Histoautoradiography of central nervous system in rats with generalized tetanus due to 125I-toxin

Summary Rats were injected i.v. with ¹²⁵I-tetanus toxin. In autoradiographs of the spinal cord radioactivity was found over the pericarya and in the surroundings of the motoneurones whereas grain density was less over their nuclear region. In addition, pericarya in the lateral horn of the thoracic region and also the bipolar cells of the spinal ganglia contained radioactivity. The central part and the dorsal horns of spinal cord, and the white substance did not show any appreciable radioactivity. Within the medulla oblongata, clusters of large cells representing motor nuclei, as well as some fibre tracts close to them, contained ¹²⁵I. Forebrain and cerebellum remained free.According to its histoautoradiographic appearance, generalized tetanus can be described best as a combination of multiple local tetani.     

Peripheral neuropathy after administration of tetanus toxoid

Summary A reversible paresis of several muscles of the right arm was observed in a 20-year-old female, 3 weeks after the intragluteal injection of tetanus toxoid and 6–7 weeks after multiple injuries. The EMG findings indicated a transitory conduction block of nerve fibers to the deltoid and pectoralis muscles and a more severe axonal-demyelinating lesion of fibers to the biceps muscle. The cause of the palsy was probably a reaction to the administration of tetanus toxoid; other possible causes are discussed.

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Zusammenfassung Bei einer 20jährigen Patientin wurde 3 Wochen nach einer intraglutealen Injektion von Tetanus-Toxoid und 6–7 Wochen nach einem Polytrauma eine Lähmung mehrerer Muskeln des rechten Armes beobachtet. Die EMG-Befunde zeigten vorübergehend eine Leitungsblockade der Nerven zum M. deltoides und pectoralis und eine schwerere, axonale und Markscheidenläsion der Fasern zum M. biceps. Die Ursache der Lähmung ist möglicherweise in der Applikation des Tetanus-Toxoid zu sehen, andere mögliche Ursachen werden diskutiert.

     

Metabolic fate of 125I-tetanus toxin in the spinal cord of rats and cats with early local tetanus

Summary Local tetanus was elicited in rats and cats by intramuscular injection of ¹²⁵I-tetanus toxin. After different times spinal radioactivity was extracted with either non-ionic (Lubrol PX) or ionic (sodium dodecyl sulfate, SDS) detergents and compared with native or ¹²⁵I-toxin by gel filtration, SDS-gel electrophoresis, immunological procedures, and toxicity tests. In double-isotope experiments, ¹³¹I-toxin was added to the extracts as standard.In rats, the bulk of extracted material was indistinguishable from native toxin. However, there was a slight shift of the extracted material towards smaller molecular weights in gel filtration with Lubrol. In gel filtration with SDS, the toxin peak was followed by some tailing of ¹²⁵I radioactivity. Accordingly a small part of extracted radioactivity moves faster than the standard in SDS disc gel electrophoresis. These findings taken together indicate some degradation in vivo.Adsorption to solid-phase antibodies indicated that more than 80% of the radioactivity extracted from rats was still immunoreactive. It yielded a zone confluent with extrinsic toxin in immunodiffusion.The spinal cord Lubrol extract from rats was still toxic in the expected range. Due to the very small amounts of toxin present, no precise toxicity data could be given.In cats, there was also some evidence for radioactive split products in both SDS gel filtration and disc gel electrophoresis. The patterns closely resembled those obtained with extracts from rat spinal cord.SDS extracts from rat and cat spinal cords, poisoned with ¹²⁵I tetanus toxin in vivo, were also subjected to SDS disc gel electrophoresis followign reduction with dithioerythritol (DTE). They yielded large and small chains of the same size as did native toxin.In vitro, extensive degradation with brain homogenate from rats took place at pH 3.65, but not at pH 7.5. This indicates that lysosomal degradation is not a major metabolic pathway of tetanus toxin in vivo, although it is possible in principle.It is concluded that a) unlike other toxins, tetanus toxin is not necessarily degraded during its cellular uptake, b) the bulk of radioactive material is indistinguishable, following its neuronal ascent, from native or labeled toxin, c) a part of the radioactivity is recovered as split products.     

Distribution of tetanus toxin in rat peripheral nerve trunks during the development of local tetanus

Summary Mouse bioassay studies were made on the tetanus toxin content of three different segments of both sciatic nerve trunks after unilateral gastrocnemius muscle injections. Multiple toxin dosages, sampling times, nerve transections, and nerve dissections to produce three samples per segment (undissected nerve, epineurium, and perineurium plus endoneurium) were used.The results showed that after i.m. injections a dosage dependent bilateral toxin distribution could be detected in the undissected nerve or its epineurium. The characteristics of this epineurial toxin distribution are discussed.A unilateral toxin distribution was detected only in the stripped (perineurium plus endoneurium) nerve segment from the injected extremity. This unilateral toxin distribution had the characteristics of centripetal toxin transport along the tissue spaces or axons to the spinal cord. This unilateral centripetal toxin gradient was temporally related to the onset of local tetanus. Both the toxin gradient pattern and local tetanus due to i.m. injected toxin could be prevented by nerve transection.These data suggest that peripheral nerves do transport tetanus toxin to the spinal cord and that this transport in an important factor in the pathogenesis of local tetanus.     

Advances in tetanus research

Summary Due to the use of advanced biochemical and neurophysiological techniques, our knowledge of the pathogenesis of tetanus has considerably improved during the past years.Radio-labelled tetanus toxin has been traced within different nerves up to the anterior horn of the spinal cord where its localization down to the cellular level has been achieved. The distribution of labelled toxin depends on time and is influenced by antitoxin. The longer the duration of poisoning, the smaller the effect of antitoxin on the spinal enrichment of toxin and on the onset of toxic symptoms. The neural ascent of toxin into a spinal cord segment is enhanced by stimulation of the segmental nerves. Not only the motor nerves, but also sensory and vegetative nerves are able to serve as guide-rails for the toxin. The generalized tetanus has been understood as a special kind of local tetanus.For a long time, disinhibition of the alpha motor system was considered to be the characteristic action of tetanus toxin, but recent evidence is in favour of an additional disinhibition of the gamma motor system (perhaps even preceding the alpha disinhibition) and also of the sympathetic spinal reflexes. This finding should have therapeutic implications. The detection of inhibitory effects of tetanus toxin on peripheral cholinergic synapses points again to the close similarity between tetanus toxin and botulinum A toxin.The trends of future research will presumably lead to the elementary processes at the molecular and cellular level which are the basis of the clinical picture of tetanus.     

Human Lymphocyte Populations in Blood after Tetanus Vaccination

The content of circulating T and B lymphocytes in 9 healthy volunteers was estimated before and after secondary vaccination with tetanus toxoid. E, Fc, and C3 receptors were demonstrated by rosette techniques and surface Ig by fluorescence technique. The absolute number of T and non-T (B) lymphocytes was simultaneously increased during the first 48 h and at 144 h after vaccination. The correlation between paired T and non-T (B) lymphocyte counts was enhanced after vaccination, prolbably reflecting mechanisms governing the traffic of lymphocytes.     

B-cell exposure to self-antigen induces IL-10 producing B cells as well as IL-6- and TNF-α-producing B-cell subsets in healthy humans

Human B cells are able to secrete IL-10 after stimulation with mitogens, but their ability to produce IL-10 and regulate T-cell responses after stimulation with self-antigens is unclear. We co-cultured thyroglobulin-pulsed B cells from healthy donors with autologous T cells and observed production of IL-10 and TGF-β, in addition to TNF-α and IL-6. Pulsing with foreign antigen, tetanus toxoid (TT), induced a Th1-response with minimal IL-10 production. After thyroglobulin-pulsing, 1.10±0.50% of B cells and 1.00±0.20% of CD4(+) T cells produced IL-10, compared to 0.29±0.19% of B cells (P=0.01) and 0.13±0.15% of CD4(+) T cells (P=0.006) following TT-pulsing. Thyroglobulin-stimulated, IL-10-secreting B cells were enriched within CD5(+) and CD24(high) cells. While thyroglobulin-pulsed B cells induced only modest proliferation of CD4(+) T cells, B cells pulsed with TT induced vigorous proliferation. Thus, B cells mediate self-antigen-specific IL-10, TNF-α and IL-6 production in co-cultures with T cells and contribute actively to these cytokine secretions.     

Alginate Nanoparticles as a Promising Adjuvant and Vaccine Delivery System

During last decades, diphtheria has remained as a serious disease that still outbreaks and can occur worldwide. Recently, new vaccine delivery systems have been developed by using the biodegradable and biocompatible polymers such as alginate. Alginate nanoparticles as a carrier with adjuvant and prolong release properties that enhance the immunogenicity of vaccines. In this study diphtheria toxoid loaded nanoparticles were prepared by ionic gelation technique and characterized with respect to size, zeta potential, morphology, encapsulation efficiency, release profile, and immunogenicity. Appropriate parameters (calcium chloride and sodium alginate concentration, homogenization rate and homogenization time) redounded to the formation of suitable nanoparticles with a mean diameter of 70±0.5 nm. The loading studies of the nanoparticles resulted in high loading capacities (>90%) and subsequent release studies showed prolong profile. The stability and antigenicity of toxoid were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and ouchterlony test and proved that the encapsulation process did not affect the antigenic integrity and activity. Guinea pigs immunized with the diphtheria toxoid-loaded alginate nanoparticles showed highest humoral immune response than conventional vaccine. It is concluded that, with regard to the desirable properties of nanoparticles and high immunogenicity, alginate nanoparticles could be considered as a new promising vaccine delivery and adjuvant system.     

Polymer conjugates for drug targeting. From inspired to inspiration!

One goal of gene therapy is the targeted delivery of therapeutic genes to defined tissues. One attractive target is the central nervous system as there are several neuronal degenerative diseases which may be amenable to gene therapy. At present there is a lack of delivery systems that are able to target genes specifically to neuronal cells. Multi-domain proteins were designed and constructed to facilitate the delivery of exogenous genes to neuronal cells. Neuronal targeting activity of the proteins was achieved by inclusion of the H_C fragment of tetanus toxin (TeNT), a protein with well-characterised tropism for the central nervous system. The yeast Gal4 DNA-binding domain enabled specific binding of DNA while the translocation domain from diphtheria toxin (DT) was included to facilitate crossing of the endosomal vesicle. One multi-domain protein, containing all three of these domains, was found to transfect up to 8% of neuroblastoma N18-RE105 cells with marker genes. Monitoring the transfection by confocal microscopy indicated that this protein-DNA transfection complex is to some extent localised at the cell surface, suggesting that further improvements to translocating this membrane barrier may yield higher transfection levels. The demonstration that this multi-domain protein can target genes specifically to neuronal cells is a first step in the development of novel vectors for the delivery of genes with therapeutic potential to diseased neuronal tissues.