Rozprawy doktorskie na temat „Tetanus”

This thesis describes the immunization protocols for the production of antibodies against tetanus toxin and toxoid in guinea pigs and mice. Antibodies were successfully raised against the toxin without mortalities in either species. The murine sera obtained, were isotyped by ELISA and the toxin was proven to be a superior antigen in eliciting production of IgG$\rm\sb{2a}$ and IgG$\sb3$. The two isotypes which have demonstrated antitumor activity. The anti-toxoid sera exhibited a lower reactivity towards the toxin and toxoid when compared with anti-toxin sera. The reactivity of recombinant tetanus toxin fragment C was studied and the results indicated that in the murine serum, 72% of anti-toxin or anti-toxoid antibodies were directed against epitopes on fragment C. The study of the guinea pig sera suggested that similar to mouse serum, it can develop in response to toxin as an antigen, antibodies against toxin which are mostly directed against the fragment C portion. On the other hand. guinea pigs seem to respond to the toxoid as an antigen by producing antibodies to more than fragment C. (Abstract shortened by UMI.)

Although efforts have been made towards improving the health of children across the globe with notable results, neonatal tetanus (NNT) remains a major contributor to the neonatal death rates in Nigeria. This problem calls for a concerted effort by the government to achieve the revised global NNT elimination deadline of 2015. The purpose of this cross-sectional quantitative study using secondary data was to establish the prevalence of NNT in Nigeria's northeast region and to ascertain if there was any significant difference in frequency of antenatal care (ANC), trained traditional birth attendants (TBAs), and umbilical cord treatments, using single sample proportions test and chi-squared tests of independence. The framework for this research was the theory of planned behavior. The participants (N = 312) were mothers of NNT babies. In spite a continual decline in the NNT cases between 2010 (26%) and 2013 (9%), the prevalence rate of NNT was unacceptably high at 28.815%. Also, significant differences existed as mothers who gave birth to NNT babies received significantly fewer or no ANC (p < 0.001), received significantly fewer or no attention from TBAs (p < 0.001), and reported significantly fewer incidences of proper umbilical cord treatments (p < 0.001). The chi-squared tests of independence resulted in significant differences in the frequencies of mothers who received ANC between Nigerian provinces (p < 0.001) and mothers who had their baby's umbilical cord treated (p = 0.005). This study will contribute to social change by guiding health care policy makers and immunization program managers on maternal and newborn health care services and indicate ways to build capacity of the TBAs for safe home delivery/hygienic handling of umbilical cord of newborns.

Transcutaneous immunisation (TCI) is a novel needle-free method of vaccine delivery, which involves the application of soluble antigens onto the surface of intact skin. In this thesis, the immunogenicity and neutralising potency of tetanus neurotoxin (TeNT) fragments were compared to that of tetanus toxoid (TTxd) following TCI. In addition, to understand the mechanisms of induction of immune response by TCI, in vitro and in vivo interaction of tetanus proteins with skin immune cells was also investigated. The 50kDa recombinant carboxyl-terminal fragment of tetanus toxin (HCWT) and a 50kDa HC mutant(HCM115) which is devoid of neuronal binding properties, were expressed and purified by affinity chromatography, and excess endotoxin removed by size exclusion chromatography. Mice immunized with HCWT, in the absence of adjuvant, induced the highest anti-toxoid and anti-HCWT antibody titers, with significant increases in the toxin neutralising antibody response when compared with TTxd. In vitro studies demonstrated that both HC fragments and TTxd were capable of up regulating the surface expression of activation marker ICAM-1 on murine bone-marrow derived dendritic cells (DC), but not on the human keratinocyte cell line HaCaT. Real-time reverse transcriptase-PCR (RealtimeRT-PCR) analysis showed that TNF-α expression was up regulated in vivo as early as 10 minutes following TCI, and this was caused by shaving rather than by tetanus proteins themselves. Immunohistochemistry staining to monitor the translocation of HCWT fragment and TTxd through the skin following TCI showed that HCWT protein could be detected in both the epidermis and dermis within four hours post-application. However, TTxd translocation appeared to be much slower in comparison. Collectively, the results presented in this thesis suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens, which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunisation.

The incidence and mortality rates of neonatal tetanus (NNT) remain underreported in Nigeria. This cross-sectional study was guided by the Mosley and Chen's model for the elements of child survival in developing countries. The goals of the study were twofold: (a) to compare the NNT prevalence and the mortality rates from the existing surveillance system and active surveillance of health facility records in 7 selected health facilities from 2010 to 2014 in Katsina state, Nigeria and (b) to assess the associations between selected NNT risk factors, number of maternal tetanus toxoid injections, frequency of antenatal visits, place of delivery, and cord care, and neonatal mortality as the outcome variable. Data from 332 NNT records were extracted through retrospective records review and analyzed using a logistic regression model. The prevalence of NNT and mortality rate were 336 cases and 3.4 deaths per 100,000 population, respectively, while the prevalence of NNT and mortality rate reported through the IDSR system were 111 cases and 1.0 death per 100,000 population, respectively. Only neonates whose mothers had 1 dose of tetanus toxoid vaccine were significantly associated with NNT mortality, (p < 0.05), OR = 4.12, 95% CI [1.04, 16.29]. Frequency of antenatal visits, place of delivery, and cord care were all not significant predictors of NNT mortality. Implications for positive social change include gaining knowledge on associations between NNT risk factors and neonatal mortality, and strengthening the NNT surveillance system with the capacity for early detection of potential risk factors to develop specific public health interventions aimed at improving the outcome of neonatal tetanus.

Tetanus neurotoxin (TeNT) is among the most poisonous substances on Earth and a major cause of neonatal death in non-vaccinated areas. There are approximately 300,000 cases reported worldwide each year, and the mortality rate is between 10-20%. In this work, I identified an extracellular matrix protein receptor for TeNT at the neuromuscular junction (NMJ) and developed a peptide inhibitor, which prevents tetanic paralysis in vivo in mice. TeNT binds to the NMJ with an extremely high affinity, yet the nature of its receptor complex was poorly understood. I showed that the presence of nidogens (also known as entactins) at the NMJ is the main determinant for TeNT binding. Nidogens are extracellular matrix (ECM) proteins, which are taken up into the endosomal carriers containing tetanus toxin binding fragment (HCT) in motor neurons. Inhibition of the HCT-nidogen interaction using a peptide originating from nidogen-1 abolishes HCT binding on these cells. TeNT causes slowly progressing local tetanus when it is injected intramuscularly into the triceps surae muscle in a low dose. When preincubated with the peptide originating from nidogen-1, TeNT injection does not alter the coordination of mice and the muscle force remains largely unchanged. Genetic ablation of nidogens prevented the binding of TeNT to neurons and the intact NMJ and protected mice from TeNT induced spastic paralysis. In my thesis I demonstrated for the first time, that an ECM protein accumulates and presents a neurotropic pathogen to the presynapse. This study follows recent studies showing that growth factors trigger downstream signalling more efficiently if they bind to certain ECM components – a new and rising concept in neuroscience.

Tetanus toxin exerts its primary biological effect by impairing the release of inhibitory neurotransmitters in the central nervous system; the mechanism of this blockade, however, remains unknown. Studies using adrenal medullary chromaffin cells, which are closely related to the toxin's target neurones but much more accessible to biochemical investigation, have the potential to provide information on various aspects of the intoxication process of tetanus. Nicotine-evoked (but not basal) secretion of catecholamines from intact chromaffin cells was inhibited by tetanus toxin in a dose-dependent fashion up to a maximum of 75%, half-maximal inhibition being achieved at 0.7 nM toxin (in single- or double-chain form). The time course of this inhibition was long, approximately 16 hours. Catecholamine release evoked by Ba²⁺ ions was not affected by the toxin, while its effect on K⁺ -evokedsecretion was never greater than 30%. Pre-incubation ofcells with ganglioside GT1, a specific ligand of the toxin, in the absence of toxin itself inhibited nicotine-evoked release of catecholamines (50&37 inhibition with 24 uM GT1), making it impossible to evaluate the role of GT1 in mediating the action of the toxin. Tetanus toxin (radiolabelled) was also shown to bind in a specific fashion to chromaffin cells. Toxin binding under less physiological conditions of pH and ionic strength was a higher capacity (B_max 0.7-1.2 pmol/mg protein) than that found under more physiological conditions (B_max 0.2-0.3 pmol/mg protein); this is also characteristic of toxin binding to synaptic membrances. In both cases there appeared to be at least two components to toxin binding, a higher affinity component with a K_d value of approximately 1nM (which did not account for more than 20% of total binding capacity), and a lower affinity binding with a K_d value of 10-25 nM. As expected, pre-incubationof chromaffin cells with ganglioside GT1 enhanced their toxin-binding capacity, but did not noticeably affect K_d value, perhaps indicating that gangliosides mediate binding of tetanus toxin to untreated chromaffin cells. This was further suggested by the finding that neuraminidase treatment of cells markedly reduced toxin-binding capacity (by 50% at pH 7.4, 90% at pH 6.0) and also by the observation that pre-incubation of ¹²⁵I-labelled tetanus toxin with GT1 led to a reduction in its binding to chromaffin cells. Mild trypsinisation of chromaffin cells completely abolished toxin-binding under both sets of conditions, suggesting the involvement of a protein component in toxin binding also.

Chelating Recombinant Antibodies (CRAbs) are a form of high affinity tandem single chain antibody (scFv) with two component scFvs which target non‐overlapping epitopes on the same antigen molecule. The optimised inter‐scFv linker allows simultaneous engagement of the scFvs resulting in a synergistic improvement in affinity. High affinity is a desirable characteristic in therapeutic antibodies since it strongly correlates with improved potency. Building upon previous work, this thesis describes the development of a phage‐display based approach toward isolating CRAbs with both the optimal scFv pairing and inter‐scFv linker using tetanus toxin as a model antigen. This would circumvent the need for structural data and complement the traditional approaches used to enhance antibody affinity. Fifteen scFvs specific for the C‐terminal sub‐domain of the tetanus toxin heavy chain (TT‐Hc) were characterised in terms of overall and soluble expression levels, sequence and bioinformatics features, phage‐display propensity and toxin neutralisation potency. Seven clones were identified for further analysis by affinity measurements, oligomerisation analysis and quantitative toxin neutralisation capacity. These characteristics varied significantly between clones including the affinity (KD) which varied, from 10nM to over 1000nM. ScFv characterisation revealed a strong positive correlation between phage display propensity and overall expression levels as well as a strong correlation between clone affinity and potency. Multiple scFv pairings hypothetically capable of chelation were identified by competition binding analysis and many of these clones exhibited cooperative binding effects when added in combination to TT‐Hc. A tandem‐scFv library was constructed linking seven of the characterised scFv clones (C1, C2, C4, J2, J4, N4 and N5) in all 49 possible combinations and permutations each with 7 inter‐scFv linker lengths. The inter‐scFv linkers harboured some randomised codons giving a total diversity of 5x107 clones. The library contained a mixture of hypothetically chelating and competing scFv pairings. Carefully optimised affinity driven phage display which included off‐rate selection enriched the anti‐TT‐Hc tandem‐scFv library to up to 90% chelating clones from under 50% after just two‐rounds of selection with a single clone, C4‐N4, dominating around 30% of the output. This clone was classified as hypothetically chelating from the scFv competition binding experiments. Despite some modest biases in the constructed library, the results clearly indicated that phage display can enrich for an optimal and chelating scFv pairing. This approach for isolating CRAbs will allow for the facile isolation of very high affinity antibodies against any target of interest given a sufficiently diverse panel of scFvs circumventing the need for prior scFv characterisation or elucidation of antibody‐antigen structural data.

Tetanus and botulinum neurotoxins cause neuroparalysis by inhibiting neuroexocytosis. They are composed by two main chains: the 100 kDa heavy chain (H) mediates the neurospecific binding to target cells and chaperons the entry of the 50 kDa light chain (L). After binding on the plasma membrane, these neurotoxins enter into nerve terminals via endocytosis inside synaptic vesicles, as shown here for the first time by immuno-electron microscopy. The lumenal acidic pH induces a structural change of the neurotoxin molecule that becomes capable of translocating its L chain into the cytosol, via a transmembrane protein-conducting channel made by the H chain. This is the least understood step of the intoxication process primarily because it takes place inside vesicles within the cytosol. In the present study, we describe how this passage can be made accessible to investigation by making it to occur at the plasma membrane of neurons. The neurotoxin, bound to the plasma membrane of cerebellar granular neurons in the cold, was exposed to a low pH extracellular medium and the entry of the L chain was monitored by measuring its specific metalloprotease activity with a ratiometric method. We found that the neurotoxin has to be bound to the membrane via at least two anchorage sites in order for a productive low-pH induced structural change to take place. In addition, this process can only occur if the single inter-chain disulfide bond is intact. The pH dependence of the conformational change of tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) /B, /C and /D is similar and takes place in the same slightly acidic range, which comprises that present inside synaptic vesicles. Thanks to this reliable method we have also studied the temperature dependence and the time course of TeNT, BoNT/C and BoNT/D L chain entry across the plasma membrane. The time course of translocation of the L chain varies for the three neurotoxins, but remains in the range of minutes at 37 °C, whilst it takes much longer at °20 C. BoNT/C does not enter neurons at 20 °C. Translocation also depends on the dimension of the pH gradient. These data are discussed with respect to the contribution of the membrane translocation step to the total time to paralysis and to the low toxicity of these neurotoxins in cold-blood vertebrates. Another fundamental event along CNTs neuron intoxication process is the reduction of the interchain disulphide bond. This is a conditio sine qua non to free the catalytic part of the molecule in the cytosol of neurons. By using specific inhibitors of the various cytosolic protein disulfides reducing systems, we show here that the NADPH-Thioredoxin reductase-Thioredoxin redox system is the main responsible for this disulfide reduction. In addition, we indicate auranofin, as a possible basis for the design of novel inhibitors of these neurotoxins. BoNT/A is the most frequent cause of human botulism and at the same time is largely used in human therapy. Some evidences indicate that it enters inside nerve terminals via endocytosis of synaptic vesicles, though this has not been formally proven. The metalloprotease L chain of the neurotoxin then reaches the cytosol in a process driven by low pH, but the acidic compartment wherefrom it translocates has not been identified. Using immunoelectron microscopy, we show that BoNT/A does indeed enter inside synaptic vesicles and that each vesicle contains either one or two toxin molecules. This finding indicates that it is the BoNT/A protein receptor SV2, and not its polysialoganglioside receptor that determines the number of toxin molecules taken up by a single vesicle. In addition, by rapid quenching the vesicle transmembrane pH gradient, we show that translocation of the neurotoxin into the cytosol is a fast process. Taken together, these results strongly indicate that translocation of BoNT/A takes place from synaptic vesicles, and not from endosomal compartments, and that the translocation machinery is operated by one or two neurotoxin molecules. Another important aspect regarding CNTs research is their employment in human therapy. Accordingly, BoNT/A is almost invariably used in the treatment of many human diseases characterized by hyperactivity of peripheral cholinergic nerve terminals. Unfortunately, some patients are or become resistant to it. This drawback can be overcome by using other botulinum toxins, and pre-clinical studies have been performed with different toxin serotypes. Botulinum neurotoxin type D has never been tested in human muscles in vivo. Here we show that BoNT/D is very effective upon injection in mice, on the mouse hemidiaphragm preparation and on different rat primary neuronal cultures. From these experiments, doses to be injected in human volunteers were determined. The effect of the injection into the human Extensor Digitorum Brevis muscle was assayed by measuring the compound muscle action potential at different times after injection. Botulinum toxin type D was found to be very uneffective in inducing human skeletal muscle paralysis. These results are interpreted in terms of recent reports on neuronal surface receptors of this neurotoxin and of the established double receptor model of binding.
Le Neurotossine clostridiali (CNT), sono esotossine di origine batterica che causano le due note sindromi neuroparalitiche tetano e botulismo attraverso il blocco della neuroesocitosi. Sono composte da due catene principali legate covalentemente da un unico ponte disolfuro. La catena pesante di 100 kDa (H) fornisce il legame neurospecifico e media l'ingresso della catena leggera (L) di 50 kDa nei neuroni bersaglio. Dopo il legame sulla membrana plasmatica, queste neurotossine entrano nei terminali nervosi all'interno di vescicole sinaptiche tramite endocitosi. Qui il pH acido induce un cambiamento strutturale della molecola che diventa capace di traslocare la catena L nel citosol, grazie ad un canale predisposto dalla catena H. Questo è il passaggio meno conosciuto lungo tutto il processo perlopiù a causa della sede dove avviene, ovvero piccoli compartimenti endocitici scarsamente manipolabili dall’esterno. Nel presente studio si descrive come questo passaggio sia stato reso accessibile all'indagine facendolo verificare sulla superficie dei neuroni. La neurotossina, legata a freddo alla membrana plasmatica di neuroni primari di cervelletto, è stata esposta ad un mezzo tamponato a basso pH per simulare quanto avviene nelle vescicole sinaptiche. L'ingresso della catena L è stato monitorato misurando l'attività metalloproteasica specifica con un metodo raziometrico. Abbiamo trovato che la neurotossina deve essere necessariamente legata alla membrana con almeno due siti di ancoraggio al fine di andare incontro ad un cambiamento strutturale funzionale alla traslocazione. Inoltre, questo processo può avvenire solo se il disolfuro intercatena è intatto. La pH-dipendenza del riarrangiamento conformazionale della tossina tetanica (TeNT) e delle tossine botuliniche (BoNT) B, C e D è simile e avviene nello stesso intervallo, in una condizione di media acidità, tuttosommato simile a quella che si pensa esistere all'interno vescicole sinaptiche. Grazie a questo affidabile metodo di indagine, abbiamo proceduto studiando la dipendenza dalla temperatura e la cinetica della traslocazione di TeNT, BoNT/C e BoNT/D. A 37 °C, la traslocazione delle tre diverse tossine varia nel tempo, ma rimane sostanzialmente nell'intervallo di minuti minuti, mentre ne richiede molto più a 20 °C. BoNT/C non trasloca a 20 °C. La traslocazione, come precedentemente visto, dipende anche dalla dimensione del gradiente di pH. Questi dati vengono discussi considerando l’intero arco di tempo necessario alla tossine per intossicare i neuroni, così come la scarsa tossicità delle tossine nei vertebrati a sangue freddo. Un altro evento fondamentale lungo il processo di intossicazione è la riduzione del legame disolfuro intercatena. Questa è una conditio sine qua non per liberare la parte catalitica della molecola nel citosol dei neuroni. Utilizzando inibitori specifici dei diversi sistemi ossidoreduttivi citoplasmatici, si è dimostrato che il sistema NADPH-tioredossina reduttasi-tioredossina, è il principale responsabile di questo evento. Inoltre, auranofin viene indicato come possibile molecola lead per la progettazione di nuovi inibitori di queste neurotossine. BoNT/A è responsabile della maggior parte dei casi di botulismo nell’uomo e allo stesso tempo è indicata, quasi senza alternative, come agente terapeutico per il trattamento di numerose condizione patologiche. Alcune prove indicano che essa penetra all’interno dei terminali nervosi via endocitosi di vescicole sinaptiche, ma questo non è mai stato formalmente provato. La subunità catalitica accede quindi al citosol grazie ad un cambiamento conformazionale guidato da un gradient di pH. Tuttavia, quale sia il compartimento acido sfruttato dalla tossina per innescare il cambiamento conformazionale non è stato ancora determinato. Attraverso esperimenti di immuno detezione e miscroscopia elettronica, abbiamo dimostrato che BoNT/A sfrutta il riciclo di vescicole sinaptiche per essere internalizzata e che il numero massimo di tossine per vescicola è dettato dal numero di recettori proteici, nella fattispecie SV2, presenti all’interno della stessa, piuttosto che dal recettore glicolipidico presente sulla membrane esterna. A suffragio di ciò, la rapida inibizione dell’acidificazione dei compartimenti acidi attraverso specifici inibitori, mostra una cinetica di traslocazione di BoNT/A talmente rapida da poter escludere con certezza che tale evento possa avvenire a livello di organelli acidificabili diversi dalle vescicole sinaptiche, quali possono essere ad esempio gli endosomi. Presi nel loro insieme, questi risultati propendono per un funzionamento di BoNT/A come una nanomacchina capace di traslocare la subunità catalitica attraverso l’impiego di una o tuttalpiù due molecole. Un altro aspetto per cui la ricerca sulle CNT è importante è sicuramente il loro impiego in terapia umana. BoNT/A è usata nel trattamento di molte malattie umane caratterizzate da iperattività dei terminali nervosi periferici colinergici. Purtroppo, alcuni pazienti sono o diventano resistenti alla terapia. Questo inconveniente può essere superato utilizzando altre tossine botuliniche ed infatti studi pre-clinici sono stati condotti con differenti sierotipi di tossina. La neurotossina botulinica di tipo D non è mai stato testata in vivo su muscoli umani. Qui viene mostrato che BoNT/D rappresenta la tossina più efficace in preparazioni in vivo ed ex vivo nei topi. Da questi esperimenti preliminari, sono state determinate le dosi da testare in volontari umani. L'effetto della iniezione nel muscolo delle dita umano Extensor Digitorum Brevis è stata saggiata misurando la risposta del potenziale d’azione evocato nei muscoli a diversi tempi dopo l'iniezione. BoNT/D è risultata essere scarsamente efficace nella neuroparalisi del muscolo scheletrico umano ed è per cui poco appetibile per l’uso umano. Questi risultati sono stati interpretati considerando i recenti risultati in merito ai recettori sfruttati da tale sierotipo e il modo con cui la stessa si lega ad essi.

Vaccination coverage in adults remains suboptimal. Health organisations have only recently begun to recognise the role of socio-psychological factors in vaccination decisions. These factors are particularly important, given that they are inherently amenable to policy and behaviour change. This thesis employs a mixed-methods approach to investigate the determinants of adult seasonal influenza and tetanus vaccination in the UK general adult population. It focuses on socio-psychological factors and draws upon health behaviour models, heuristics and biases and customer journey mapping theoretical approaches to guide research and elucidate findings. A narrative and a systematic review and meta-analysis reveal there are a number of socio-psychological factors frequently associated with vaccination, particularly influenza and influenza vaccine risk perception, perceived vaccine effectiveness and reported physician recommendation, and show that most of the evidence in this area is produced in the US. They also highlight the importance of some vaccine risk perceptions, such as influenza-like symptoms and unspecific side-effects, and demonstrate that the existing evidence is highly heterogeneous and often lacking in quality, further supporting a case for robust empirical research on this topic. Two qualitative studies show that vaccine uptake is largely driven by people’s risk perception of influenza and tetanus, and that the tetanus vaccine is perceived as safe, unlike the influenza vaccine. They also reveal how specific healthcare ‘touchpoints’ across the immunisation journey can facilitate or hinder uptake. A novel finding is that certain childhood experiences can influence adult vaccination decisions. Two cross-sectional survey studies show that a compact set of variables can predict 91% of influenza and 75% of tetanus vaccination behaviour. They also demonstrate that socio-psychological factors are the most important determinants of vaccination behaviour. This thesis shows that incorporating socio-psychological dimensions in all aspect of immunisation policy, from surveillance systems to policy evaluation, is critical to improve vaccination rates.

Правець є актуальною проблемою національної системи охорони здоров’я, що пов’язано насамперед із низьким охопленням вакцинацією населення України.
Tetanus is a serious, potentially life-threatening infectious disease, occurrence of which is limited to post-vaccination immunity. This abstract clearly shows the clinical case of tetanus in unvaccinated 6-year-old child with favorable outcome. The disease duration was 64 days. This case is an objective indicator of the strength of epidemic process and system state immunization in the region.Targeting primary care physicians to provide immunization to "risk groups" will help to prevent tetanus.

Injection of tetanus toxin (TeNT), systemically or directly into the brain, has long been known to cause spastic paralysis or seizures respectively: thought to be due to disruption of inhibitory neurons and cleavage of vesicle associated membrane protein 2 (VAMP2). Here we investigate mechanisms involved in TeNT-induced chronic epilepsy in the first 16 days following injections in vivo and focally onto organotypic hippocampal slice cultures. Immunohistochemical analysis identified a spatial and temporal cleavage of both VAMP1 and VAMP2 progressing from day 2 post injection through to days 8 and 16. This was concentration dependent in slice cultures. VAMP1 has been shown to co-localise predominantly with inhibitory and VAMP2 with excitatory neurons. Contradicting previous results we have shown cleavage of both VAMP1 and VAMP2, disruption of both inhibition and excitation and direct effects of the toxin in the contralateral hippocampus. This indicates that inhibitory neurons and VAMP2 are not specifically targeted by TeNT. This project benefits from the combination of electrophysiological and immunohistochemical techniques to uncover functional changes induced by TeNT. It is also the first study of focally injected TeNT onto slice cultures and offers benefits for future long term studies of the effects of the toxin and drug screening.

Tetanus toxin is internalised at the neuromuscular junction into vesicular carriers undergoing fast retrograde transport to the spinal cord. We determined the pH regulation of this compartment in living motor neurons using a chimera of the tetanus toxin binding fragment (TeNT He) and a pH-sensitive variant of the green fluorescent protein (ratiometric pHluorin). We have demonstrated that moving retrograde carriers display a narrow range of neutral pHs, which is kept constant during transport. Stationary TeNT Hc-positive organelles instead exhibit a wide spectrum of pHs, ranging from acidic to neutral. This distinct pH regulation is due to a differential targeting of the vacuolar (H*) ATPase (vATPase), which is not present on moving TeNT He compartments. Accordingly, inhibition of the vATPase does not affect axonal retrograde transport of TeNT He. However, a functional vATPase is required for early steps of TeNT He trafficking following endocytosis, and it is localised to axonal vesicles containing TeNT He- Altogether, these findings indicate that the vATPase plays a specific role in early sorting events directing TeNT He to axonal carriers, but not in their subsequent progression along the retrograde transport route. This novel regulatory role for vATPase in a sorting event linked to retrograde transport adds to the numerous functions of this protein complex. Hydrogen peroxide inhibits the vATPase localised to synaptic vesicles, which leads to a disturbed trafficking of neurotransmitters. Increased levels of toxic oxygen radicals have been detected in mice overexpressing human mutant Cu/Zn superoxide dismutase (SOD 1093A) found in patients with familial amyotrophic lateral sclerosis (ALS). These mice develop motor neuron degeneration and muscle paralysis as observed in ALS patients. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. We show that retrograde axonal transport defects are already present in motoneurons of S0D1G93A mice during embryonic development. Surprisingly, crossing S0D1G93A mice with mice, which have a single point mutation in the dynein motor complex (Loa) delays disease progression and significantly increases life span of Loa/SODlG93A mice. Moreover, we observed a complete recovery in axonal transport of these mice, which may be responsible for amelioration of the symptoms. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.

A toxina tetânica é uma proteína sintetizada pelo bacilo Clostridium tetani que após destoxificação através da ação do formol, continua apresentando propriedades antigênicas e imunogênicas, obtendo a denominação toxóide tetânico. A síntese dessa proteína ocorre quando esse bacilo encontra-se na sua forma vegetativa e em meio de cultura específico relativamente complexo contendo glicose e peptonas. O efeito simultâneo de diferentes níveis de glicose (Go) e N-Z Case TT® (NZo) como fontes de carbono e nitrogênio, respectivamente, na produção de toxina tetânica foi investigada nesta primeira parte do trabalho em cultivo estático por meio de planejamento fatorial em estrela com cinco níveis e avaliado por metodologia de superfície de resposta, com a finalidade de otimização do processo. O valor mais alto de toxina tetânica encontrado, correspondente a Go = 9,7 g/L e NZo = 43,5 g/L, foi 79% maior que aqueles obtidos em condições padrões de cultivo (Go = 8,0 g/L e NZo = 25,0 g/L). Também foram realizados cultivos de C. tetani utilizando o processo descontínuo alimentado com diferentes protocolos para a correção da concentração de glicose no meio de cultivo ao longo do tempo em diferentes concentrações iniciais de N-Z Case TT®. Dois grupos de ensaios foram executados: a) experimentos realizados com a correção da concentração de glicose para 3,0 g/L nos instantes 16, 56 e 88 h e b) experimentos com correção inicial da concentração de glicose para 3,0 g/L e após esta cair para 1-1,5 g/L. O primeiro protocolo de correção da concentração de glicose e NZo = 50,0 g/L foram as melhores condições para obtenção de toxina tetânica. Nestas condições, o título de toxina tetânica foi 300% maior que aqueles obtidos em cultivos padrão.
The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivations of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (Go) and N-Z Case TT® (NZo) as carbon and nitrogen sources, respectively, on the production of tetanus toxin, have been investigated in this work in static cultivations by means of a five-levels star-shaped experimental design and evaluated by Response Surface Methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin, achieved at Go = 9.7 g/L and NZo = 43.5 g/L, was 79% higher than that obtained with standard cultivations (Go = 8.0 g/L and NZo = 25.0 g/L). Also, there were carried out cultivations of C. tetani using fed-batch process at different protocols to correct the glucose concentration in the cultivation medium along the time at different initial N-Z Case TT® concentrations (NZo). Two series of runs were performed: a) experiments with the correction of the glucose concentration to 3.0 g/L in the times 16, 56 and 88 hours and b) experiments with initial correction of the glucose concentration to 3.0 g/L and after it to drop to 1-1,5 g/L. The former protocol to correct the glucose concentration and NZo = 50.0 g/L were the best condition to obtain tetanus toxin. In these conditions, the yield of tetanus toxin was 300% higher than that obtained with standard cultivations.

Class of 2014 Abstract
Specific Aims: Tetanus, diphtheria, and pertussis are diseases, which are preventable through proper vaccination. In spite of the availability these vaccines, however, there has recently been a surge in the number of pertussis cases in the United States. The objective of this study is to determine provider adherence to tetanus, diphtheria and pertussis guidelines set forth by the Advisory Committee on Immunization Practices in a primary care setting before and after a clinical pharmacist intervention. Methods: A retrospective cohort of chart reviews was conducted between January 1 – September 30, 2013 to determine immunization adherence to tetanus, diphteria, and pertussis vaccination guidelines. A clinical pharmacist then preformed a series of cross-sectional chart reviews as an intervention. Following the intervention, a retrospective chart review was conducted to evaluate if Tdap vaccination rates improved between March 17-23, 2014. Main Results: Overall immunization rates greatly improved following the intervention (p<0.0001; x2=44.988). For non-pregnant adults between the ages of 19-64 the vaccination rate improved from 26% to 61.1% (p<0.0001; x2=47.07). A statistically significant improvement was not seen in the groups with patients 65 or older or pregnant women (p>0.05). Tdap vaccination status was appropriately evaluated and vaccinations given by primary doctors improved from 17.7% to 61.2% and those prescribed by nurse practitioners improved from 22.4% to 56.3%. Conclusion: Intervention by a Clinical Pharmacist helped improve overall provider adherence to the tetanus, diphteria, and pertussis vaccination guidelines.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
As neurotoxinas produzidas por espécies do género Clostridium, responsáveis pelo tétano e botulismo, são classificadas como potentes metaloproteases constituídas por três domínios funcionais dotados de diferentes funções: ligação neuroespecífica; internalização neuronal; translocação membranar; atividade proteolítica do complexo proteico soluble N-ethylmaleimide fusion attachment protein Receptor (SNARE). Os sete serotipos de neurotoxinas botulínicas (BoNTs) inibem a libertação de acetilcolina ao nível dos terminais colinérgicos periféricos. A neurotoxina tetânica (TeNT), após ligação e internalização aos terminais colinérgicos periféricos sofre um transporte axonal reverso até à espinal medula onde inibe a libertação de ácido λ-aminobutírico (GABA) e glicina nos interneurónios inibitórios. A sinaptobrevina representa o local proteolítico das BoNTs dos serotipos B, D, F e G e da TeNT, enquanto que a SNAP-25 constitui o alvo de ação das BoNTs dos serotipos E, A e C. Para além da SNAP-25 a sintaxina 1 representa outro alvo proteolítico da BoNT do serotipo C. O botulismo é uma doença rara que tem como principal agente etiológico a espécie C. botulinum responsável pela síntese e secreção de BoNTs. A intoxicação alimentar e a colonização intestinal de crianças entre uma semana e um ano de idade representam as principais vias de exposição da doença, que se manifesta por uma paralisia muscular flácida generalizada associada a uma inibição do sistema parassimpático podendo na fase mais avançada da levar à morte por insuficiência respiratória. O tétano nos dias de hoje é uma doença endémica apenas para alguns países subdesenvolvidos. C. tetani representa o agente etiológico do tétano pela produção da TeNT. A contaminação de feridas com esporos bacterianos constitui a fonte de propagação da doença que se traduz numa hiperatividade generalizada dos músculos esqueléticos associada a espasmos e rigidez muscular. O comprometimento generalizado do sistema simpático representa a principal causa de morte da doença. Atualmente as BoNTs estão aprovadas para o tratamento de uma vasta gama de patologias associadas à hiperfunção dos terminais colinérgicos periféricos.
The neurotoxins produced by species of the genus Clostridium, responsible for botulism and tetanus are potent metalloprotease classified as consisting of three functional domains endowed with different functions: neuro specific bond; neuronal internalization; membrane translocation; proteolytic activity of the SNARE (soluble N-ethylmaleimide fusion attachment protein Receptor) protein complex. Seven serotypes of botulinum neurotoxins (BoNTs) inhibit the release of acetylcholine at the level of peripheral cholinergic terminals. The tetanus neurotoxin (TeNT) and internalization following binding to peripheral cholinergic terminals undergoes a reverse axonal transport to the spinal cord, where it inhibits the release of λ-aminobutyric acid (GABA) and glycine in inhibitory interneurons. Synaptobrevin is the site of proteolytic BoNTs serotypes B, D, F and G and TeNT, while SNAP-25 is the action target of the BoNTs serotype E, A and C. In addition to SNAP-25 and syntaxin 1 represents another target for proteolytic BoNT serotype C. Botulism is a rare disease whose main etiologic agent C. botulinum responsible for the synthesis and secretion of BoNTs. Food poisoning and intestinal colonization in children between one week and one year of age represent the main ways of exposure of the disease, manifested by a widespread flaccid muscular paralysis associated with an inhibition of the parasympathetic system, may in advanced stage lead to death by respiratory failure. Tetanus today is endemic only some underdeveloped countries. C. tetani is the causative agent of tetanus for the production of TeNT. The contamination of wounds with bacterial spores is the source of spread of disease which translates into a general hyperactivity of skeletal muscles associated with muscle spasticity. The widespread involvement of the sympathetic nervous system is the leading cause of death from the disease. BoNTs are the currently approved for the treatment of a wide range of conditions associated with cholinergic hyperfunction of peripheral terminals.

Background: Adult immigrants may have been under-immunized in their countries of origin. We estimated the seroprevalence and examined risk factors for susceptibility to diphtheria and tetanus.
Methods: 1480 newly-arrived adult immigrants were recruited in Montreal, 2002-2004. We collected socio-demographic information and vaccination history, and measured serum antibodies to tetanus and diphtheria by EIA to identify susceptibility, defined as ≤0.15 IU/ml and ≤0.10 IU/ml, respectively.
Results: 45% (95% CI, 42%-48%) of immigrants, mean age 31 +/- 8.6 years, lacked immunity to either tetanus or diphtheria, ranging by region of origin (23%-79%). 32% (95% CI, 30%-34%) were susceptible to tetanus and susceptibility increased with age. Susceptibility to diphtheria was less prevalent [26% (95% CI, 24%-28%)] and decreased with age.
Conclusions: Many adult immigrants would benefit from diphtheria/tetanus vaccine. Susceptibility to tetanus suggests under-vaccination, while decreasing susceptibility to diphtheria with age suggests that immunity has been boosted from ongoing transmission in some countries of origin.
Introduction: Les immigrants adultes sont peut-être sous-immunisés dans leurs pays d'origine. Nous avons effectué une étude de la prévalence de la susceptibilité au tétanos et à la diphtérie et nous avons examiné les indicateurs potentiels de cette susceptibilité.
Méthodes: 1480 adultes ayant immigré récemment ont été recrutés à Montréal entre 2002 et 2004. Nous avons obtenus des informations sociodémographiques et l'histoire de vaccination et nous avons mesuré les anticorps sériques contre le tétanos et la diphtérie avec le test EIA; la susceptibilité était définie comme ≤0.15 IU/ml et ≤0.10 IU/ml, respectivement.
Résultats: Quarante-cinq pour cent (95% IC, 42%-48%) des immigrants, avec un âge moyen de 31 +/- 8.6 ans, n'étaient pas immuns soit contre le tétanos ou la diphtérie, avec des variations selon la région d'origine (23%-79%). Trente-deux pour cent (95% IC, 30%-34%) étaient susceptibles au tétanos et la susceptibilité augmentait avec l'âge. La susceptibilité à la diphtérie était moins prévalente [26% (95% IC, 24%-28%)] et diminuait avec l'âge.
Conclusions: Plusieurs immigrants adultes bénéficieraient du vaccin contre le tétanos et la diphtérie. La susceptibilité au tétanos suggère la présence de sous-vaccination, tandis que la susceptibilité à la diphtérie, qui diminue avec l'âge, suggère que l'immunité a été stimulée par la transmission continue de l'infection dans certains pays d'origine.

JESSICA MASTRODOMENICO An Examination of the Socio-Demographic Characteristics Associated with Adult Vaccination Prevalence for Preventable Diseases in the United States Background: An estimated 50,000 adults in the United States (U.S.) die each year from one of 10 vaccine preventable diseases. For those who survive vaccine preventable infections, health care costs and loss of income become more significant. While children in the U.S. aged 0-2 exhibit vaccine prevalence rates of almost 90%, some adult vaccine prevalence rates in the U.S. population are reported to be nearly 30-40% less than the goals set forth by Healthy People 2010. The purpose of this study was to examine the associations between socio-demographic characteristics of U.S. adults and adult vaccination prevalence for pneumococcal, hepatitis A, hepatitis B, tetanus, and pertussis. Methods: Data from the 2008 National Health Interview Survey were assessed examining various health indicators and characteristics of non-institutionalized adults and children. The sample was restricted to adults ≥18 years of age. Odds ratios were calculated and multivariate logistic regression was also conducted. P-values of <0.05 and 95% confidence intervals were used to determine statistical significance. Results: There were 21781 total observations; 19.3% received the pneumococcal vaccine, 9.4% received the hepatitis A vaccine, 27.2% received the hepatitis B vaccine, 55.1% received the tetanus vaccine, and 15.2% received the pertussis vaccine. Of the socio-demographic characteristics examined, age, health insurance, marital status, and education were significant for either all five or at least four of the vaccines included in this study. As one might expect those who reported health insurance and those who had a higher level of education usually had a higher likelihood of vaccine receipt as compared to those without health insurance and those with less than a high school education. Age associations varied due to age-related recommendations for certain vaccines such as pneumococcal (recommended for adults ≥65). Compared to the married population (referent), marital status results varied, but for reasons unclear. Whites, the referent group, were the most likely to be vaccinated as compared to Blacks, Hispanics/Latinos, and Asians. Hispanics/Latinos typically had the lowest likelihood of vaccination in this examination. Conclusions: This study further explores the impact of socio-demographic disparities on vaccination status and adds new information to the literature regarding adult vaccination rates for preventable diseases. While research exists related to strengthening interventions such as patient reminder systems, those who do not see the same health care providers on a regular basis remain at risk for lower vaccination prevalence. It is important to better understand the role of social determinants of health, specifically in terms of vaccinations. Future research is needed to further characterize the association of socio-demographic factors with receipt of optional vaccines in adults.

Anticorpos monoclonais (AcMos) para uso terapêutico correspondem a uma área importante na indústria de biofármacos, em especial os AcMos humanos, que apresentam menor probabilidade de elicitar imunogenicidade. O objetivo deste trabalho consistiu em obter AcMos humanos antitetânicos através da separação de linfócitos B produtores de anticorpos específicos utilizando o antígeno ou de plasmablastos. As células foram coletadas de doadores após vacinação e separadas por equipamento de cell sorter. As regiões variáveis dos anticorpos foram amplificadas e clonadas em vetores de expressão, que foram usados para transfectar transitoriamente células HEK293-F. O uso da toxina tetânica conjugada independentemente com dois marcadores, biotina e Alexa Fluor® 647, possibilitou a separação específica de linfócitos B produtores de AcMos antitetânicos, que foram avaliados por ELISA, western blotting e pela inibição da ligação da toxina ao gangliosídio GT1b. O ensaio in vivo mostrou proteção total dos animais contra a toxina tetânica quando três AcMos foram usados em conjunto.
Monoclonal antibodies (mAbs) for therapeutic use correspond to a major area of the biopharmaceutical industry, especially human mAbs that are less prone to elicit immunogenicity. The objective of this work was to obtain anti-tetanus human mAbs through separation of memory B lymphocytes producing specific antibodies stained with the antigen or plasmablasts. Cells were collected from peripheral blood of donors after vaccination and separated through cell sorting. The variable regions of the antibodies were amplified and cloned in expression vectors for transient transfection of HEK293-F cells. The staining with the tetanus toxin labeled independently with two markers, biotin and Alexa Fluor® 647 allowed the separation of specific B lymphocytes producing anti-tetanus mAbs. The antibodies expressed were evaluated by ELISA, western blotting and the inhibition of the binding of the tetanus toxin to the ganglioside GT1b. The in vivo neutralization assay showed that a pool of three different mAbs were able to protect mice against the tetanus toxin.

This study examines the possible mechanisms by which radiation and the bacterial toxin, Tetanus toxoid (TT), suppress the murine splenic mononuclear cell (SMNC) response to mitogen. This study demonstrates that the lymphocyte proliferation response of SMNC to the mitogenic lectin PHA can be suppressed by TT in a dose-dependant manner in vitro, without affecting the viability of the cells, in the anti-proliferative concentrations used (0.5-5 $\mu$g/ml). SMNC pre-incubated with TT could suppress the pHA blastogenic response of fresh autologous cells during co-incubation suggestion the involvement of activated suppressor cells. Flow cytometric analysis demonstrated that TT does not produce an alteration in the cellular balance, indicating that the suppression would appear to be dependant upon a change in T cell function. TT down-regulated the expression of class II MHC antigens on antigen-presenting cells which may represent an inappropriate costimulatory signal required for T cell activation. Whole body irradiation has been reported to induce active immune suppression. In the present study, ionizing radiation (0-700 cGy) produced decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. There was no evidence, however, to indicate that irradiation (100 cGy) activated suppressor cells during the first 7 days post-irradiation. Similarly, radiation did not seem to interact with TT to increase the amount of TT-induced suppression. (Abstract shortened by UMI.)

Previous studies have shown that certain antigens can down-regulate immune responses of human peripheral blood mononuclear cells (PBMC) both in vivo and in vitro. This study demonstrates that tetanus toxoid (TT), in a dose-dependent fashion, suppresses the induction of a blastogenic response of PBMC by phytohemagglutinin (PHA), monoclonal anti-CD3, and anti-CD4 antibody. Pokeweed mitogen (PWM)-induction of IgG and IgM is suppressed as well. The suppression is partially reversed by indomethacin and IL-2, but not by IL-1 or tumor necrosis factor (TNF-$\alpha$). PBMC pre-incubated with TT could suppress the PHA blastogenic response of fresh autologous cells during co-incubation. The removal of CD4$\sp+$ cells prior to induction of suppression greatly diminished the suppression of PHA blastogenic response, whereas the elimination of CD8$\sp+$ cells had no effect. Therefore it is concluded that TT induces non-specific suppressor cells that can strongly dampen mitogenic responses, and that CD4$\sp+$ cells plays an important role in this suppression. CD4 $\sp+$ cell, together with monocytes, may suppress mitogenic responses involving a prostaglandin-dependent pathway.

A eficácia da associação quádrupla (DPT + BCG) foi estudada através da proteção e comparação da soro conversão das vacinas, usando como adjuvante o hidróxido de alumínio e ou BCG. A potência da vacina pertussis foi avaliada pelo teste de proteção em camundongos e o BCG pelo teste de consumo de oxigênio e de vitalidade, sendo considerada satisfatória. Sendo os toxóides diftérico e tetânico considerados proteínas inertes e estáveis, suas potências não foram determinadas depois de associadas. Os níveis de anticorpos foram determinados para difteria e tétano pela reação imunoenzimática, para vacina pertussis pela reação de imunofluorescência e para o BCG pela conversão tuberculínica. Os níveis de conversão foram satisfatórios, revelando ser possível a associação sem prejuízo para nenhum dos antígenos. A associação DPT + BCG não causou reação local ou geral significante, possibilitando uma simplificação operacional.
The efficacy of the quadruple association (DPT + BCG) was studied though protection and comparison ot the conversion serum in vaccines using aluminium hydroxide or BCG as adjuvant. Both the protection power of the Pertussis vaccine evaluated by the protection test in mice and BCG by the oxygen uptake and counts of viable particles were considered satisfactory. Being difteria and tetanus toxoids considered inert and stable proteins, their protection wasn\'t determined after their combination. The antibody levels produced by the antigens were determined by the enzyme-linked immunosorbent assay to the difteria and tetanus toxoids, by the fluorescent antibody technique to the Pertussis vaccine and by the tuberculin conversion to the BCG. The conversion levels were satisfatory and there was no damage in their association. There was no meaningful local or general reaction in this association making an operational simplification possible.

O presente relatório surge na sequência da realização do estágio curricular na coo-perativa Fomento Extremeño Ovino Sociedad Agraria de Transformación no âmbito do Mestrado Integrado em Medicina Veterinária na Universidade de Évora. Este relatório apresenta-se dividido em duas partes. Na primeira são descritas as atividades desenvolvi-das no controlo reprodutivo, profilaxia médica e clínica médica. A segunda parte é cons-tituída por uma revisão bibliográfica sobre o tétano em ovinos, patologia que tem especial importância nesta espécie devido aos procedimentos realizados no maneio ainda muito tradicionais, e no final são apresentados dois casos clínicos e a sua respetiva discussão à luz do estado de arte; Ruminants clinics Abstract: This report follows up on the completion of the internship at the Fomento Extre-meño Ovino Sociedad Agraria de Transformación as part of the Master Degree in Vete-rinary Medicine at the University of Évora. Thereby the present repor is divided into two parts. The first part describes the activities performed in reproductive control, medical prophylaxis and medical clinic. The second part is composed by a bibliographical review about tetanus in sheeps, a pathology that has special importance in this species due to animal management procedures performed wich are still very tradicional. Lastly, this re-port presents two clínical cases and its respective discussion according to presente knowledge.