, which required intensive care management, muscle relaxation, intramuscular and intrathecal administration of tetanus immu-noglobulin, with satisfactory outcomes in all the cases. DISCUSSION: Its treatment is divided into two main sections; the first part, the control of infection and elimination of the causative agent, with washing and debridement of wounds, administration of antibiotics and neutralization of the neurotoxin. The second part is life support in the intensive care unit, with the administration of sedation, muscular relaxation and control of dysautonomia and the management of complications. CONCLUSIONS: Despite the advances in vaccination, tetanus is still a present disease, whose diagnosis and rapid and adequate treatment allows patients to survive, as in the cases reported here.

Tetanus arises from wound contamination with Clostridium tetani, but approximately one fifth of patients have no discernable entry wound. Clostridium tetani is culturable from animal feces, suggesting the gastrointestinal tract could be an endogenous reservoir or direct-entry portal, but human data are lacking. In this study of 101 Vietnamese adults with tetanus and 29 hospitalized control subjects, admission stool samples were cultured for C. tetani. Anti-tetanus toxin antibodies were measured by ELISA. Clostridium tetani toxigenicity was evaluated using polymerase chain reaction and sequencing. Toxigenic C. tetani was cultured from stool samples in 50 of 100 (50%) tetanus cases and 12 of 28 (42.9%) control subjects (P = 0.50), and stool samples of 44 of 85 (52.4%) tetanus cases with clinically identified wounds compared with 6 of 15 (47.6%) patients without clinically identified wounds (P = 0.28). Nine of 12 (75%) control subjects with toxigenic C. tetani in their stool samples lacked protective antibody concentrations. These findings fail to show evidence of an association between gastrointestinal C. tetani and tetanus infection, but emphasize the importance of increasing vaccination coverage.

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (

We review some of the precursor works of the Pasteurians in the field of bacterial toxins. The word “toxin” was coined in 1888 by Ludwig Brieger to qualify different types of poison released by bacteria. Pasteur had identified the bacteria as the cause of putrefaction but never used the word toxin. In 1888, Émile Roux and Alexandre Yersin were the first to demonstrate that the bacteria causing diphtheria was releasing a deadly toxin. In 1923, Gaston Ramon treated that toxin with formalin and heat, resulting in the concept of “anatoxin” as a mean of vaccination. A similar approach was performed to obtain the tetanus anatoxin by Pierre Descombey, Christian Zoeller and G. Ramon. On his side, Elie Metchnikoff also studied the tetanus toxin and investigated the cholera toxin. His colleague from Odessa, Nikolaï GamaleÏa who was expected to join Institut Pasteur, wrote the first book on bacterial poisons while other Pasteurians such as Etienne Burnet, Maurice Nicolle, Emile Césari, and Constant Jouan wrote books on toxins. Concerning the endotoxins, Alexandre Besredka obtained the first immune antiserum against lipopolysaccharide, and André Boivin characterized the biochemical nature of the endotoxins in a work initiated with Lydia Mesrobeanu in Bucharest.

Botulism and tetanus are the 2 primary manifestations of neurologic disease caused by clostridial toxins. Only a small dose of clostridial toxin is required to induce severe, and often fatal, disease. Consequently, definitive diagnosis of either disease is nearly impossible to achieve antemortem or postmortem; presumptive diagnosis is usually made based on physical and neurologic examination findings. Because the severity of clinical signs can worsen rapidly, prognosis worsens when therapeutic intervention is delayed. Highly effective vaccines are available against both botulism and tetanus and are critical in preventative approaches to control.

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/Met

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.

The central amygdala (CeA) is critically involved in a range of adaptive behaviors, including defensive behaviors. Neurons in the CeA send long-range projections to a number of extra-amygdala targets, but the functions of these projections remain elusive. Here, we report that a previously neglected CeA-to-globus pallidus external segment (GPe) circuit plays an essential role in classical fear conditioning. By anatomic tracing,in situhybridization and channelrhodopsin (ChR2)-assisted circuit mapping in both male and female mice, we found that a subset of CeA neurons send projections to the GPe, and the majority of these GPe-projecting CeA neurons express the neuropeptide somatostatin. Notably, chronic inhibition of GPe-projecting CeA neurons with the tetanus toxin light chain (TeLC) completely blocks auditory fear conditioning.In vivofiber photometry revealed that these neurons are selectively excited by the unconditioned stimulus (US) during fear conditioning. Furthermore, transient optogenetic inactivation or activation of these neurons selectively during US presentation impairs or promotes, respectively, fear learning. Our results suggest that a major function of GPe-projecting CeA neurons is to represent and convey US-related information through the CeA-GPe circuit, thereby regulating learning in fear conditioning.SIGNIFICANCE STATEMENTThe central amygdala (CeA) has been implicated in the establishment of defensive behaviors toward threats, but the underlying circuit mechanisms remain unclear. Here, we found that a subpopulation of neurons in the CeA, which are mainly those that express the neuropeptide somatostatin, send projections to the globus pallidus external segment (GPe), and this CeA-GPe circuit conveys unconditioned stimulus (US)-related information during classical fear conditioning, thereby having an indispensable role in learning. Our results reveal a previously unknown circuit mechanism for fear learning.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.

Formaldehyde detoxification is a process for converting tetanus toxin (TT) and diphtheria toxin (DT) into tetanus toxoid (TTd) and diphtheria toxoid (DTd), respectively. The mechanism of this detoxification process has been investigated by several previous studies based on lab-scale toxoids. To obtain greater insights of the effects induced by formaldehyde, industrial TTd and DTd batches obtained from different detoxification processes were studied in this work. Using liquid chromatography-mass spectrometry (LC-MS), 15 and 20 repeatable formaldehyde-induced modification sites of TTd and DTd were identified, respectively. Toxoid which had a higher formaldehyde-induced modification rate observed by LC-MS, also had larger bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aggregates which were observed on size exclusion chromatogram (SEC) were confirmed by SDS-PAGE and LC-MS. Formaldehyde detoxification also led to a decrease of isoelectric point (pI) values and an increase of retention on weak anion exchange (WAX) column. Specific toxicity tests were conducted to evaluate toxicity of the TTd and DTd samples obtained with different detoxification conditions. Results from the specific toxicity tests showed that all toxoids used in this study were qualified, including toxoids obtained from mild and drastic detoxification conditions. However, obtained from mild detoxification conditions had less aggregates and may lead to a higher degree of glycosylation in conjugate vaccines than the ones obtained from drastic detoxification conditions. Thus, we suggest that mild detoxification conditions should be used to obtain TTd and DTd. Furthermore, as well as studying the formaldehyde-induced modifications and toxicity in TTd and DTd, the effects of the detoxification process on foreign proteins were also investigated. An increase in foreign proteins were observed in the aggregate than in the monomer of the toxoids. Additionally, some foreign proteins in the monomer of the toxins transferred to the aggregate of toxoids due to the formation of cross-linking. To eliminate the risk of cross-linking foreign proteins to toxoids in vaccination programs, a purification process is necessary before the detoxification process and/or the use of toxoids in vaccines.

The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20 % mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored., Comment: manuscript accepted for publication by talanta (DOI 10.1016/j.talanta.2021.122883). In total

Objective To determine electrical changes in the heart in a chronic, nonstatus model of epilepsy. Methods Electrocorticography (ECoG) and electrocardiography (ECG) of nine animals (five made epileptic by intrahippocampal injection of tetanus neurotoxin (TeNT) and four controls), are monitored continuously by radiotelemetry for up to 7 weeks. Results Epileptic animals develop a median of 168 seizures, with postictal tachycardias reaching a mean of 487 beats/min and lasting a mean of 661 seconds. Ictal changes in heart rate include tachycardia and in the case of convulsive seizures, bradyarrhythmias resembling Mobitz type 1 second‐degree atrioventricular block; notably the P‐R interval increased before block. Postictally, the amplitude of T wave increases. Interictally, QT dependence on RR is modest and conventional QT corrections prove ineffective. Interictal QT intervals, measured at a heart rate of 400 bpm, increased from 65 to 75 ms, an increase dependent on seizure incidence over the preceding 10‐14 days. Significance Repeated seizures induce a sustained tachycardia and increase in QT interval of the ECG and evoke arrhythmias including periods of atrioventricular block during Racine type 4 and 5 seizures. These changes in cardiac function may predispose to development in fatal arrhythmias and sudden death in humans with epilepsy.

Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.

IntroductionIntratumoral injections of novel therapeutics can activate tumor antigen-specific T cells for locoregional tumor control and may even induce durable systemic protection (against distant metastases) via recirculating T cells. Here we explored the possibility of a universal immunotherapy that promotes T-cell responses in situ and beyond, upon intratumoral injection of nanoparticles formulated with micron-sized crystals.MethodsCucumber mosaic virus-like particles containing a tetanus toxin peptide (CuMVTT) were formulated with microcrystalline tyrosine (MCT) adjuvant and injected directly in B16F10 melanoma tumors. To further enhance immunogenicity, we loaded the nanoparticles with a TLR7/8 ligand and incorporated a universal tetanus toxin T-helper cell peptide. We assessed therapeutic efficacy and induction of local and systemic immune responses, including RNA sequencing, providing broad insight into the tumor microenvironment and correlates of protection.ResultsMCT crystals were successfully decorated with CuMVTT nanoparticles. This ‘immune-enhancer’ formed immunogenic depots in injected tumors, enhanced polyfunctional CD8+ and CD4+ T cells, and inhibited B16F10 tumor growth locally and systemically. Local inflammation and immune responses were associated with upregulation of genes involved in complement activation and collagen formation.ConclusionsOur new immune-enhancer turned immunologically cold tumors into hot ones and inhibited local and distant tumor growth. This type of immunotherapy does not require the identification of (patient–individual) relevant tumor antigens. It is well tolerated, non-infectious, and affordable, and can readily be upscaled for future clinical testing and broad application in melanoma and likely other solid tumors.

Background Tetanus remains common in many low- and middle-income countries, but as critical care services improve, mortality from tetanus is improving. Nevertheless, patients develop severe syndromes associated with autonomic nervous system disturbance (ANSD) and the requirement for mechanical ventilation (MV). Understanding factors associated with worse outcome in such settings is important to direct interventions. In this study, we investigate risk factors for disease severity and long-term physical outcome in adults with tetanus admitted to a Vietnamese intensive care unit. Methods Clinical and demographic variables were collected prospectively from 180 adults with tetanus. Physical function component scores (PCS), calculated from Short Form Health Survey (SF-36), were assessed in 79 patients at hospital discharge, 3 and 6 months post discharge. Results Age, temperature, heart rate, lower peripheral oxygen saturation (SpO2) and shorter time from first symptom to admission were associated with MV (OR 1.03 [ 95% confidence interval (CI) 1.00, 1.05], p = 0.04; OR 2.10 [95% CI 1.03, 4.60], p = 0.04; OR 1.04 [ 95% CI 1.01, 1.07], p = 0.02); OR 0.80 [95% CI 0.66, 0.94], p = 0.02 and OR 0.65 [95% CI 0.52, 0.79, p Heart rate, SpO2 and time from first symptom to admission were associated with ANSD (OR 1.03 [95% CI 1.01, 1.06], p p = 0.04 and OR 0.64 [95% CI 0.48, 0.80], p Median [interquartile range] PCS at hospital discharge, 3 and 6 months were 32.37 [24.95–41.57, 53.0 [41.6–56.3] and 54.8 [51.6–57.3], respectively. Age, female sex, admission systolic blood pressure, admission SpO2, MV, ANSD, midazolam requirement, hospital-acquired infection, pressure ulcer and duration of ICU and hospital stay were associated with reduced 0.25 quantile PCS at 6 months after hospital discharge. Conclusions MV and ANSD may be suitable endpoints for future research. Risk factors for reduced physical function at 3 months and 6 months post discharge suggest that modifiable features during hospital management are important determinants of long-term outcome.

Purpose Tetanus is a life-threatening disease characterized by muscle spasm caused by neurotoxin of Clostridium tetani. Given the current passive immunotherapy of tetanus with human anti-toxin polyclonal antibodies (PAbs) and the limitations of such preparations, neutralizing monoclonal antibodies (MAbs), especially chimeric or human antibodies with reduced immunogenicity might be considered as an alternative source. Methods A mouse-human chimeric MAb, designated c-1F2C2, was generated and its binding specificities to various recombinant fragments of tetanus toxin, generated in E. coli, were determined. In vivo toxin neutralizing activity of c-1F2C2 was evaluated and compared with that of a commercially available human anti-toxin PAb in a mouse model. The possible mechanisms of toxin neutralizing activity of c-1F2C2 were investigated by assessing its inhibitory effects on toxin receptors binding, including GT1b ganglioside receptor and those expressed on PC12 cells. Results In vivo neutralizing assay showed that c-1F2C2 was able to protect mice against tetanus toxin with an estimated potency of 7.7 IU/mg comparing with 1.9 IU/mg of the commercial human anti-toxin PAb for 10 MLD toxin and 10 IU/mg versus 1.9 IU/mg of the PAb for 2.5 MLD toxin. c-1F2C2 recognized fragment C of the toxin, which is responsible for binding of the toxin to its receptor on neuronal cells. Accordingly, the chimeric MAb partially prevented the toxin from binding to its receptors on PC12 cells (37% inhibition). Conclusion The chimeric MAb c-1F2C2 displayed similar structural and functional characteristics compared to its murine counterpart and might be useful for passive immunotherapy of tetanus.

The rationale for a formal study of the LAL (kinetic chromogenic method) assay for anticrotallic (SAC), antirabies (SAR), antitetanus (SAT) and antiscorpion (SAE) sera involved the determination of parameters required by the Brazilian National Health Surveillance Agency (ANVISA), the USA Food and Drug Administration (FDA), USP (United States Pharmacopeia, 39) and ICH (International Conference on Harmonization). The curve correlation coefficients obtained with the standard endotoxin control ranged from -0.980 to -1.000 in all experiments. Endotoxin recovery added to the SAC, SAR, SAT and SAE samples, at the working dilutions (1:10, 1:10, 1:10 and 1, 100 respectively), met the criteria required by the FDA, USP and Brazilian ANVISA for the Inhibition-Potentiation test. The applied methodology for the four analyzed sera fulfilled the required criteria for all performance parameters. Thus, the present study demonstrated that the in vivo pyrogen test can be potentially replaced by the LAL assay for all assessed sera samples displaying higher sensitivity and following the 3 Rs principle, in addition to maintaining quality control in Sanitary Surveillance.

Multiepitope cancer vaccines are announcing themselves as the future of melanoma treatment. Herein, high immunogenic regions of transmembrane protein 31 (TMEM31) antigen were selected according to cytotoxic T lymphocytes' (CTL) epitopes and major histocompatibility complex (MHC) binding affinity through in silico analyses. The 32-62, 77-105, and 125-165 residues of the TMEM31 were selected as the immunodominant fragments. They were linked together by RVRR and HEYGAEALERAG motifs to improve epitopes separation and presentation. In addition, to activate helper T lymphocytes (HTL), Pan HLA DR-binding epitope (PADRE) peptide sequence and tetanus toxin fragment C (TTFrC) were incorporated into the final construct. Also, the Beta-defensin conserved domain was utilized in the final construct as a novel adjuvant for Toll-like receptor 4/myeloid differentiation factor (TLR4-MD) activation. The CTL epitopes, cleavage sites, post-translational modifications, TAP transport efficiency, and B cells epitopes were predicted for the peptide vaccine. The final construct contained multiple CTL and B cell epitopes. In addition, it showed 93.55% and 99.13% population coverage in the world for HLA I and HLA II, respectively. According to these preliminary results, the multiepitope cancer vaccine can be an appropriate choice for further experimental investigations.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

Our previous study demonstrated an indispensable role of Peyer’s patches (PPs) for the induction of antigen-specific secretory (S)IgA antibody responses after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella-Tox C). In this study, we defined the PP lymphoid structures and immune cells required for the induction of mucosal SIgA antibody responses. Adoptive transfer of mononuclear cells (MNCs) from PPs into PP-deficient (PP-null) mice failed to elicit tetanus toxoid (TT)-specific mucosal immunity. However, when the same PP MNCs were transferred into lethally irradiated PP-normal recipient mice, PP MNCs preferentially emigrated to recipient PPs, leading to PP lymphoid structures and TT-specific SIgA antibody responses. Significantly reduced numbers of TT-specific IgA antibody-forming cells were detected in the mesenteric lymph nodes (MLNs) and intestinal lamina propria of mice when surface expression of the sphingosine 1-phosphate receptor on lymphocytes was inhibited by its agonist FTY720. However, FTY720 treatment did not alter dendritic cell migration or Salmonella dissemination into these tissues. When rSalmonella-Tox C-stimulated CD4+ T cells isolated from PPs, MLNs and the spleen were co-cultured with B cells from these tissues, significantly increased levels of TT-specific IgA antibody responses were exclusively induced in cultures containing PP B cells. Furthermore, surface IgA+ PP B cells produced TT-specific IgA antibody responses in vitro. These findings suggest that PP lymphoid structures and surface IgA+ PP B cells are essential elements for the induction of antigen-specific intestinal SIgA antibody responses to oral Salmonella.

Along with robust immunogenicity, an ideal vaccine candidate should be able to produce a long lasting protection. In this regard, the frequency of memory B-cells is possibly an important factor in memory B-cell persistency and duration of immunological memory. On this basis, binding domains of tetanus toxin (HcT), botulinum type A1 toxin (HcA), and heat-labile toxin (LTB) were selected as antigen models that induced long-term, midterm and short-term immune memory, respectively. In the present study, the frequency of total memory B-cells after immunization with HcT, HcA and LTB antigens after 90 and 180 days, and also after one booster, in 190 days, was evaluated. The results showed a significant correlation between frequency of total memory B-cells and duration of humoral immunity. Compared to other antigens, the HcT antibody titers and HcT total memory B-cell populations were greater and persistent even after 6 months. At 6 months after the final immunization, all HcT- and HcA-immunized mice survived against tetanus and botulinum toxins, and also LT toxin binding to GM1 ganglioside was blocked in LTB-immunized mice. We conclude the frequency of memory B-cells and their duration are likely a key factor for vaccine memory duration.

Transmembrane member 16A (TMEM16A) is the Ca2+-activated chloride channel in airways and intestine. It has been associated with goblet cell metaplasia, as expression of TMEM16A is strongly up-regulated in cystic fibrosis and asthma during mucus hypersecretion. However, the possible role of TMEM16A for mucus production or mucus secretion remains obscure, and whether TMEM16A controls the function of intestinal goblet cells is entirely unknown. Basal mucus secretion in lungs occurs through low levels of ATP in the airway surface liquid. Here, we report for the first time that TMEM16A is essential for basal secretion of mucus in airways and intestine. Airway-ciliated and intestinal epithelial-specific knockout of TMEM16A ( TMEM16Aflox/floxFoxJ1, TMEM16Aflox/floxVil1) leads to accumulation of mucus in airway club (Clara) cells and intestinal goblet cells, respectively. Acute ATP-induced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly as a result of compromised release of prosecretory cytokines. Knockdown or inhibition of TMEM16A in human Calu3 airway epithelial cells indicates compromised IL-8 release. In intestinal goblet cells lacking expression of TMEM16A, mucus accumulates as a result of compromised ATP-induced secretion. In contrast, cholinergic mucus secretion by compound exocytosis is independent of TMEM16A. The data demonstrate a previously unrecognized role of TMEM16A for membrane exocytosis and describe a novel, ATP-driven pathway for intestinal mucus secretion. We conclude that ATP-dependent mucus secretion in both airways and intestine requires TMEM16A. The present results may form the basis for a novel, therapeutic approach for the treatment of mucus hypersecretion in inflammatory airway and intestinal disease.-Benedetto, R., Cabrita, I., Schreiber, R., Kunzelmann, K. TMEM16A is indispensable for basal mucus secretion in airways and intestine.

Tetanus is a fatal disease caused by tetanus neurotoxin (TeNT). TeNT is composed of a light chain (Lc) and a heavy chain, the latter of which is classified into two domains, N-terminus Hn and C-terminus Hc. Several TeNT-neutralizing antibodies have been reported, but it remains unclear which TeNT domains are involved in neutralization. To further understand the mechanism of these antibodies, we isolated TeNT-reactive human antibody clones from peripheral blood mononuclear cells. We then analyzed the reactivity of the isolated antibody clones to each protein domain and their inhibition of Hc-ganglioside GT1b binding, which is critical for TeNT toxicity. We also investigated the TeNT-neutralizing ability of isolated antibody clones and showed that an Hn-reactive clone protected strongly against TeNT toxicity in mice. Furthermore, combination treatment of Hn-reactive antibody clones with both Hc-reactive and TeNT mix (the mixture of Hc, Hn, and Lc proteins)–reactive antibody clones enhanced the neutralizing effect. These results indicated that antibody clones targeting Hn effectively neutralized TeNT. In addition, the use of a cocktail composed of Hc-, Hn-, and TeNT mix–reactive antibodies provided enhanced protection compared to the use of each antibody alone.

The seemingly random and unpredictable nature of seizures is a major debilitating factor for people with epilepsy. An increasing body of evidence demonstrates that the epileptic brain exhibits long-term fluctuations in seizure susceptibility, and seizure emergence seems to be a consequence of processes operating over multiple temporal scales. A deeper insight into the mechanisms responsible for long-term seizure fluctuations may provide important information for understanding the complex nature of seizure genesis. In this study, we explored the long-term dynamics of seizures in the tetanus toxin model of temporal lobe epilepsy. The results demonstrate the existence of long-term fluctuations in seizure probability, where seizures form clusters in time and are then followed by seizure-free periods. Within each cluster, seizure distribution is non-Poissonian, as demonstrated by the progressively increasing inter-seizure interval (ISI), which marks the approaching cluster termination. The lengthening of ISIs is paralleled by: increasing behavioral seizure severity, the occurrence of convulsive seizures, recruitment of extra-hippocampal structures and the spread of electrographic epileptiform activity outside of the limbic system. The results suggest that repeated non-convulsive seizures obey the ‘seizures-beget-seizures’ principle, leading to the occurrence of convulsive seizures, which decrease the probability of a subsequent seizure and, thus, increase the following ISI. The cumulative effect of repeated convulsive seizures leads to cluster termination, followed by a long inter-cluster period. We propose that seizures themselves are an endogenous factor that contributes to long-term fluctuations in seizure susceptibility and their mutual interaction determines the future evolution of disease activity.

In the aging process, the brain presents biochemical and morphological alterations. The neurons of the limbic system show reduced size dendrites, in addition to the loss of dendritic spines. These disturbances trigger a decrease in motor and cognitive function. Likewise, it is reported that during aging, in the brain, there is a significant decrease in neurotrophic factors, which are essential in promoting the survival and plasticity of neurons. The carboxyl-terminal fragment of the heavy chain of the tetanus toxin (Hc-TeTx) acts similarly to neurotrophic factors, inducing neuroprotection in different models of neuronal damage. The aim here, was to evaluate the effect of Hc-TeTx on the motor processes of elderly mice (18 months old), and its impact on the dendritic morphology and density of dendritic spines of neurons in the limbic system. The morphological analysis in the dendrites was evaluated employing Golgi-Cox staining. Hc-TeTx was administered (0.5 mg/kg) intraperitoneally for three days in 18-month-old mice. Locomotor activity was evaluated in a novel environment 30 days after the last administration of Hc-TeTx. Mice treated with Hc-TeTx showed significant changes in their motor behavior, and an increased dendritic spine density of pyramidal neurons in layers 3 and 5 of the prefrontal cortex in the hippocampus, and medium spiny neurons of the nucleus accumbens (NAcc). In conclusion, the Hc-TeTx improves the plasticity of the brain regions of the limbic system of aged mice. Therefore, it is proposed as a pharmacological alternative to prevent or delay brain damage during aging.

We present the hypothesis to the scientific community actively designing clinical trials and recommending public health guidelines to control the pandemic that – “Tetanus vaccination may be contributing to reduced severity of the COVID-19 infection” – and urge further research to validate or invalidate the effectiveness of the tetanus toxoid vaccine against COVID-19. This hypothesis was revealed by an explainable artificial intelligence system unleashed on open public biomedical datasets. As a foundation for scientific rigor, we describe the data and the artificial intelligence system, document the provenance and methodology used to derive the hypothesis and also gather potentially relevant data/evidence from recent studies. We conclude that while correlations may not be reason for causation, correlations from multiple sources is more than a serendipitous co-incidence that is worthy of further and deeper investigation.

International audience; Tetanus vaccination is of major importance for public health in most countries in the world. The World Health Organization indicated that 15,000 tetanus cases were reported in 2018 (Organization, World Health, 2019). Currently, vaccine manufacturers use tetanus toxin produced by Clostridium tetani fermentation in complex media. The complex components, commonly derived from animal sources, introduce potential variability in cultures. To achieve replicable fermentation and to avoid toxic or allergic reactions from animal-source compounds, several studies have tried to switch from complex to chemically defined media without affecting toxin titers. The present review introduces the current knowledge on i) C. tetani strain diversity, whole-genome sequences and metabolic networks; ii) toxin regulation and synthesis; and iii) culture media, cultivation processes and growth requirements. We critically reviewed the reported data on metabolism in C. tetani and completed comparative genomic and proteomic analyses with other Clostridia species. We integrated genomic data based on whole-genome sequence annotation, supplemented with cofactor specificities determined by protein sequence identity, in a new map of C. tetani central metabolism. This is the first data review that integrates insights from omics experiments on C. tetani. The overview of C. tetani physiology described here could provide support for the design of new chemically defined media devoid of complex sources for toxin production.

Summary Four potent native human monoclonal antibodies (mAbs) targeting distinct epitopes on tetanus toxin (TeNT) are isolated with neutralization potency ranging from approximately 17 mg to 6 mg each that are equivalent to 250 IU of human anti-TeNT immunoglobulin. TT0170 binds fragment B, and TT0069 and TT0155 bind fragment AB. mAb TT0067 binds fragment C and blocks the binding of TeNT to gangliosides. The co-crystal structure of TT0067 with fragment C of TeNT at a 2.0-A resolution demonstrates that mAb TT0067 directly occupies the W pocket of one of the receptor binding sites on TeNT, resulting in blocking the binding of TeNT to ganglioside on the surface of host cells. This study reveals at the atomic level the mechanism of action by the TeNT neutralizing antibody. The key neutralization epitope on the fragment C of TeNT identified in our work provides the critical information for the development of fragment C of TeNT as a better and safer tetanus vaccine.

Patient: Male, 59-year-old Final Diagnosis: Tetanus Symptoms: Acute respiratory failure • facial paralysis • neck stiffness • trismus Medication: — Clinical Procedure: Bronchoscopy Specialty: Critical Care Medicine • Pulmonology Objective: Unusual clinical course Background: Tetanus is a potentially fatal infectious disease which, during its evolution, creates multiple complications, usually requiring intensive management and care. Case Report: We present a clinical case of a 59-year-old male patient with generalized tetanus admitted to the intensive care unit. Flexible bronchoscopy revealed contraction of the bronchial demonstrating that tetany existed at the respiratory level, which rarely becomes evident. The clinical manifestations included trismus, facial paralysis, neck stiffness, and compromised respiratory function. The patient presented a state of respiratory failure that required invasive mechanical ventilation which was evaluated by bronchoscopy and that showed spasms of the bronchial musculature. The patient presented generalized tetanus in which the bronchial affectation was evaluated by bronchoscopy in the intensive care unit. In developed countries, the anti-tetanus toxoid vaccine has ostensibly decreased its incidence, while it is endemic in developing countries, and although there are measures such as vaccination that try to reduce its incidence, in Ecuador there is an increase in incidences. In this patient case, contraction of the bronchial rings was observed, demonstrating that tetany existed at the respiratory level, which rarely becomes evident. Conclusions: Although muscular contractions are widespread, this clinical case evidences bronchial spams reported and visualized by bronchoscopy.

Tetanus is an acute, fatal disease caused by exotoxin produced by Clostridium tetani. The current vaccine against tetanus is based on inactivated tetanus toxin (TeNT). To develop a recombinant TeNT vaccine suitable for replacement of full-length tetanus toxoid (TT) vaccine for use in humans, a recombinant non-tagged isoform of the Hc domain of the tetanus toxin (THc) was expressed in Escherichia coli and purified by sequential chromatography steps. The immunogenicity and protective effect of the THc antigen were explored and compared with those of TT in Balb/c mice. The THc-based subunit vaccine provided complete protection against TeNT challenge following a high dosage as a toxoid vaccine. While the anti-THc and neutralising antibody titres were higher for the THc-based vaccine than the TT vaccine because protective epitopes are located on the THc domain. Frequency- and dose-dependent immunoprotection were also observed in THc-immunised mice. Mice immunised with one injection of 1 μg or 4 μg THc antigen were completely protected against 102 or 103 50% mouse lethal dose (LD50) of TeNT, respectively. Furthermore, the THc protein was found to recognise and bind to ganglioside GT1b in a dose-dependent manner, and anti-THc sera antibodies also inhibited binding between THc and GT1b. Antigen on the form of recombinant non-tagged THc domain expressed in E. coli achieved strong immunoprotective potency, suggesting that it could be developed into a candidate subunit vaccine against tetanus as an alternative to the current TT vaccine.

Tetanus is a fatal disease caused by Clostridium tetani infections. To prevent infections, a toxoid vaccine, developed almost a century ago, is routinely used in humans and animals. The vaccine is listed in the World Health Organisation list of Essential Medicines and can be produced and administered very cheaply in the developing world for less than one US Dollar per dose. Recent developments in both analytical tools and frameworks for systems biology provide industry with an opportunity to gain a deeper understanding of the parameters that determine C. tetani virulence and physiological behaviour in bioreactors. Here, we compared a traditional fermentation process with a fermentation medium supplemented with five heavily consumed amino acids. The experiment demonstrated that amino acid catabolism plays a key role in the virulence of C. tetani. The addition of the five amino acids favoured growth, decreased toxin production and changed C. tetani morphology. Using time-course transcriptomics, we created a "fermentation map", which shows that the tetanus toxin transcriptional regulator BotR, P21 and the tetanus toxin gene was downregulated. Moreover, this in-depth analysis revealed potential genes that might be involved in C. tetani virulence regulation. We observed differential expression of genes related to cell separation, surface/cell adhesion, pyrimidine biosynthesis and salvage, flagellar motility, and prophage genes. Overall, the fermentation map shows that, mediated by free amino acid concentrations, virulence in C. tetani is regulated at the transcriptional level and affects a plethora of metabolic functions.

Clostridium tetani produces a potent neurotoxin, the tetanus toxin (TeNT), which is responsible for an often-fatal neurological disease (tetanus) characterized by spastic paralysis. Prevention is efficiently acquired by vaccination with the TeNT toxoid, which is obtained by C. tetani fermentation and subsequent purification and chemical inactivation. C. tetani synthesizes TeNT in a regulated manner. Indeed, the TeNT gene (tent) is mainly expressed in the late exponential and early stationary growth phases. The gene tetR (tetanus regulatory gene), located immediately upstream of tent, encodes an alternative sigma factor which was previously identified as a positive regulator of tent. In addition, the genome of C. tetani encodes more than 127 putative regulators, including 30 two-component systems (TCSs). Here, we investigated the impact of 12 regulators on TeNT synthesis which were selected based on their homology with related regulatory elements involved in toxin production in other clostridial species. Among nine TCSs tested, three of them impact TeNT production, including two positive regulators that indirectly stimulate tent and tetR transcription. One negative regulator was identified that interacts with both tent and tetR promoters. Two other TCSs showed a moderate effect: one binds to the tent promoter and weakly increases the extracellular TeNT level, and another one has a weak inverse effect. In addition, CodY (control of dciA (decoyinine induced operon) Y) but not Spo0A (sporulation stage 0) or the DNA repair protein Mfd (mutation frequency decline) positively controls TeNT synthesis by interacting with the tent promoter. Moreover, we found that inorganic phosphate and carbonate are among the environmental factors that control TeNT production. Our data show that TeNT synthesis is under the control of a complex network of regulators that are largely distinct from those involved in the control of toxin production in Clostridium botulinum or Clostridium difficile.

We illuminate a possible explanatory pathophysiologic mechanism for retinal cellular neuropathy by means of a novel diagnostic method using ophthalmoscopic imaging and a molecular imaging agent targeted to fast axonal transport. The retinal neuropathies are a group of diseases with damage to retinal neural elements. Retinopathies lead to blindness but are typically diagnosed late, when substantial neuronal loss and vision loss have already occurred. We devised a fluorescent imaging agent based on the non-toxic C fragment of tetanus toxin (TTc), which is taken up and transported in neurons using the highly conserved fast axonal transport mechanism. TTc serves as an imaging biomarker for normal axonal transport and demonstrates impairment of axonal transport early in the course of an N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinopathy model in rats. Transport-related imaging findings were dramatically different between normal and retinopathic eyes prior to presumed neuronal cell death. This proof-of-concept study provides justification for future clinical translation.

During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; GlyVMMneurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of GlyVMMneurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; GluSLDneurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcolepticorexin-ataxin-3mice. We also showed that GlyVMMneurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENTWe identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy.

During World War I (WWI), infectious diseases including tetanus were among the most important causes of death. Even though its efficacy was somewhat controversial before the war, tetanus antiserum played a key role in reducing the mortality of this disease. A vial of tetanus antiserum dating back from WWI, left behind on the French battlefield by the US Army, was borrowed from a private collection and opened. The serum contained within was characterized by orthogonal biochemical techniques to determine if any neutralizing IgGs could remain after 100 years of storage. In vitro analysis by Size Exclusion Chromatography and Serum Protein Electrophoresis suggested the presence of residual IgG. In spite of our hopes, these IgGs were not able to protect mice against tetanus toxin challenge in a neutralizing assay. Even though our results indicate the presence of remaining IgGs inside the serum, they were functionally disabled. These results show that obscurity alone is insufficient to protect IgGs from degradation over very long periods of time at room temperature. HIGHLIGHTS: Tetanus antiserum found its place in the therapeutic arsenal during World War I A century-old vial of tetanus antiserum was opened for biochemical and in vivo characterization Biochemical assays revealed the presence of proteins having all the characteristics of IgGs The serum was unable to protect mice against toxinic challenge.

Cervical cancer is the fourth most common cancer in women with an estimated 570,000 new cases in 2018 which constitute about 6. 6% of all cancers in women according to WHO report 2018. Approximately 90% of the 270,000 deaths from cervical cancer in 2015 occurred in low- and middle-income countries. In cervical cancers, which is caused by human papillomavirus (HPV) infection, the expression of HPV 16 E6 and E7 proteins are essential for tumor cell transformation and maintenance of malignancy. Prophylactic vaccines against cervical cancer caused by human papillomavirus have not proven successful. Although virus-like particle-based (VLPs) vaccines have been developed with prophylactic activities to prevent most HPV infections, the therapeutic effect of VLP vaccines has yet to be demonstrated for those who were already infected. A recent study showed that pre-conditioning mice with a potent antigen such as tetanus toxoid significantly improves lymph node homing and efficacy of dendritic cells. Tetanus toxoid has also been used in combination with DNA vaccines designed from tumor based antigens. In the present study, we pre-conditioned mice with tetanus toxoid followed by vaccination with a Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) overexpressing tumor-cell based vaccine (GVAX). We observed that pre-conditioning with tetanus toxoid followed by vaccination with GVAX regressed tumor growth and enhanced the overall survival of the mice. Pre-conditioning with tetanus toxoid enhanced the immune response which was observed by enlarged spleen size, higher proliferation rate of lymphocytes, a higher level of IFN-γ, TNF-α, and IL-4 antigen-specific secretions by the splenocytes. Pre-conditioning with tetanus toxoid increased memory T cell migration into the tumor site and spleen. The antigen-specific cytotoxic T cell lysis percentage was also found to be higher in the group of mice vaccinated with the combination of tetanus toxoid and GVAX. Hence, pre-conditioning with tetanus toxoid prior to vaccination with a tumor-cell based vaccine overexpressing GM-CSF might be an effective strategy for targeting E7-specific HPV-associated cervical malignancy.

International audience; Tetanus is a neurologic disease of humans and animals characterized by spastic paralysis. Tetanus is caused by tetanus toxin (TeNT) produced by Clostridium tetani, an environmental soilborne, gram-positive, sporulating bacterium. The disease most often results from wound contamination by soil containing C. tetani spores. Horses, sheep, and humans are highly sensitive to TeNT, whereas cattle, dogs, and cats are more resistant. The diagnosis of tetanus is mainly based on the characteristic clinical signs. Identification of C. tetani at the wound site is often difficult.

Study design Case series on four dogs. Objectives To determine the alleviation of motor symptoms in spinal cord injury (SCI) by tetanus neurotoxin (TeNT). Setting Different Berlin veterinary clinics, Germany. Methods We report on the effect of intramuscular injections of low-dose TeNT into paretic hind limb muscles 2–157 weeks after SCI due to lumbar disc herniation in a clinical case series on four dogs. All dogs underwent unsuccessful or incomplete surgical decompression prior to TeNT treatment. TeNT was injected on a compassionate basis. Stance, gait ability and the diameter of the rectus femoris muscle were assessed as parameters. Results All four dogs improved their stance and three of these dogs improved in gait at 4 and 6 weeks after TeNT injections without evidence of side effects or spreading of TeNT effects. At the same time, the size of the rectus femoris muscle diameter increased considerably as compared with baseline (baseline: 100%; 4 weeks: 148.7% ± 10.9%; 6 weeks: 137.1% ± 7.9%). Conclusions Facilitation of α-motor neurons by TeNT injections into paretic hind limb muscles of four dogs improved standing and/or gait abilities and partly reversed muscle atrophy after SCI. The absence of generalized or painful muscle spasms supports the safety of low-dose TeNT. Therefore, TeNT might evolve as a promising therapeutic option for muscle paresis of central origin, e.g. in individuals with SCI, stroke or multiple sclerosis.

We built a regression model to describe the progress of epileptogenesis in a rat intrahippocampal tetanus toxin (TeNT) epilepsy model by identifying informative neural features from hippocampal local field potentials (LFPs). The LFPs were recorded from the awake and freely behaving animals during the latent period and the active-seizure period. Frequency domain neural features including power spectral density, coherence and phase coherence were calculated from the hippocampal LFPs. A least angle regression with elastic net regularization (LARS-ENR) model successfully predicted a relative day from the first seizure in multiple rats (R2 test = 0.724±0.025). By leveraging a characteristic of LARS-ENR which reduces unnecessary features, we identified the neural features related to epileptogenesis in a TeNT model.

Background: Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine. Objective: In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems. Methods: The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice. Results: The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity. Conclusion: The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

The carboxyl-terminal domain of the heavy chain of tetanus toxin (Hc-TeTx) exerts a neuroprotective effect in neurodegenerative diseases via the activation of signaling pathways related to neurotrophins, and also through inhibiting apoptotic cell death. Here, we demonstrate that Hc-TeTx preserves motoneurons from chronic excitotoxicity in an in vitro model of amyotrophic lateral sclerosis. Furthermore, we found that PI3-K/Akt pathway, but not p21ras/MAPK pathway, is involved in their beneficial effects under chronic excitotoxicity. Moreover, we corroborate the capacity of the Hc-TeTx to be transported retrogradely into the spinal motor neurons and also its capacity to bind to the motoneuron-like cell line NSC-34. These findings suggest a possible therapeutic tool to improve motoneuron preservation in neurodegenerative diseases such as amyotrophic lateral sclerosis.

Background Spore-forming bacteria of the Bacillus genus are widely used probiotics known to exert their beneficial effects also through the stimulation of the host immune response. The oral delivery of B. toyonensis spores has been shown to improve the immune response to a parenterally administered viral antigen in mice, suggesting that probiotics may increase the efficiency of systemic vaccines. We used the C fragment of the tetanus toxin (TTFC) as a model antigen to evaluate whether a treatment with B. toyonensis spores affected the immune response to a mucosal antigen. Results Purified TTFC was given to mice by the nasal route either as a free protein or adsorbed to B. subtilis spores, a mucosal vaccine delivery system proved effective with several antigens, including TTFC. Spore adsorption was extremely efficient and TTFC was shown to be exposed on the spore surface. Spore-adsorbed TTFC was more efficient than the free antigen in inducing an immune response and the probiotic treatment improved the response, increasing the production of TTFC-specific secretory immunoglobin A (sIgA) and causing a faster production of serum IgG. The analysis of the induced cytokines indicated that also the cellular immune response was increased by the probiotic treatment. A 16S RNA-based analysis of the gut microbial composition did not show dramatic differences due to the probiotic treatment. However, the abundance of members of the Ruminiclostridium 6 genus was found to correlate with the increased immune response of animals immunized with the spore-adsorbed antigen and treated with the probiotic. Conclusion Our results indicate that B. toyonensis spores significantly contribute to the humoral and cellular responses elicited by a mucosal immunization with spore-adsorbed TTFC, pointing to the probiotic treatment as an alternative to the use of adjuvants for mucosal vaccinations.

Oligoclonal combinations of several monoclonal antibodies (MAbs) are being considered for the treatment of various infectious pathologies. These combinations are less sensitive to antigen structural changes than individual MAbs; at the same time, their characteristics can be more efficiently controlled than those of polyclonal antibodies. The main goal of this study was to evaluate the binding characteristics of six biclonal equimolar preparations (BEP) of tetanus toxin (TeNT)-specific MAbs and to investigate how the MAb combination influences the BEPs' protective capacity. We show that a combination of TeNT-specific MAbs, which not only bind TeNT but also exert positive cooperative effects, results in a BEP with superior binding characteristics and protective capacity, when compared with the individual component MAbs. Furthermore, we show that a MAb with only partial protective capacity but positive effects on the binding of the other BEP component can be used as a valuable constituent of the BEP. (C) 2018 Elsevier Ltd. All rights reserved.

Transient instruction changes migration The brain neocortex is built by waves of neurons migrating from deep within the brain to the surface layers. Ohtaka-Maruyama et al. found that a layer of neurons that multipolar neurons encounter on their travels instructs the migrating neurons to change phenotype and direction (see the Perspective by Schinder and Lanuza). These subplate neurons form transient glutamatergic synapses with the immature migrants. This results in the migrating multipolar neurons becoming bipolar, more directed, and faster in their final migrations. Science , this issue p. 313 ; see also p. 265

The in vitro potency of botulinum neurotoxin (BoNT) serotypes is often measured by monitoring cleavage of their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein substrates. A frequently used method is Western blot, whereby the full-length protein and cleaved form migrate at different molecular weights. Until now, it has been extremely difficult to detect the cleaved cellular form of the SNARE protein vesicle associated membrane protein 1, 2 or 3 (VAMP1, 2 or 3) by Western blot. These VAMP isoforms are the substrates of BoNT serotypes BoNT/B, D, F and G as well as tetanus neurotoxin. Using custom made anti-VAMP antibodies against epitopes either side of the cleavage sites for BoNT/B, BoNT/D and BoNT/F, we have successfully detected the cleaved C-terminal VAMP fragment in cortical neurons. These new antibodies enable quantitative assessment of the potency of VAMP-cleaving neurotoxins by a gain of signal Western blot assay.