Tétanos generalizado: Una amenaza persistente, reporte de tres casos y revisión de la literatura / Generalized tetanus: A persistent threat, review of the literature and report of three cases

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.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Synthetic Cannabinoid-Induced Immunosuppression Augments Cerebellar Dysfunction in Tetanus-Toxin Treated Mice

Synthetic cannabinoids are one of most abused new psychoactive substances. The recreational use of abused drug has aroused serious concerns about the consequences of these drugs on infection. However, the effects of synthetic cannabinoid on resistance to tetanus toxin are not fully understood yet. In the present study, we aimed to determine if the administration of synthetic cannabinoids increase the susceptibility to tetanus toxin-induced motor behavioral deficit and functional changes in cerebellar neurons in mice. Furthermore, we measured T lymphocytes marker levels, such as CD8 and CD4 which against tetanus toxin. JWH-210 administration decreased expression levels of T cell activators including cluster of differentiation (CD) 3ε, CD3γ, CD74p31, and CD74p41. In addition, we demonstrated that JWH-210 induced motor impairment and decrement of vesicle-associated membrane proteins 2 levels in the cerebellum of mice treated with tetanus toxin. Furthermore, cerebellar glutamatergic neuronal homeostasis was hampered by JWH-210 administration, as evidenced by increased glutamate concentration levels in the cerebellum. These results suggest that JWH-210 may increase the vulnerability to tetanus toxin via the regulation of immune function.

Tetanus toxin fragment C fused to flagellin makes a potent mucosal vaccine

PURPOSE: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus. MATERIALS AND METHODS: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed. RESULTS: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin. CONCLUSION: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.

Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus / 한국실험동물학회지

In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.

Dimensionamiento de los sistemas de microfiltración y ultrafiltración para la producción de la toxina tetánica elaborada en el Instituto Nacional de Higiene Rafael Rangel / Microfiltration and ultrafiltration systems dimension for using in tetanus toxin production in the Instituto Nacional de Higiene "Rafael Rangel"

Se evaluó el uso de la tecnología de Flujo de Filtración Tan gencial (FFT), para obtener la toxina tetánica a partir de cultivos de la bacteria Clostridium tetani, usando el proceso de Micro filtración (MF), para eliminar el paquete celular y posteriormente, a partir del filtrado obtenido, concentrar y diafiltrar la Toxina Tetánica usando el proceso de Ultrafiltración (UF). Se determinaron las características de los filtros, condiciones de trabajo y el dimensionamiento de los equipos a adquirir para la nueva producción industrial de Toxina Tetánica. Se evaluaron el flujo, tiempo, rendimiento del proceso y las características del producto obtenido. Utilizando cultivos con Toxina Tetánica en un equipo de filtración de laboratorio, diseñado para producir el efecto de FFT. Se seleccionó las membranas tipo cassettes, formato Suspended Screen, porosidad 0,2μm, como las adecuadas para el proceso de MF, ya que mostraron un 100% de transmisión de la Toxina Tetánica, ausencia de restos celulares y flujo promedio de filtrado de 73.30 L/m2h. Así mismo, se seleccionaron las membranas tipo cassettes, formato Omega, porosidad 50 y 70 kDa, como las adecuadas para el proceso de UF, ya que mostraron 100% de recuperación de la toxina, ausencia de toxina en el filtrado y adecuados flujos de filtrado (106,7 y 104,4 L/m2h, respectivamente). Estos resultados permitieron dimensionar, considerando las variables a utilizar en la producción industrial (Volumen 650 a 950 litros, tiempo de procesos, 3 horas), el área de filtración de los equipos de MF y UF a adquirir, estimados en 20m2 y 5m2, respectivamente.

Tangential Flow Filtration (TFF) technology was evaluated to process tetanus toxin which is produced by Clostridium tetani bacterium. Microfiltration (MF) is used to retain cells while allowing passage of the toxin to the filtrate stream. The filtrate is co - llected and further processed by Ultrafiltration (UF) to concentrate the toxin and to maximize the wash of small species by a Dia filtration step. Both, MF and UF processes were evaluated to specify the filters and corresponding critical process parameters to scale-up the application. As part of the evaluation, flow ra te, processing time, yield and product attributes were characterized. The cell harvest containing the tetanus toxin was processed using a laboratory scale TFF system designed to product the TFF effect. The evaluation demonstrated that a cassette in sus pended screen format and membrane with 0.2μm pore is the right selection for the MF step. It showed 100% of toxin transmission without the presence of cellular debris and average process flux of 73.30 L/m2h. The UF step was conducted using the same system with cassettes in me dium screen format with pores of 50 and 70kDa. It showed 100% retention of the toxin with a process flux of 106,7 and 104,4L/m2h, respectively. To maximise product retention during UF, the 50 kDa membrane was selected. These results were used to scale-up the application to process the industrial volume of 650 a 950 liters in 3 hours of processing time. Membrane area sizing of MF and UF to be acquired is estimated in 20m2 and 5m2, respectively.

Production and immunogenicity analysis of conformation-stable fragment-C mutant of tetanus toxin / 生物工程学报

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to A1a and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to > 95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1 x 10(3) 50% lethal doses (LD50s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.

Tetanus Developed in Gangrenous Perforation of Small Bowel

Tetanus is a neurologic disorder caused by a tetanospasmin released from Clostridium tetani and usually occurs following a stab wound or dirty abrasion. Tetanus is uncommon in Korea due to the introduction of vaccination programs. Furthermore, tetanus associated with a gangrenous perforation of the small bowel is extremely rare. We report a case of tetanus developed in a patient who was diagnosed with a gangrenous perforation of the small bowel. This is the first reported case in Korea.

Neurotoxinas de invertebrados como alternativas terapéuticas y herramientas en investigación básica / Neurotoxins from invertebrates as alternative therapeutic agents and tools in basic research

Los venenos que producen los animales son una mezcla compleja de proteínas, péptidos, enzimas y trazas deelementos no proteicos tales como carbohidratos y sales, cuya finalidad es inmovilizar la presa y comenzar a digerirla. Las toxinas son sustancias aisladas de venenos, con una o varias acciones específicas sobre las víctimas. Entre estos compuestos, son numerosos los que tienen acción sobre receptores específicos ubicados en el sistema nervioso central y/o periférico, mientras que otros ejercen sus efectos actuando sobre otras proteínas. Desde el descubrimiento en 1971, del péptido que dio origen al Captopril, y teniendo en cuenta que muchas toxinas son útiles como herramientas para el estudio de procesos fisiológicos, se comenzó a mirar los venenos de animales como fuentes ricas en compuestos bioactivos y a pensar en su uso potencial como agentes terapéuticos. Así pues, en la actualidad disponemos de diferentes medicamentos y herramientas diagnósticas o de investigación básica derivados de toxinas. Esta revisión, basada en publicaciones realizadas en los últimos 10 años, busca proporcionar una visión actual del uso de algunas de estas moléculas como herramientas en diferentes campos de la biomedicina y la farmacia, y en su aplicación como nuevas alternativas terapéuticas o como modelos en el diseño de las mismas.

Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein, target delivery to CNS and neurotrophy biology ability / 中华外科杂志

<p><b>OBJECTIVE</b>Expression, purification of tetanus toxin C fragment/cardiotrophin-1 recombinant fusion protein (CT-1/TTC) in BL21 (DE3) E. coli, examined whether tetanus toxin C fragment mediate the cardiotrophin-1 target delivery to the central nervous system and the cardiotrophin-1 has the neurotrophic ability.</p><p><b>METHODS</b>Induction by IPTG, the fusion protein was expressed and then purified by GST affinity agarose. The interest protein was viewed by SDS-PAGE, further characterized by Western Blot Rat sciatic nerve transected model was selected. Using drug by nerve-regeneration-chamber and intramuscular injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained with immunohistochemistry method. And select the new born SD rat sciatic nerve transected model, using CT-1/TTC fusion protein by muscle injection. Execute these animals one week after the operation. The L4-L6 segments of the spinal cord were harvested after transaortic perfusion with 4% paraformaldehyde. The freeze sections of spinal tissues were stained by Nissl's staining.</p><p><b>RESULTS</b>After induction, the fusion protein was about 15% of the total protein and the soluble part was predominant. Purified by GST fusion protein column, the interest protein's concentration is 2.7 g/L. The CT-1/TTC fusion protein was found in lumbar intumescentia by immunohistochemistry method. And after sciatic nerve transected, the numbers of cornu anterius medullae spinalis motoneurons in L4-L6 segments, compared to CT-1/TTC protein grope, have a lower survival rate.</p><p><b>CONCLUSIONS</b>The recombinant CT-1/TTC protein can be expressed and purified in BL21 (DE3) E. coli. This fusion protein has two biological activities of targeting delivery to central nervous system and protecting the cornu anterius medullae spinalis motoneurons.</p>

Comparación cinética y bioquímica de tres cepas de clostridium tetani, para la producción de toxina tetánica / Kinetic and biochemical comparison between three strains of clostridium tetani for production of titanic toxin

En este estudio se comparó el comportamiento bioquímico y cinético de tres cepas de Clostridium tetani, Harvard (Ha), Massachusetts (Ms) y Caracas (Ca); cepas utilizadas comúnmente en la producción del toxoide tetánico. Se realizaron pruebas bioquímicas para la identificación de los microorganismos anaerobios y se desarrollaron cultivos en reactor de 5L, bajo las siguientes condiciones: Medio Latham Mueller "Con" y "Sin" glutamato de sodio; 1 por ciento (v/v) de inóculo, pH inicial, 7.1 ± 0.2 (variable), 35ðC y 100 r.p.m. Los resultados mostraron características bioquímicas similares entre las tres cepas y la cepa control ATCC 9441. El comportamiento cinético de las cepas Ms y Ha fue similar para todos los parámetros evaluados, obteniéndose mayor productividad en cultivos suplementados con 2.5g/L de glutamato de sodio (Ms: Sin: 625Lf/L.h y Con: 658Lf/L.h; Ha: Sin: 610Lf/L.h y Con: 658Lf/L.h). La cepa Ca, produjo mayor cantidad de biomasa (Ca: 0.5420g/L; Ms: 0.2721g/L y Ha: 0.2009g/L); sin embargo, la productividad de toxina fue menor (Sin: 174Lf/L.h y Con: 417Lf/L.h). Estos resultados descartan la utilización de la cepa Ca para la producción del toxoide, bajo las condiciones de fermentación ensayadas.

One Case of Tetanus after Taking Acupuncture

Tetanus is a neurologic disease which features the muscle spasm as the hallmark. It is an infectious disease with high mortality rate triggered by tetanospasmin produced by Clostridium tetani. This report concerns incidence of tetanus consequent to oriental medical care such as acupuncture and moxibustion. Although the tetanus occurrence has shown a remarkable decline since nationwide vaccinations in some of the developed countries, including Korea, it still remains to be an important issue, to be dealt within Korea, as the majority of the patients are old aged and Korean population is rapidly becoming an aging society. Furthermore, since more elders are coming to rely on Oriental medicine in Korea, the Korean elders are at a higher risk than elsewhere. The lack of medical experiences, including those in oriental medical field, has been hindering early diagnosis of Tetanus. This study aims to encourage rapid and accurate decisions in diagnosis and treatment through reviewing symptoms particularly specific to tetanus, and also to arouse attention to the riskiness of invasive procedures involving skin puncture.

PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos).</p><p><b>METHODS</b>To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing.</p><p><b>RESULTS</b>Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene.</p><p><b>CONCLUSION</b>PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.</p>

Gene cloning, optimized expression and immunogenicity evaluation of tetanus toxin fragment C / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain highly purified tetanus toxin fragment C (TTC) with good immunogenicity.</p><p><b>METHODS</b>The gene fragment encoding TTC was amplified from Clostridium tetani plasmid DNA by PCR, inserted into the vector pET43.1a (+) and expressed in E. coli BL21(DE3)plysS. After purification using Ni2+-chelate affinity chromatography, the expressed fusion protein was digested by thrombin and the resultant TTC protein was purified with Ni2+-chelate affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purifed TTC protein was then used to immunize mice to test its immunogenecity.</p><p><b>RESULTS</b>The 1373-bp gene fragment encoding TTC was obtained, and the constructed recombinant expression vector pET43.1a (+)-TTC was successfully expressed in E. coli BL21(DE3)plysS. SDS-PAGE identified a recombinant fusion protein with relative molecular mass (Mr) of 117 000, which accounted for 22% of the total bacterial protein. The TTC protein with Mr of 50 000 was obtained after purification of the thrombin digestion products of the fusion protein, with a purity reaching 95.5%. Both the fusion protein and TTC protein could be recognized by anti-tetanus toxin antibody as shown by Western blotting. The titer of the anti-serum from mice immunized with the TTC protein was 1:25 600, and the anti-serum could specifically bind to tetanus toxin.</p><p><b>CONCLUSION</b>Highly purified and immunogenetic TTC protein has been successfully obtained, which provides a good model antigen for studying antigen presentation and immune responses in vivo.</p>

Electrophysiological Tests in Generalized Tetanus

BACKGROUND: Tetanus toxin selectively blocks inhibitory synapses in the brainstem as well as the spinal cord. Therefore, in contradiction to Stiff Person syndrome, patients with generalized tetanus usually show abnormal masseter silent periods as well as abnormal F/M amplitude or H/M amplitude ratios. This study aimed to verify the characteristics of electrophysiological findings of generalized tetanus. METHODS: The authors retrospectively studied clinical and electrophysiological characteristics of 7 patients with generalized tetanus, who were admitted to the neurology department of Hallym Medical Center from 1995 to 2005. RESULTS: All the seven patients showed abnormal masseter silent periods. Three of them showed somewhat improvement in the silent period at follow-up study as trismus was improving. Full NCSs done in two patients did not show any abnormalities except an increased F/M amplitude ratio. One patient with a wound site in his left finger showed an abnormal F/M amplitude ratio only in the right upper extremity without involvement of other extremities. Another patient showed an increased H/M amplitude ratio without an increased F/M amplitude ratio. (In this patient we did not conduct full NCS tests.) CONCLUSIONS: The Masseter silent period could be used as a diagnostic tool and parameter of clinical improvement in patients with generalized tetanus.

Detección inmunoenzimática de la toxina de la bacteria Clostridium tetani: una alternativa a las pruebas biológicas con ratones / Immunoenzimatic detection of the Clostridium tetani bacterial toxin: an alternative to mice bioassays

Cell-free extracts from 20 strains of Clostridium tetani isolated from soil samples, were tested for tetanus toxin production using an enzyme immunoassay. All the extracts were classified as positive for the toxin presence, and eight of them showed absorbance values corresponding to tetanus toxin concentrations between 3.2 and 88 ng/ml; thus, they fell within the linear absorbance range (0.135-0.317). All dilutions of toxin used to obtain the calibration curve (0.0071 to 1.1 ng) were lethal for mice.

Effect of the Heat Shock Protein 70 on the Adjuvanticity Induced by a Bacterial Flagellin of Vibrio ulnificus

Recently we have shown that a bacterial flagellin, Vibrio vulnifiucs FlaB (Vv-FlaB), has a strong adjuvant activity to induce protective immune response. In order to investigate the adjuvanticity of Vv-FlaB, we prepared highly purified recombinant protein by using an intein fusion protein purification system. However, in the process of the purification, we unexpectedly encountered a contamination with a 70 kDa protein. We proved the 70 kDa protein as the heat shock protein 70 (HSP70) by Western blotting. Unfortunately, it was reported that the HSP70 has a strong adjuvanticity. In this study we investigated the role of contaminating HSP70 on the Vv-FlaB-mediated adjuvanticity. We separated Vv-FlaB and HSP70 by using a high performance protein purification chromatography and compared adjuvant activities of Vv-FlaB, HSP70 and Vv-FlaB/HSP70 mixture. Using an intranasal immunization mouse model, we observed that co-administration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific antibody (Ig) responses. However contaminating doses of HSP70 did not affect the adjuvanticity of Vv-FlaB and furthermore HSP70 alone did not enhance TT-specific Ig response and protective immunity against lethal challenge with tetanus toxin. These results show that the HSP70 contaminating Vv-FlaB preparations did not affect the adjuvanticity of Vv-FlaB.

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved

Analysis of the reported symptoms that preceded the death of King Rajasinghe of Sithawake (1592 AD).

A copy of an ola leaf manuscript, the original of which was written around the late 1590s, was published recently. It describes the sequence of events leading to the death of the warrior King Rajasinghe of the Sithawake Kingdom (1521-1593). A study of the contents of this letter is presented. The dressing applied to a wound on the foot of the King is described in that letter. It is likely that this dressing would have been an ideal medium to produce the highly potent tetanus exotoxin. The toxin would have diffused into the open wound and produced the sequence of symptoms and signs mentioned in the letter. An analysis of the symptoms and signs noted during the King's last illness as described in the letter is presented. Laryngospasm, and tonic and clonic spasms are easily identified. In addition, a group of symptoms attributed in the 1960s to sympathetic over-activity in tetanus are also recognisable. The conclusion is drawn that the King died of tetanus. The intriguing possibility of the wilful use of a lethal dressing on an open wound as a biological contact poison is left open for discussion.

Age Related Serosurvey of Immunity to Tetanus in Korean Populations / 감염

BACKGROUND: The incidence of tetanus in adults and neonatal tetanus have been markedly reduced by world-wide use of DTP vaccines. But, tetanus is still one kind of major health problems in many developing countries, and several serosurvey stduies in developed countries revealed that substantial proportions of adult population may lack immunity against tetanus and immunity level against tetanus is continuously decreasing by age. In Korea, tetanus outbreaks have been disappeared since the 1980s by high acceptant DTaP vaccination rates. Annually, few tetanus patient has been reported since 1990s. But, there have been no seroepidemiological studies to tetanus, no trials to assess tetanus immunizations. And we do not use Td vaccine in adults for maintaning tetanus immunity. In this aspect, we conducted age related survey of immunity to tetanus and indirectly assessed the immunogenecity of tetanus vaccines, used in Korea. METHODS: For the evaluation of age related serosurvey of tetanus immunity in Korean population, study subjects were classified into 16 groups (A~J group; below 10 years with one year interval, K~O group; 11~60 yrs with 10 years interval, P group; over 60 yrs). The numbers of each group were 100, and sex distributions of each group were almostly equal. And for the indirect assessment of tetanus immunization in Korean children, children under 15 years old age were classified into 6 groups (I~VI) according to the status of DTaP vaccination. The numbers of this each group were 50, and sex ratio was almostly equal. Specific IgG antibody to tetanus toxin were detected by ELISA. And the ANOVA repeated t-test was used to compare antibody levels in study groups. RESULTS: In age related groups, the antibody levels to tetanus toxin were well maintained until 20 years old age group (L group), but thereafter the titers abruptly decreased below 0.1 IU/mL and over 75% populations among the groups over 30 years old age needed maintenance of protective immunity to tetanus. The antibody level of male was statistically higher than that of female in P group. In the groups related DTaP vaccination status, the antibody titer was very low in prevaccination group (I), but the titers after primary vaccinations were sharply increased and highly maintained until 15 years. CONCLUSOIN: The results of our study revealed that the immunity to tetanus was dramatically decreased in age groups over 30 years old. This result indicates that Td vaccination program in adults should be considered for maintenance of immunity to tetanus. And our study indicate that DTaP vaccination programs and vaccines, used in Korea, are effective for acquisition and maintenance of tetanus immunity in Korean children.