References


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Ref 1: Murrell TG, O'Donoghue PJ, Ellis T. (1986)

A review of the sheep-multiple sclerosis connection. (Medical Hypotheses) https://www.ncbi.nlm.nih.gov/pubmed/2871478 This paper reviews a notion that the prevalence of multiple sclerosis is high in global areas where sheep populations are concentrated. Pilot studies are reported to serum antibodies in humans to three sheep diseases; focal symmetrical encephalomalacia (FSE), maedi visna and sarcocystis. In MS patients and controls antibodies were not found to the epsilon neurotoxin of the FSE organism, Clostridium welchii type D and to a caprine retrovirus that is closely related to maedi-visna virus. However, 34% of MS and control patients had antibodies to the protozoan parasite Sarcocystis spp., tissue cysts of which contain a powerful neurotoxin, sarcocystin. It is suggested that epidemiological MS prevalence rates for country areas of southern Australia require further study along with an examination for the prevalence of MS in vegetarians.
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Ref 2: Osamu Miyamoto, Kazunori Sumitani, Takehiro Nakamura, Shin-ichi Yamagami, Shigeru Miyata, Toshifumi Itano, Tetsuro Negi, Akinobu Okabe (2000)

Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus (FEMS Microbiology Letters) https://academic.oup.com/femsle/article/189/1/109/659425 The mechanism of neurotoxicity of Clostridium perfringens epsilon toxin to the mouse brain was investigated. Intravenous injection in mice with the toxin caused seizure and excited hippocampal neurons. Microdialysis revealed that epsilon toxin induced excessive glutamate release in the hippocampus. Both the seizure and glutamate release were attenuated by prior injection with riluzole, an inhibitor of pre-synaptic glutamate release, suggesting that this toxin enhances glutamate efflux, leading to seizure and hippocampal neuronal damage.
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Ref 3: Amjad A. Ilyas, Zie-Wie Chen, Stuart D. Cook (2003)

Antibodies to sulfatide in cerebrospinal fluid of patients with multiple sclerosis (Neuroimmunology) https://www.jni-journal.com/article/S0165-5728(03)00131-0/abstract The identity of target antigen(s) in multiple sclerosis (MS) remains elusive despite much effort to identify it. We analyzed cerebrospinal fluid (CSF) from patients with MS, other neurological diseases (OND), other diseases (OD) and healthy controls for antibodies against purified sulfatide, a major glycosphingolipid of human myelin, by an enzyme-linked immunosorbent assay (ELISA) and a thin-layer chromatogram (TLC)-immunostaining technique. Elevated anti-sulfatide antibodies were significantly higher in MS patients as compared with the OND group (p<0.05) and all controls combined (P<0.025). Binding of high titer antibodies to sulfatide was confirmed with TLC-immunostaining. Anti-sulfatide antibodies were detected in all subtypes of MS although the frequency was higher in patients with secondary progressive MS (SPMS) than in patients with primary progressive (PPMS) and relapsing–remitting MS (RRMS). The data demonstrate a humoral response to sulfatide in the CSF of patients with MS.
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Ref 4: Skrivanová E, Marounek M, Dlouhá G, Kanka J. (2004)

Susceptibility of Clostridium perfringens to C-C fatty acids. (Letters in Applied Microbiology) https://www.ncbi.nlm.nih.gov/pubmed/15960756 AIMS: To determine susceptibility of Clostridium perfringens strains CCM 4435(T) and CNCTC 5459 to C(2)-C(18) fatty acids, and evaluate influence of pH in cultures grown on glucose. Straw particles were added to cultures to simulate the presence of solid phase of the digestive tract milieu. METHODS AND RESULTS: Antimicrobial activity of fatty acids was expressed as a concentration at which only 50% of the initial glucose was utilized. Lauric acid showed the highest antimicrobial activity, followed by myristic, capric, oleic and caprylic acid. Only strain CNCTC 5459 was susceptible to linoleic acid. Neither caproic acid and acids with a shorter carbon chain nor palmitic and stearic acid influenced substrate utilization. The antimicrobial activity of myristic, oleic and linoleic acid decreased when clostridia were grown in the presence of straw particles. In cultures of both strains treated with capric and lauric acid at pH 5.0-5.3, the number of viable cells was <10(2) ml(-1). Only lauric acid reduced number of viable cells of both strains below 10(2) ml(-1) at pH > 6. Transmission electron microscopy revealed separation of inner and outer membranes and cytoplasma disorganization in cells treated with lauric acid. CONCLUSIONS: Lauric acid had the highest activity towards C. perfringens among fatty acid tested. Its activity was not influenced by the presence of solid particles and did not cease at pH > 6. SIGNIFICANCE AND IMPACT OF THE STUDY: Lauric acid might be a means for control of clostridial infections in farm animals.
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Ref 5: Michael H. Barnett, MBBS and John W. Prineas, MBBS (2004)

Relapsing and Remitting Multiple Sclerosis: Pathology of the Newly Forming Lesion (Annals of Neurology) http://cpnhelp.org/files/Ref1_Annals04.pdf The pathological changes associated with the formation of new symptomatic lesions in patients with relapsing and remitting multiple sclerosis (MS) have remained largely unknown, because such lesions are rarely fatal. Among previous reports of newly forming lesions of known clinical duration, the earliest appear to be two “7-day” brainstem lesions reported by Adams and colleagues in 1989.1 Such “acute” lesions generally are described as actively demyelinating lesions in which myelin phagocytes directly engage normal-appearing myelin sheaths in the presence of infiltrating T cells. Because similar changes are observed in some forms of experimental allergic encephalomyelitis (EAE), this has encouraged the view that tissue destruction in MS is the result of a T-cell–mediated cellular immune response directed against myelin. This study describes the clinical and pathological findings in a young patient with relapsing and remitting MS who died within 24 hours of the onset of a new symptomatic brainstem lesion. In view of the unusual findings, we reviewed tissue obtained at autopsy from other patients with rapidly deteriorating MS. Nine lesions were found that were essentially identical to the fatal brainstem lesion in the first case. The earliest structural change shared by all 10 lesions was extensive oligodendrocyte apoptosis in tissue exhibiting early microglial activation but few or no infiltrating lymphocytes or myelin phagocytes. This is unlike any current laboratory model of the disease, in particular, experimental allergic encephalomyelitis, which raises the possibility of some novel disease process underlying lesion formation in relapsing and remitting MS.
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Ref 6: Vissia Viglietta, Clare Baecher-Allan, Howard L. Weiner, and David A. Hafler (2004)

Loss of Functional Suppression by CD4+CD25+ Regulatory T Cells in Patients with Multiple Sclerosis (Journal of Experimental Medicine) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211881/ CD4+CD25+ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4+ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4+CD25+ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4+CD25hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4+CD25hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4+CD25hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4+CD25hi regulatory T cell function in patients with MS.
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Ref 7: Sriram S, Steiner I. (2005)

Experimental allergic encephalomyelitis: a misleading model of multiple sclerosis (Annals of Neurology) https://www.limav.org/italia/wp-content/uploads/2016/10/Siram-2005-Experimental-Allergic-Encephalomyelitis-a-misleading-model-of-multiple-sclerosis.pdf Despite many years of intensive research, multiple sclerosis (MS) defies understanding and treatment remains suboptimal. The prevailing hypothesis is that MS is immune mediated and that experimental allergic encephalomyelitis (EAE) is a suitable model to elucidate pathogenesis and devise therapy. This review examines critically the validity that EAE is an adequate and useful animal model of MS and finds credible evidence lacking. EAE represents more a model of acute central nervous system inflammation than the counterpart of MS. We propose to reconsider the utilization of EAE, especially when this model is used to define therapy. This will also force us to examine MS without the restraints imposed by EAE, as to what it is, rather than what it looks like. Introduction Although the cause and pathogenesis of multiple sclerosis (MS) are unknown, current prevailing hypothesis favors MS to represent an autoimmune disorder directed against nervous system antigens. 1–3 The basic concept proposes that exposure to environmental pathogens activates autoreactive T cells that recognize central nervous system (CNS) autoantigens, leading to inflammation and demyelination.4 –7 This belief is promoted by some similarities between MS and the various animal models of experimental allergic encephalitis (EAE).8 Since the initial experiments by Rivers, the stage was set for the use of experimental animal models to study CNS inflammation and demyelination.9 Over the last 30 years, the number of EAE-cited publications in English has quadrupled; a Medline search identifies a total of 678 articles on EAE between the years 1970 and 1980, 1,860 articles between 1990 and 2000, and approximately 1,600 publications since 2001. Besides the utilization of EAE to study MS, it has also been harnessed for developing therapeutic strategies for MS.10 –12 Indeed, the majority of the current therapies being planned for phase II and III trials in MS were first examined in EAE. Thus, EAE has become a central player in the arena of MS. Is it indeed a suitable and relevant research tool for MS? It has improved our understanding of acute inflammatory demyelinating syndromes, advanced our knowledge of the genetic susceptibility to autoimmunity, and helped uncover mechanisms of lymphocyte trafficking and the role of blood–brain barrier in CNS inflammation. We propose, however, that although EAE is a useful model of acute human CNS demyelination such as acute disseminated encephalomyelitis (ADEM), its contribution to the understanding of MS has been limited. We focus here on the lack of resemblance of the EAE model with MS and examine its shortcomings when attempting to extrapolate the findings from the model to the human disease.
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Ref 8: Srinivasan R, Sailasuta N, Hurd R, Nelson S, Pelletier D. (2005)

Evidence of elevated glutamate in multiple sclerosis using magnetic resonance spectroscopy at 3 T. (Brain) https://www.ncbi.nlm.nih.gov/pubmed/15758036 Histopathological reports of multiple sclerosis and its animal models have shown evidence of a link between axonal injury in active lesions and impaired glutamate metabolism. Mature oligodendrocytes play a role in glutamate uptake to maintain glutamate homeostasis but in multiple sclerosis white matter the loss of expression of glutamate transporters in the lesion vicinity results in ineffective glutamate removal. Using a magnetic resonance spectroscopy technique that isolates the glutamate resonance at 3 T, we compared glutamate levels between normal subjects and multiple sclerosis patients in different brain areas. Metabolite concentrations (glutamate, glutamine, N-acetyl-aspartate, myo-inositol, choline, creatine) were derived from LCmodel and corrected for T1 relaxation time. Glutamate concentrations were found to be elevated in acute lesions (P = 0.02) and normal-appearing white matter (P = 0.03), with no significant elevation in chronic lesions (P = 0.77). The N-acetyl-aspartate level in chronic lesions was significantly lower (P < 0.001) than in acute lesions and normal-appearing white matter. The choline level in acute lesions was significantly higher (P < 0.001) than in chronic lesions. Evidence was also found for increased glial activity in multiple sclerosis, with significantly higher (P < 0.001) myo-inositol levels in acute lesions compared with control white matter. These in vivo results support the hypothesis that altered glutamate metabolism is present in brains of multiple sclerosis patients.
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Ref 9: James J. Campbell (2006)

Tregs control B-cell life and death (Blood) http://www.bloodjournal.org/content/107/10/3818.2 In this issue, Zhao and colleagues describe the surprising finding that CD4+CD25+ regulatory T cells (Tregs) abrogate B-cell proliferation by direct induction of B-cell death through granzyme- and perforin-dependent pathways. Regulatory T cells have received much attention in recent years for their ability to dampen otherwise severe immune responses. Understanding the mechanisms by which such cells control immune responses could lead to innovative clinical interventions: The ability to increase specific regulatory T-cell (Treg) activities could potentially control a variety of autoimmune diseases. Further, inhibition of specific Treg activities could break immunotolerance to cancer cells, allowing normal immune responses to eliminate tumors. It has been clear since 2001 that activated CD4+CD25+ T cells could inhibit proliferation and Ig secretion of LPS-activated B cells, but the mechanisms of action remained unclear. Zhao and colleagues have found that activated T cells are able to inhibit B-cell proliferation by directly inducing apoptosis of the proliferating B cells themselves. This mechanism is quite different from how Tregs control T-cell proliferation, in which Tregs prevent IL-2 production by proliferating T cells. The authors demonstrate convincingly that CD4+CD25+ T cells do not induce B-cell apoptosis through the pathways that would normally be associated with lymphocyte-lymphocyte interactions (ie, Fas/FasL, TNF/TNFR, or TRAIL/TRAILR). Instead, the apoptotic event requires a combination of granzyme B and perforin activities. The requirement for these 2 pathways is reminiscent of the manner in which CD8 cytotoxic T cells kill their targets through class I–dependent antigen recognition. In fact, the authors demonstrate that activated CD4+CD25+ T cells produce and release granzyme B similarly to CD8 T cells, an activity not found in CD4+CD25– T cells. The authors further demonstrate that B-cell apoptosis mediated by CD4+CD25+ T cells can be antigen dependent. CD4+CD25+ T cells from mice bearing a transgenic TCR that recognizes an ovalbumin peptide were used for these experiments. Proliferating B cells were divided into 2 separate groups, one pulsed with ovalbumin and one unpulsed. Ovalbumin-specific CD4+CD25+ T cells were dramatically more efficient at killing antigen-pulsed B cells, demonstrating that this Treg activity is indeed antigen dependent. Thus, an entirely new mechanism of Treg activity has been discovered. B-cell proliferation is controlled in a completely different manner from T-cell proliferation. This mechanism involves direct, antigen-selective induction of B-cell apoptosis. These findings are an important advance in our knowledge of Treg function.
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Ref 10: Juneja VK, Bari ML, Inatsu Y, Kawamoto S, Friedman M. (2007)

Control of Clostridium perfringens spores by green tea leaf extracts during cooling of cooked ground beef, chicken, and pork (Journal of Food Protection) https://www.ncbi.nlm.nih.gov/pubmed/17612073 We investigated the inhibition of Clostridium perfringens spore germination and outgrowth by two green tea extracts with low (green tea leaf powder [GTL]; 141 mg of total catechins per g of green tea extract) and high (green tea leaf extract [GTE]; 697 mg of total catechins per g of extract) catechin levels during abusive chilling of retail cooked ground beef, chicken, and pork. Green tea extracts were mixed into the thawed beef, chicken, and pork at concentrations of 0.5, 1.0, and 2.0% (wt/ wt), along with a heat-activated (75 degrees C for 20 min) three-strain spore cocktail to obtain a final concentration of approximately 3 log spores per g. Samples (5 g) of the ground beef, chicken, and pork were then vacuum packaged and cooked to 71 degrees C for 1 h in a temperature-controlled water bath. Thereafter, the products were cooled from 54.4 to 7.2 degrees C in 12, 15, 18, or 21 h, resulting in significant increases (P < 0.05) in the germination and outgrowth of C. perfringens populations in the ground beef, chicken, and pork control samples without GTL or GTE. Supplementation with 0.5 to 2% levels of GTL did not inhibit C. perfringens growth from spores. In contrast, the addition of 0.5 to 2% levels of GTE to beef, chicken, and pork resulted in a concentration-and time-dependent inhibition of C. perfringens growth from spores. At a 2% level of GTE, a significant (P < 0.05) inhibition of growth occurred at all chill rates for cooked ground beef, chicken, and pork. These results suggest that widely consumed catechins from green tea can reduce the potential risk of C. perfringens spore germination and outgrowth during abusive cooling from 54.4 to 7.2 degrees C in 12, 15, 18, or 21 h of cooling for ground beef, chicken, and pork.
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Ref 11: (2007)

Carvacrol, cinnamaldehyde, oregano oil, and thymol inhibit Clostridium perfringens spore germination and outgrowth in ground turkey during chilling. (Journal of Food Protection) https://www.ncbi.nlm.nih.gov/pubmed/17265885 Inhibition of Clostridium perfringens by plant-derived carvacrol, cinnamaldehyde, thymol, and oregano oil was evaluated during abusive chilling of cooked ground turkey. Test substances were mixed into thawed turkey product at concentrations of 0.1, 0.5, 1.0, or 2.0% (wt/wt) along with a heat-activated three-strain C. perfringens spore cocktail to obtain final spore concentrations of ca. 2.2 to 2.8 log CFU spores per g of turkey meat. Aliquots (5 g) of the ground turkey mixtures were vacuum packaged and then cooked in a water bath, where the temperature was raised to 60 degrees C in I h. The products were cooled from 54.4 to 7.2 degrees C in 12, 15, 18, or 21 h, resulting in 2.9-, 5.5-, 4.9-, and 4.2-log CFU/g increases, respectively, in C. perfringens populations in samples without antimicrobials. Incorporation of test compounds (0.1 to 0.5%) into the turkey completely inhibited C. perfringens spore germination and outgrowth (P < or = 0.05) during exponential cooling in 12 h. Longer chilling times (15, 18, and 21 h) required greater concentrations (0.5 to 2.0%) to inhibit spore germination and outgrowth. Cinnamaldehyde was significantly (P < 0.05) more effective (<1.0-log CFU/g growth) than the other compounds at a lower concentration (0.5%) at the most abusive chilling rate evaluated (21 h). These findings establish the value of the plant-derived antimicrobials for inhibiting C. perfringens in commercial ground turkey products.
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Ref 12: J W Finnie, Jim Manavis, Peter Blumbergs (2008)

Aquaporin-4 in Acute Cerebral Edema Produced by Clostridium perfringens Type D Epsilon Toxin (Veterinary Pathology) https://www.researchgate.net/publication/5363991_Aquaporin-4_in_Acute_Cerebral_Edema_Produced_by_Clostridium_perfringens_Type_D_Epsilon_Toxin Sheep, particularly lambs, with high circulating levels of Clostridium perfringens type D epsilon toxin develop severe neurologic signs and often die suddenly. On microscopic examination, in the brain, there is microvascular endothelial injury and diffuse vasogenic edema. The aquaporin (AQP) family of membrane water-channel proteins, especially AQP-4, is important in the regulation of water balance in the brain and facilitates reabsorption of excess fluid. In rats given epsilon toxin, generalized cerebral edema was demonstrated by marked albumin extravasation and was correlated with widespread upregulation of AQP-4 in astrocytes. These results suggest that AQP-4 has a role in the clearance of edema fluid from brains damaged by this clostridial toxin.
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Ref 13: L. Timbermont et al (2010)

Control of Clostridium perfringens-induced necrotic enteritis in broilers by target-released butyric acid, fatty acids and essential oils (Avian Pathology) https://tandfonline.com/doi/full/10.1080/03079451003610586 The efficacy of target-released butyric acid, medium-chain fatty acids (C6 to C12 but mainly lauric acid) and essential oils (thymol, cinnamaldehyde, essential oil of eucalyptus) micro-encapsulated in a poly-sugar matrix to control necrotic enteritis was investigated. The minimal inhibitory concentrations of the different additives were determined in vitro, showing that lauric acid, thymol, and cinnamaldehyde are very effective in inhibiting the growth of Clostridium perfringens. The in vivo effects were studied in two trials in an experimental necrotic enteritis model in broiler chickens. In the first trial, four groups of chickens were fed a diet supplemented with butyric acid, with essential oils, with butyric acid in combination with medium-chain fatty acids, or with butyric acid in combination with medium-chain fatty acids and essential oils. In all groups except for the group receiving only butyric acid, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. In the second trial the same products were tested but at a higher concentration. An additional group was fed a diet supplemented with only medium-chain fatty acids. In all groups except for that receiving butyric acid in combination with medium-chain fatty acids and essential oils, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. These results suggest that butyric acid, medium-chain fatty acids and/or essential oils may contribute to the prevention of necrotic enteritis in broilers.
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Ref 14: Popoff MR (2011)

Epsilon toxin: a fascinating pore-forming toxin (FEBS Journal) https://www.ncbi.nlm.nih.gov/pubmed/21535407 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.
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Ref 15: Antón OM, Andrés-Delgado L, Reglero-Real N, Batista A, Alonso MA. (2011)

MAL protein controls protein sorting at the supramolecular activation cluster of human T lymphocytes. (Journal of Immunology) https://www.ncbi.nlm.nih.gov/pubmed/21508261 T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.
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Ref 16: Susan E. Ivie and Mark S. McClain (2012)

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1 (Biochemistry) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534817/#!po=78.1250. Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.
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Ref 17: LaetitiaWioland, Jean-LucDupont, Jean-LouisBossu, Michel R.Popoff, Bernard Poulain (2013)

Attack of the nervous system by Clostridium perfringens Epsilon toxin: From disease to mode of action on neural cells (Toxicon) https://www.sciencedirect.com/science/article/pii/S0041010113001335 Epsilon toxin (ET), produced by Clostridium perfringens types B and D, ranks among the four most potent poisonous substances known so far. ET-intoxication is responsible for enterotoxaemia in animals, mainly sheep and goats. This disease comprises several manifestations indicating the attack of the nervous system. This review aims to summarize the effects of ET on central nervous system. ET binds to endothelial cells of brain capillary vessels before passing through the blood–brain barrier. Therefore, it induces perivascular oedema and accumulates into brain. ET binding to different brain structures and to different component in the brain indicates regional susceptibility to the toxin. Histological examination has revealed nerve tissue and cellular lesions, which may be directly or indirectly caused by ET. The naturally occurring disease caused by ET-intoxication can be reproduced experimentally in rodents. In mice and rats, ET recognizes receptor at the surface of different neural cell types, including certain neurons (e.g. the granule cells in cerebellum) as well as oligodendrocytes, which are the glial cells responsible for the axons myelination. Moreover, ET induces release of glutamate and other transmitters, leading to firing of neural network. The precise mode of action of ET on neural cells remains to be determined.
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Ref 18: Kareem Rashid Rumah,Jennifer Linden,Vincent A. Fischetti,Timothy Vartanian (2013)

Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease (PlosOne) https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076359 We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin’s tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate
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Ref 19: TITBALL, Richard W.; BOKORI-BROWN, Monika; NAYLOR, Claire; (2013)

EPSILON TOXIN EPITOPES FROM CLOSTRIDIUM PERFRINGENS WITH REDUCED TOXICITY () https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2013144636&recNum=278&docAn=GB2013050821&queryString=(CL/antibod*)%20OR%20(DE/antibod*)%20OR%20(ET/antibod*)%20&maxRec=161737 There is provided an epsilon toxin epitope polypeptide comprising a sequence of at least 10 contiguous amino acids from SEQ ID NO:3, the sequence comprising a mutation of at least one tyrosine residue compared to the equivalent sequence in SEQ ID NO:3, the polypeptide being capable of binding an antibody which binds to SEQ ID NO:5 and having reduced toxicity compared with the toxicity of SEQ ID NO:5. The polypeptide is useful in a method of vaccinating a subject against developing a disease caused by Clostridium perfringens and/or caused by (or associated with the presence of) active epsilon toxin.
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Ref 20: Kapil Gaur, Kshitija Iyer, Sumalatha Pola, Richa Gupta, Anil K Gadipelli, Praveen Suddala, Prakash Acharya, Shounak Pal and Vishal Tripathy (2014)

The Clostridium perfringens Epsilon Toxin as a Bioterrorism Weapon (Journal of Microbial & Biochemical Technology) https://www.longdom.org/open-access/the-clostridium-perfringens-epsilon-toxin-as-a-bioterrorism-weapon-1948-5948.S8-009.pdf The Clostridium perfringens epsilon toxin made by the microorganism true bacteria could be a doubtless fatal bioterrorism weapon. If used effectively, this agent might cause important morbidity and mortality and, because of a scarcity of existing analysis into the result of this poison in humans, no therapeutic or preventive measures exist as medical countermeasures. Notable well for manufacturing severe medicine malady in domestic mammals, Clostridium perfringens epsilon toxin might be promptly weaponized and dispersed via AN orally ingestible or aerosol route. This critical review considers the present literature and tries to position stress on understanding this organism and this poison in its rightful place as AN agent of goodly concern to be used in act of terrorism attacks. The review includes a discussion of the bacteria genus, the precise toxins of C. perfringens in addition as focusing on Clostridium perfringens epsilon toxin above all. Discussion The bacterial toxin of eubacteria prefringens could be a probably dangerous terrorist act weapon. Intoxication through oral body process in animals of the microorganism in reproductive structure type produces severe and infrequently fatal sickness related to cerebral and different organ puffiness and a chop-chop fatal clinical syndrome. If effectively weaponized via associate degree aerosol or oral body process route, the toxic and deadly doses in humans don’t seem to be farfamed. No human immunizing agent or specific therapeutic is available for treatment of bacterial toxin created sickness in humans, creating it all the additional attractive as a possible weapon of mass morbidity and mortality. Continued analysis and understanding of this agent are necessary to confirm protection against this agent.
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Ref 21: Oscar G. Vidaurre, Jeffery D. Haines, (2014)

Cerebrospinal fluid ceramides from patients with multiple sclerosis impair neuronal bioenergetics (Brain) https://academic.oup.com/brain/article/137/8/2271/2848013 Axonal damage is a prominent cause of disability and yet its pathogenesis is incompletely understood. Using a xenogeneic system, here we define the bioenergetic changes induced in rat neurons by exposure to cerebrospinal fluid samples from patients with multiple sclerosis compared to control subjects. A first discovery cohort of cerebrospinal fluid from 13 patients with multiple sclerosis and 10 control subjects showed that acute exposure to cerebrospinal fluid from patients with multiple sclerosis induced oxidative stress and decreased expression of neuroprotective genes, while increasing expression of genes involved in lipid signalling and in the response to oxidative stress. Protracted exposure of neurons to stress led to neurotoxicity and bioenergetics failure after cerebrospinal fluid exposure and positively correlated with the levels of neurofilament light chain. These findings were validated using a second independent cohort of cerebrospinal fluid samples (eight patients with multiple sclerosis and eight control subjects), collected at a different centre. The toxic effect of cerebrospinal fluid on neurons was not attributable to differences in IgG content, glucose, lactate or glutamate levels or differences in cytokine levels. A lipidomic profiling approach led to the identification of increased levels of ceramide C16:0 and C24:0 in the cerebrospinal fluid from patients with multiple sclerosis. Exposure of cultured neurons to micelles composed of these ceramide species was sufficient to recapitulate the bioenergetic dysfunction and oxidative damage induced by exposure to cerebrospinal fluid from patients with multiple sclerosis. Therefore, our data suggest that C16:0 and C24:0 ceramides are enriched in the cerebrospinal fluid of patients with multiple sclerosis and are sufficient to induce neuronal mitochondrial dysfunction and axonal damage.
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Ref 22: Kareem Rashid Rumah (2014)

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/178/ (The Origin of Multiple Sclerosis Revisited: The Case for a Soluble Toxin) https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/178/ Weill Cornell
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Ref 23: Kareem Rashid Rumah ,Yinghua Ma ,Jennifer R. Linden ,Myat Lin Oo,Josef Anrather,Nicole Schaeren-Wiemers,Miguel A. Alonso,Vincent A. Fischetti,Mark S. McClain,Timothy Vartanian (2015)

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin (Plos Pathogens) https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004896 Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.
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Ref 24: Anne-Katrin Pröbstel, Nicholas S. R. Sanderson and Tobias Derfuss (2015)

B Cells and Autoantibodies in Multiple Sclerosis (International Journal of Molecular Sciences) https://www.mdpi.com/1422-0067/16/7/16576/htm While over the past decades T cells have been considered key players in the pathogenesis of multiple sclerosis (MS), it has only recently become evident that B cells have a major contributing role. Our understanding of the role of B cells has evolved substantially following the clinical success of B cell-targeting therapies and increasing experimental evidence for significant B cell involvement. Rather than mere antibody-producing cells, it is becoming clear that they are team players with the capacity to prime and regulate T cells, and function both as pro- and anti-inflammatory mediators. However, despite tremendous efforts, the target antigen(s) of B cells in MS have yet to be identified. The first part of this review summarizes the clinical evidence and results from animal studies pointing to the relevance of B cells in the pathogenesis of MS. The second part gives an overview of the currently known potential autoantigen targets. The third part recapitulates and critically appraises the currently available B cell-directed therapies. B Cell-Directed Therapies—Towards Personalized Treatment While substantial evidence for B cell involvement in MS comes from animal studies, the success of B cell-directed therapies illustrates a triumphant example of how translational medicine promotes basic findings into clinical applications, allowing for more diverse and personalized treatment options for MS patients. A variety of currently available treatments such as natalizumab, fingolimod, and alemtuzumab, as well as several anti-CD20 and anti-CD19 therapies currently being tested in clinical trials (anti-CD20: rituximab, ocrelizumab, ofatumumab; anti-CD19: MEDI-551; anti-BAFF-R: VAY736), have a substantial targeting effect on B cells (Table 1). Although natalizumab exhibits its primary effect on the reduction of T cells in the CNS by blocking their egress into the CNS via alpha 4-integrin blockade, it has also been shown to reduce B cell counts in the CNS of MS patients [109]. In contrast, the effect of fingolimod is most pronounced in the peripheral blood, leading to a marked decrease of circulating B cells by sequestration in the secondary lymphoid organs, and it also directly alters proportions of B cell subpopulations in treated patients with MS [110]. Alemtuzumab, which has been recently approved for the treatment of active multiple sclerosis, is a monoclonal anti-CD52 antibody with a direct depleting effect on both B and T cells
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Ref 25: Hayriye Cetin-Karaca,Melissa Newman (2015)

Antimicrobial Efficacy of Natural Phenolic Compounds against Gram Positive Foodborne Pathogens (Journal of Food Research) https://www.researchgate.net/publication/283685523_Antimicrobial_Efficacy_of_Natural_Phenolic_Compounds_against_Gram_Positive_Foodborne_Pathogens Protection of food from pathogens and spoilage organisms has been achieved by a variety of methods. Due to consumer preference, health and economic concerns in recent years, there is considerable interest to employ natural antimicrobials as an alternative to control the growth of microorganisms. This study evaluates the antimicrobial efficacy of natural plant derived phenolic compounds (PDPC) including chlorogenic acid, coumarin, curcumin, ellagic acid, (-) epicatechin, eugenol, rosmarinic acid, rutin, tannic acid, thymol, thymoquinone, and xanthohumol) as preservatives in food products. Several strains of Bacillus, Listeria and Clostridium species were treated with 12 natural PDPCs. Concentrations of 5, 10, 15, and 20 ppm of each compound were evaluated by broth micro-dilution method and the MICs were determined by using optical density after 24 and 60 hours of incubation. Thymoquinone, xanthohumol and ellagic acid demonstrated the highest antimicrobial efficacy (MIC <20 ppm). Structural alterations in treated bacteria were observed via scanning electron microscopy. The results demonstrated that the PDPCs have varying antimicrobial activities against both aerobic and anaerobic Gram-positive foodborne pathogens following 24 hour and 60 hour incubation periods, respectively. Natural sources of phenolic compounds contain major antimicrobial components and have great potential to control the growth of pathogens and be used as natural antimicrobials and food preservatives for extended storage.This study highlighted the antimicrobial efficacy of some PDPCs which may replace the artificial antimicrobials and preservatives in food industry to partially or completely control or inhibit the growth of harmful bacteria.
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Ref 26: Carles Gil ,Jonatan Dorca-Arévalo,Juan Blasi (2015)

Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide (Plos One) https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140321 Epsilon toxin (Etx) is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx) that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction) and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3)-phosphate and phosphatidylinositol (5)-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.
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Ref 27: Jennifer R. Linden, Yinghua Ma, Baohua Zhao, Jason Michael Harris, Kareem Rashid Rumah, Nicole Schaeren-Wiemers, Timothy Vartanian (2015)

Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination (American Society for Microbiology) https://mbio.asm.org/content/6/3/e02513-14 Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody.
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Ref 28: Vidaurre OG (2015)

Cerebrospinal fluid ceramides from patients with multiple sclerosis impair neuronal bioenergetics. (Brain) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164163/ Axonal damage is a prominent cause of disability and yet its pathogenesis is incompletely understood. Using a xenogeneic system, here we define the bioenergetic changes induced in rat neurons by exposure to cerebrospinal fluid samples from patients with multiple sclerosis compared to control subjects. A first discovery cohort of cerebrospinal fluid from 13 patients with multiple sclerosis and 10 control subjects showed that acute exposure to cerebrospinal fluid from patients with multiple sclerosis induced oxidative stress and decreased expression of neuroprotective genes, while increasing expression of genes involved in lipid signalling and in the response to oxidative stress. Protracted exposure of neurons to stress led to neurotoxicity and bioenergetics failure after cerebrospinal fluid exposure and positively correlated with the levels of neurofilament light chain. These findings were validated using a second independent cohort of cerebrospinal fluid samples (eight patients with multiple sclerosis and eight control subjects), collected at a different centre. The toxic effect of cerebrospinal fluid on neurons was not attributable to differences in IgG content, glucose, lactate or glutamate levels or differences in cytokine levels. A lipidomic profiling approach led to the identification of increased levels of ceramide C16:0 and C24:0 in the cerebrospinal fluid from patients with multiple sclerosis. Exposure of cultured neurons to micelles composed of these ceramide species was sufficient to recapitulate the bioenergetic dysfunction and oxidative damage induced by exposure to cerebrospinal fluid from patients with multiple sclerosis. Therefore, our data suggest that C16:0 and C24:0 ceramides are enriched in the cerebrospinal fluid of patients with multiple sclerosis and are sufficient to induce neuronal mitochondrial dysfunction and axonal damage.
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Ref 29: Takagishi T, Oda M, Takehara M, Kobayashi K, Nagahama M. (2016)

Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase. (Biochimica et Biophysica Acta.) https://www.ncbi.nlm.nih.gov/pubmed/27453200 BACKGROUND: Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization. METHODS: We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining. RESULTS: Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide. CONCLUSIONS: These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin. GENERAL SIGNIFICANCE: Inhibitors of nSMase may confer protection against infection.
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Ref 30: Freedman JC, McClane BA, Uzal FA. (2016)

New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia. (Anaerobe) https://www.ncbi.nlm.nih.gov/pubmed/27321761 Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells.
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Ref 31: Baker D, Marta M, Pryce G, Giovannoni G, Schmierer K (2017)

Memory B Cells are Major Targets for Effective Immunotherapy in Relapsing Multiple Sclerosis (Ebio Medicine) https://www.ncbi.nlm.nih.gov/pubmed/28161400 Although multiple sclerosis (MS) is considered to be a CD4, Th17-mediated autoimmune disease, supportive evidence is perhaps circumstantial, often based on animal studies, and is questioned by the perceived failure of CD4-depleting antibodies to control relapsing MS. Therefore, it was interestingly to find that current MS-treatments, believed to act via T cell inhibition, including: beta-interferons, glatiramer acetate, cytostatic agents, dimethyl fumarate, fingolimod, cladribine, daclizumab, rituximab/ocrelizumab physically, or functionally in the case of natalizumab, also depleted CD19+, CD27+ memory B cells. This depletion was substantial and long-term following CD52 and CD20-depletion, and both also induced long-term inhibition of MS with few treatment cycles, indicating induction-therapy activity. Importantly, memory B cells were augmented by B cell activating factor (atacicept) and tumor necrosis factor (infliximab) blockade that are known to worsen MS. This creates a unifying concept centered on memory B cells that is consistent with therapeutic, histopathological and etiological aspects of MS.
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Ref 32: Gheyath Al Gawwam, Inas K. Sharquie (2017)

Serum Glutamate Is a Predictor for the Diagnosis of Multiple Sclerosis (Scientific World Journal) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476900/ One neurotransmitter, glutamate, has been implicated in the autoimmune demyelination seen in multiple sclerosis (MS). Glutamate is present in many tissues in the body, so consideration should be given to whether the serum level of glutamate is likely well correlated with the activity of the disease. This research aimed to compare the serum glutamate levels from patients diagnosed with MS with those from an age-matched control population. A review of this data could shed light upon whether the serum testing of glutamate using Enzyme-Linked Immunosorbent Assay (ELISA) is a reliable indicator of MS activity. Serum samples were obtained from 55 patients with different patterns of MS and from 25 healthy adults as a control group. The ELISA technique was used to determine the glutamate levels in the serum samples. The mean serum glutamate level for patients with MS was 1.318 ± 0.543 nmol/ml and that of the controls was 0.873 ± 0.341 nmol/ml. The serum glutamate levels showed an area under the curve via the receiver operating characteristics (ROC) of 0.738, which was significant (p value = 0.001). The present study is the first to establish a strong connection between the serum glutamate levels and MS patients, where there was statistically significant elevation of serum glutamate in MS patients; hence this elevation might be used as a monitor to help in the diagnosis of MS patients.
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Ref 33: Morris WE, Goldstein J, Redondo LM, Cangelosi A, Geoghegan P, Brocco M, Loidl FC, Fernandez-Miyakawa ME (2017)

Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes. (Toxicon) https://www.ncbi.nlm.nih.gov/pubmed/28237716 Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.
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Ref 34: Disanto G, Barro C, Benkert P, Naegelin Y, Schädelin S, Giardiello A, Zecca C, Blennow K, Zetterberg H, Leppert D, Kappos L, Gobbi C, Kuhle J, Swiss Multiple Sclerosis Cohort Study Group (2017)

Serum Neurofilament light: A biomarker of neuronal damage in multiple sclerosis (Annals of Neurology) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519945/ Neurofilament light chains (NfL) are unique to neuronal cells, are shed to the cerebrospinal fluid (CSF), and are detectable at low concentrations in peripheral blood. Various diseases causing neuronal damage have resulted in elevated CSF concentrations. We explored the value of an ultrasensitive single-molecule array (Simoa) serum NfL (sNfL) assay in multiple sclerosis (MS).sNfL levels were measured in healthy controls (HC, n = 254) and two independent MS cohorts: (1) cross-sectional with paired serum and CSF samples (n = 142), and (2) longitudinal with repeated serum sampling (n = 246, median follow-up = 3.1 years, interquartile range [IQR] = 2.0-4.0). We assessed their relation to concurrent clinical, imaging, and treatment parameters and to future clinical outcomes.sNfL levels were higher in both MS cohorts than in HC (p < 0.001). We found a strong association between CSF NfL and sNfL (β = 0.589, p < 0.001). Patients with either brain or spinal (43.4pg/ml, IQR = 25.2-65.3) or both brain and spinal gadolinium-enhancing lesions (62.5pg/ml, IQR = 42.7-71.4) had higher sNfL than those without (29.6pg/ml, IQR = 20.9-41.8; β = 1.461, p = 0.005 and β = 1.902, p = 0.002, respectively). sNfL was independently associated with Expanded Disability Status Scale (EDSS) assessments (β = 1.105, p < 0.001) and presence of relapses (β = 1.430, p < 0.001). sNfL levels were lower under disease-modifying treatment (β = 0.818, p = 0.003). Patients with sNfL levels above the 80th, 90th, 95th, 97.5th, and 99th HC-based percentiles had higher risk of relapses (97.5th percentile: incidence rate ratio = 1.94, 95% confidence interval [CI] = 1.21-3.10, p = 0.006) and EDSS worsening (97.5th percentile: OR = 2.41, 95% CI = 1.07-5.42, p = 0.034).These results support the value of sNfL as a sensitive and clinically meaningful blood biomarker to monitor tissue damage and the effects of therapies in MS. Ann Neurol 2017;81:857-870.
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Ref 35: Rumah KR, Vartanian TK, Fischetti VA (2017)

Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens. (Frontier Cellular and Infection Microbiology) https://www.ncbi.nlm.nih.gov/pubmed/28180112 There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs.
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Ref 36: Barro C (2017)

Serum neurofilament as a predictor of disease worsening and brain and spinal cord atrophy in multiple sclerosis. (Brain) https://www.ncbi.nlm.nih.gov/pubmed/29860296 Neuro-axonal injury is a key factor in the development of permanent disability in multiple sclerosis. Neurofilament light chain in peripheral blood has recently emerged as a biofluid marker reflecting neuro-axonal damage in this disease. We aimed at comparing serum neurofilament light chain levels in multiple sclerosis and healthy controls, to determine their association with measures of disease activity and their ability to predict future clinical worsening as well as brain and spinal cord volume loss. Neurofilament light chain was measured by single molecule array assay in 2183 serum samples collected as part of an ongoing cohort study from 259 patients with multiple sclerosis (189 relapsing and 70 progressive) and 259 healthy control subjects. Clinical assessment, serum sampling and MRI were done annually; median follow-up time was 6.5 years. Brain volumes were quantified by structural image evaluation using normalization of atrophy, and structural image evaluation using normalization of atrophy, cross-sectional, cervical spinal cord volumes using spinal cord image analyser (cordial). Results were analysed using ordinary linear regression models and generalized estimating equation modelling. Serum neurofilament light chain was higher in patients with a clinically isolated syndrome or relapsing remitting multiple sclerosis as well as in patients with secondary or primary progressive multiple sclerosis than in healthy controls (age adjusted P < 0.001 for both). Serum neurofilament light chain above the 90th percentile of healthy controls values was an independent predictor of Expanded Disability Status Scale worsening in the subsequent year (P < 0.001). The probability of Expanded Disability Status Scale worsening gradually increased by higher serum neurofilament light chain percentile category. Contrast enhancing and new/enlarging lesions were independently associated with increased serum neurofilament light chain (17.8% and 4.9% increase per lesion respectively; P < 0.001). The higher the serum neurofilament light chain percentile level, the more pronounced was future brain and cervical spinal volume loss: serum neurofilament light chain above the 97.5th percentile was associated with an additional average loss in brain volume of 1.5% (P < 0.001) and spinal cord volume of 2.5% over 5 years (P = 0.009). Serum neurofilament light chain correlated with concurrent and future clinical and MRI measures of disease activity and severity. High serum neurofilament light chain levels were associated with both brain and spinal cord volume loss. Neurofilament light chain levels are a real-time, easy to measure marker of neuro-axonal injury that is conceptually more comprehensive than brain MRI.
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Ref 37: Michel R. Popoff,Bradley G. StilesBernard Poulain (2018)

Clostridium perfringens Epsilon Toxin: Structural and Mechanistic Insights (Microbial toxins) https://rd.springer.com/referenceworkentry/10.1007%2F978-94-007-6449-1_9 Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or D. ETX belongs to the heptameric β-pore-forming toxins including Aeromonas aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells and containing a β-barrel composed of 14 amphipathic β-strands. In contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin, which is responsible for enterotoxemia in animals, mainly in sheep. ETX induces perivascular edema in various tissues and accumulates particularly in the kidneys and in the brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier (BBB) and to stimulate the release of glutamate, which accounts for the nervous excitation symptoms observed in animal enterotoxemia. At the cellular level, ETX causes a rapid swelling followed by a cell death involving necrosis. Recently, ETX has been found to induce demyelination and could be involved in demyelinating diseases like multiple sclerosis. The precise mode of action of ETX remains undetermined.
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Ref 38: Jie Gao, Wenwen Xin, Jing Huang,Bin Ji,Shan Gao,Liang Chen,Lin Kang,Hao Yang, Xin Shen,Baohua Zhao, and Jinglin Wang (2018)

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors (Virulence) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276848/ Epsilon-toxin (ETX) is produced by types B and D strains of Clostridium perfringens, which cause fatal enterotoxaemia in sheep, goats and cattle. Previous studies showed that only a restricted number of cell lines are sensitive to ETX and ETX-induced hemolysis has not previously been reported. In this study, the hemolytic ability of ETX was examined using erythrocytes from 10 species including murine, rabbit, sheep, monkey and human. We found that ETX caused hemolysis in human erythrocytes (HC50 = 0.2 μM) but not erythrocytes from the other test species. Moreover, the mechanism of ETX-induced hemolysis was further explored. Recent studies showed that some bacterial toxins induce hemolysis through purinergic receptor (P2) activation. Hence, the function of purinergic receptors in ETX-induced hemolysis was tested, and we found that the non-selective P2 receptor antagonists PPADS inhibited ETX-induced lysis of human erythrocytes in a concentration-dependent manner, indicating that ETX-induced hemolysis requires activation of purinergic receptors. P2 receptors comprise seven P2X (P2X1–7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–P2Y14) receptor subtypes. The pattern of responsiveness to more selective P2-antagonists implies that both P2Y13 and P2X7 receptors are involved in ETX-induced hemolysis in human species. Furthermore, we demonstrated that extracellular ATP is likely not involved in ETX-induced hemolysis and the activation of P2 receptors. These findings clarified the mechanism of ETX-induced hemolysis and provided new insight into the activities and ETX mode of action.
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Ref 39: Marta Blanch, Jonatan Dorca-Arévalo, Anna Not, Mercè Cases, Inmaculada Gómez de Aranda, Antonio Martínez-Yélamos, Sergio Martínez-Yélamos, Carles Solsona, Juan Blasi (2018)

The Cytotoxicity of Epsilon Toxin from Clostridium perfringens on Lymphocytes Is Mediated by MAL Protein Expression. (Molecular Cell Biology) https://www.ncbi.nlm.nih.gov/pubmed/29987189 Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.
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Ref 40: Sariqa Wagley, Monika Bokori-Brown, Helen Morcrette, Andrea Malaspina, Caroline D’Arcy, Sharmilee Gnanapavan, Nicholas Lewis, Michel R Popoff, Dominika Raciborska, Richard Nicholas, Ben Turner, Richard W Titball (2018)

Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis (Multiple Sclerosis Journal) https://journals.sagepub.com/doi/full/10.1177/1352458518767327 It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease.
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Ref 41: D. Adler, J. Linden, Y. Ma, S. Shetty, M. Bokori-Brown, R. Titball, J. Fetcho, T. Vartanian (2018)

Clostridium perfringens induced blood-brain barrier permeability: specificity and temporal dynamics in a humanized zebrafish model (ECTRIMS 2018) https://onlinelibrary.ectrims-congress.eu/ectrims/2018/ectrims-2018/228291/drew.adler.clostridium.perfringens.induced.blood-brain.barrier.permeability.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D1%2Asearch%3Dadler
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Ref 42: S. Haigh, K. Telesford, J. Linden, S. Shetty, P. Winokur, Y. Ma, R. Rumah, V. Fischetti, D. Butler, C. Mason, T. Vartanian (2018)

Intestinal colonization by epsilon toxin-producing Clostridium perfringens strains is associated with multiple sclerosis (ECTRIMS 2018) https://onlinelibrary.ectrims-congress.eu/ectrims/2018/ectrims-2018/228290/sylvia.haigh.intestinal.colonization.by.epsilon.toxin-producing.clostridium.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D1%2Asearch%3Dhaigh
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Ref 43: J. Linden, S. Shetty, P. Winokur, K. Telesford, S. Haigh, K.R. Rumah, V. Fischetti, T. Vartanian (2018)

Analysis of CD4+ cells reveal increased exposure of multiple sclerosis patients to Clostridium perfringens epsilon toxin (ECTRIMS 2018) https://onlinelibrary.ectrims-congress.eu/ectrims/2018/ectrims-2018/228609/jennifer.linden.analysis.of.cd42B.cells.reveal.increased.exposure.of.multiple.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D1%2Asearch%3Dlinden
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Ref 44: Cinara R. A. V. Monteiro, Monique S. do Carmo, Bruna O. Melo, Matheus S. Alves, Camilla I. dos Santos, Sílvio G. Monteiro, Maria Rosa Q. Bomfim, Elizabeth S. Fernandes, and Valério Monteiro-Neto (2019)

In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium (Nutrients) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412307/ Many Clostridium species are found as commensal members of the intestinal microbiota. However, imbalances of the microbiota may lead to certain infections caused by these microorganisms, mainly Clostridium butyricum, Clostridium difficile, and Clostridium perfringens. In many cases, infection recurrence can occur after antibiotics, indicating the need for novel therapeutic options that act on the pathogens and also restore the microbiota. Herein, the in vitro antimicrobial activity and probiotic potential of clinical and reference strains of Bifidobacterium and Lactobacillus were investigated against Clostridium species. Antimicrobial activity was evaluated by the agar spot test and inhibition of gas production. Then, the probiotic potential of selected strains was assessed by analyzing their coaggregation ability, adhesive properties to host cells and mucin, tolerance to acidic pH and bile salts, and antimicrobial susceptibility profiles. Lactobacillus plantarum ATCC 8014 was the most promising strain based on its inhibitory activity against Clostridium spp. Also, this strain met criteria to be considered a probiotic based on its coaggregation ability, adhesive properties, and tolerance to harsh pH and bile acid salt conditions. The results indicate that among the studied strains, L. plantarum ATCC 8014 presents probiotic potential for controlling infections induced by the studied Clostridium species and should be further evaluated in in vivo animal models.
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Ref 45: Adler D, Linden JR, Shetty SV, Ma Y, Bokori-Brown M, Titball RW, Vartanian T (2019)

Clostridium perfringens Epsilon Toxin Compromises the Blood-Brain Barrier in a Humanized Zebrafish Model (iScience) https://www.ncbi.nlm.nih.gov/pubmed/31030181 Clostridium perfringens epsilon toxin (ETX) is hypothesized to mediate blood-brain barrier (BBB) permeability by binding to the myelin and lymphocyte protein (MAL) on the luminal surface of endothelial cells (ECs). However, the kinetics of this interaction and a general understanding of ETX's behavior in a live organism have yet to be appreciated. Here we investigate ETX binding and BBB breakdown in living Danio rerio (zebrafish). Wild-type zebrafish ECs do not bind ETX. When zebrafish ECs are engineered to express human MAL (hMAL), proETX binding occurs in a time-dependent manner. Injection of activated toxin in hMAL zebrafish initiates BBB leakage, hMAL downregulation, blood vessel stenosis, perivascular edema, and blood stasis. We propose a kinetic model of MAL-dependent ETX binding and neurovascular pathology. By generating a humanized zebrafish BBB model, this study contributes to our understanding of ETX-induced BBB permeability and strengthens the proposal that MAL is the ETX receptor.
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Ref 46: Christos G. Savva, Alice R. Clark, Claire E. Naylor, Michel R. Popoff, David S. Moss, Ajit K. Basak, Richard W. Titball & Monika Bokori-Brown (2019)

The pore structure of Clostridium perfringens epsilon toxin (Nature Communications) https://www.nature.com/articles/s41467-019-10645-8 Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of β-PFTs (aβ-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double β-barrel, a common feature of the aβ-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.
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Ref 47: Abhishek Shivappagowdar, Soumya Pati, Chintam Narayana, Rajagopal Ayana, Himani Kaushik, Raj Sah, Swati Garg, Ashish Khanna, Jyoti kumari, Lalit Garg, Ram Sagar, Shailja Singh (2019)

A small bioactive glycoside inhibits epsilon toxin and protects host cell death (Disease Models and Mechanisms) https://dmm.biologists.org/content/early/2019/09/05/dmm.040410 Clostridium perfringens Epsilon toxin is categorized as third most lethal bioterrorism agent by CDC, with no therapeutic counter measures available for humans. Here we have developed a high affinity inhibitory compound by synthesizing and evaluating the structure activity relationship (SAR) of a library of diverse Glycosides 1-12. SAR of glycosides-Etx heptamer revealed exceptionally strong H-bond interactions of Glycoside-4 with a druggable pocket in the oligomerization and β-hairpin region of Etx. Analysis of its structure suggested that Glycoside-4 might self-aggregate to form a robust micelle like supra-molecular complex due to its linear side chain architecture, which was authenticated by fluorescence spectroscopy. Further, this micelle hinders the Etx monomer-monomer interaction required for oligomerization, validated by both surface plasmon resonance (SPR) and immunoblotting. This phenomenon in-turn leads to blockage of pore formation. Downstream evaluation revealed that Glycoside-4 effectively blocked cell death of Etx treated cultured primary cells and maintained cellular homeostasis; via disrupting oligomerization, blocking pore formation, restoring calcium homeostasis, stabilizing mitochondrial membrane and impairing HMGB1 translocation from nucleus–to-cytoplasm. Furthermore, a single dosage of Glycoside-4 protected the Etx-challenged mice and restored normal function to multiple organs. This work for the first-time reports a potent, nontoxic glycoside with strong ability to occlude toxin lethality representing it as bio-arm therapeutics against Etx-based biological threat.
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Ref 48: (2019)

MS Society () https://www.mssociety.org.uk
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Ref 49: TITBALL, Richard William; BOKORI-BROWN, Monika; MORCRETTE, Helen; LEWIS, Nicholas Peter; (2019)

EPSILON TOXIN FROM CLOSTRIDIUM PERFRINGENS AS A VACCINE () https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019166830&_cid=P20-K0QUC1-54536-1 This invention relates to methods and compositions for detecting, diagnosing, preventing, treating or ameliorating the symptoms of a demyelinating condition selected from: : enterotoxemia (ET), multiple sclerosis(MS), clinically definite MS (CDMS), clinically isolated syndrome (CIS),neuromyelitis optica spectrum disorder(NMOSD),optic neuritis(ON), neuromyelitis optica (NMO), myelitis,myelitis, transverse myelitis (TM), a disease or condition characterised by the increase or presence of antibodies against aquaporin-4(AQP-4) and/or astrocyte damage,and acute disseminated encephalomyelitis (ADEM) in a human or animal subject in need. The methods comprise administering to the subject a composition comprising an effective amount of an agent that directly or indirectly interfereswith epsilontoxin (ETX) produced by Clostridium perfringens type B or type D bacterial strain, an ETX-binding receptor, or an interaction of ETX with its binding receptor so as to inhibit or suppress ETX modulated receptor signalling activities. The invention also provides novel polypeptides useful as a vaccine against diseases caused by or associated with the epsilon toxin of Clostridium perfringens.
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Ref 50: 2019 (2019)

Centers for Disease Control and Prevention (Bioterrorism Agents/Diseases) () https://emergency.cdc.gov/agent/agentlist-category.asp Epsilon Toxin Category B Agent
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Ref 51: Itzhak Brook (2019)

Clostridium species (Clostridium perfringens, C. butyricum, C. clostridioforme, C. innocuum, C. ramosum, C. septicum, C. sordellii, C. tertium) (Anti Microbe) http://www.antimicrobe.org/b90.asp C. perfringens. C. perfringens, as well as most other Clostridium spp. are generally, but not universally susceptible to penicillin-G, amoxicillin, ticarcillin, piperacillin, cefazolin, cefoxitin, cefotetan, third generation cephalosporins, chloramphenicol, clindamycin, macrolides, metronidazole, imipenem, meropenem, tetracycline, tigecycline, fluoroquinolones, vancomycin, daptomycin, quinupristin-dalfopristin, rifampin, and the combinations of penicillins and beta-lactamase inhibitors (1, 2, 4, 8, 17, 19, 21, 24, 26, 37). Antimicrobial susceptibilities were determined for 275 C. perfringens isolates of bovine, chicken, porcine, and turkey origin in Ontario Canada. Reduced susceptibility to bacitracin was identified in chicken (64%) and turkey (60%) isolates. Swine isolates had predominantly reduced susceptibility to clindamycin (28%) and erythromycin (31%), whereas bovine isolates had reduced susceptibility to clindamycin (10%) and florfenicol (10%). Reduced susceptibility to tetracycline was spread across all species. No clear reduced susceptibility, but elevated MIC (50) for virginiamycin was found in chicken isolates in comparison with isolates from other species (25). Resistance of C. perfringens isolates to tetracycline was found in strains isolated from poultry in Sweden (76%), Denmark (10%) and Norway (29%) ( 13) Resistance to tetracycline and lincomycin was observed in most C. perfringens isolates recovered from piglets with or without diarrhea in Brazil (23), and 18% of isolates recovered from dogs in Switzerland showed resistance to tetracycline and 54 % showed decreased susceptibility to metronidazole (11). Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentration (MIC) 50 of > 256 ug/ml recovered from broiler chickens in Jordan n (9) . However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, danofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC50 of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 μg/ml, respectively. Twenty-six (66%) and 24 (61%) out of the 39 tested C. perfringes isolates from broiler chickens in Belgium showed resistance to tetracycline and lincomycin, respectively (10).
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Ref 52: Helen Morcrette, Monika Bokori-Brown, Stephanie Ong, Leo Bennett, Brendan W. Wren, Nick Lewis & Richard W. Titball (2019)

Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein (NPJ Vaccines) https://www.nature.com/articles/s41541-019-0128-2 A variant form of Clostridium perfringens epsilon toxin (Y30A-Y196A) with mutations, which shows reduced binding to Madin–Darby canine kidney (MDCK) cells and reduced toxicity in mice, has been proposed as the next-generation enterotoxaemia vaccine. Here we show that, unexpectedly, the Y30A-Y196A variant does not show a reduction in toxicity towards Chinese hamster ovary (CHO) cells engineered to express the putative receptor for the toxin (myelin and lymphocyte protein; MAL). The further addition of mutations to residues in a second putative receptor binding site of the Y30A-Y196A variant further reduces toxicity, and we selected Y30A-Y196A-A168F for further study. Compared to Y30A-Y196A, Y30A-Y196A-A168F showed more than a 3-fold reduction in toxicity towards MDCK cells, more than a 4-fold reduction in toxicity towards mice and at least 200-fold reduction in toxicity towards CHO cells expressing sheep MAL. The immunisation of rabbits or sheep with Y30A-Y196A-A168F induced high levels of neutralising antibodies against epsilon toxin, which persisted for at least 1 year. Y30A-Y196A-A168F is a candidate for development as a next-generation enterotoxaemia vaccine.
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Ref 53: K. Rashid Rumah, Olawale E. Eleso & Vincent A. Fischetti (2019)

Human blood exposure to Clostridium perfringens epsilon toxin may shed light on erythrocyte fragility during active multiple sclerosis (Biorxiv) https://www.biorxiv.org/content/10.1101/789123v1 During active multiple sclerosis (MS), red blood cells (RBCs) harvested from patients reportedly display increased osmotic fragility and increased cellular volume (macrocytosis). The cause of these abnormalities remains unknown. We have previously proposed that Clostridium perfringens epsilon toxin (ETX) may be a blood-borne trigger for newly forming MS lesions based on its tropism for blood-brain barrier vasculature and CNS myelin. Recently, Gao et al. have reported that ETX binds to and damages human RBCs, leading to hemolysis. Moreover, the authors suggest that purinergic nucleotide (P2) receptor activation amplifies the hemolytic process. Here, we confirm that ETX indeed causes human-specific RBC lysis. However, our data suggest that the hemolytic process is mediated by metal-catalyzed oxidation of the swell-induced, nucleotide-sensitive ICln chloride channel. We use spectrophotometry, flow cytometry and Western blotting to show that ETX targets human RBCs and T lymphocytes via their shared expression of Myelin and Lymphocyte protein (MAL); a protein shown to be both necessary and sufficient for ETX binding and toxicity. ETX likely triggers T cells to release redox-active heavy metals, Cu+ and Fe3+, via the lysosomal exocytosis pathway, while RBCs likely release these heavy metals via ETX pore formation within the RBC membrane. Extracellular Cu+ and Fe3+ may then amplify hemolysis by oxidizing a previously identified heavy metal-binding site within the ICln channel pore, thus deregulating its normal conductance. Elucidating the precise mechanism of ETX-mediated hemolysis may shed light on the underlying etiology of MS, as it would explain why MS RBC abnormalities occur during active disease. IMPORTANCE During active MS, numerous reports suggest that circulating RBCs are larger than normal and fragment more easily. The exact trigger(s) for these RBC abnormalities and for newly forming MS lesions remains unidentified. We have proposed that ETX, secreted by the gut bacterium Clostridium perfringens, may be an environmental trigger for newly forming MS lesions. Indeed, ETX has been shown to breakdown the BBB, enter the brain and damage the myelin sheath. Because ETX is typically spread through the circulatory system, we wished to determine how the toxin affects human blood. Provocatively, there has been a recent report that ETX produces cellular abnormalities in human RBCs, reminiscent of what has been described during active MS. In our study, we sought to elucidate the precise mechanism for how ETX causes RBC damage. In addition to triggering BBB breakdown and CNS demyelination, ETX might also explain why RBCs appear abnormal during MS attacks.