Helen Louise Havard
Bio: Helen Louise Havard is an academic researcher from Salisbury University. The author has contributed to research in topics: Cytotoxicity & Formazan. The author has an hindex of 1, co-authored 2 publications receiving 74 citations.
TL;DR: Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4- sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate.
Abstract: A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay (r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining (r = 0.995).
11 Mar 1997
TL;DR: In this article, a presente invention se rapporte notamment a des proteines who sont basees sur la toxine mature du gene de the toxine epsilon de clostridium perfringens, mais qui possede une mutation de sorte que l'acide amine a la position 106 soit different de la sequence de phenotype sauvage, ainsi qu'a l'utilisation of ces proteines dans des compositions de vaccins.
Abstract: La presente invention se rapporte a des proteines destinees a etre utilisees dans des vaccins qui sont capables d'induire des anticorps protecteurs diriges contre la toxine epsilon de C. perfringens lorsque ces vaccins sont administres a l'animal ou a l'homme, permettant par consequent d'obtenir une prophylaxie ou une therapie destinee a lutter contre l'infection due a la toxine epsilon C. perfringens. La presente invention se rapporte notamment a des proteines qui sont basees sur la toxine mature du gene de la toxine epsilon de clostridium perfringens, mais qui possede une mutation de sorte que l'acide amine a la position 106 soit different de la sequence de phenotype sauvage, ainsi qu'a l'utilisation de ces proteines dans des compositions de vaccins.
TL;DR: Epsilon toxin is a powerful toxin that represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons and is responsible for enterotoxemia in animals, mainly sheep.
Abstract: Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.
TL;DR: Evidence is presented that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex and efflux of intracellular K+ without entry of the toxin into the cytosol.
Abstract: Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in animals, which is characterized by edema in several organs due to an increase in blood vessel permeability. The Madin-Darby canine kidney (MDCK) cell line has been found to be susceptible to epsilon-toxin (D. W. Payne, E. D. Williamson, H. Havard, N. Modi, and J. Brown, FEMS Microbiol. Lett. 116:161-168, 1994). Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K+ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37 labeled C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37 degrees C was more stable than that formed at 4 degrees C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.
TL;DR: Epsilon toxin induced in Madin-Darby canine kidney cells a rapid decrease of intracellular K+, and an increase of Cl− and Na+, whereas the increase of Ca2+ occurred later, which probably represents the basic mechanism of toxin action on target cells.
TL;DR: The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells, and the crystal structure of ε‐toxin reveals similarity to aerolysin from Aeromonas’hydrophila, parasporin‐2 from Bacillus’thuringiensis and a lectin from Laetiporus’sulphureus.
Abstract: Clostridium perfringens e-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of e-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of e-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, e-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.
TL;DR: In most cases, host–toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death.
Abstract: Clostridium perfringens uses its large arsenal of protein toxins to produce histotoxic, neurologic and intestinal infections in humans and animals. The major toxins involved in diseases are alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), and necrotic B-like (NetB) toxins. CPA is the main virulence factor involved in gas gangrene in humans, whereas its role in animal diseases is limited and controversial. CPB is responsible for necrotizing enteritis and enterotoxemia, mostly in neonatal individuals of many animal species, including humans. ETX is the main toxin involved in enterotoxemia of sheep and goats. ITX has been implicated in cases of enteritis in rabbits and other animal species; however, its specific role in causing disease has not been proved. CPE is responsible for human food-poisoning and non-foodborne C. perfringens-mediated diarrhea. NetB is the cause of necrotic enteritis in chickens. In most cases, host–toxin interaction starts on the plasma membrane of target cells via specific receptors, resulting in the activation of intracellular pathways with a variety of effects, commonly including cell death. In general, the molecular mechanisms of cell death associated with C. perfringens toxins involve features of apoptosis, necrosis and/or necroptosis.