One critical project that Mind Brain Philanthropic Foundation and StandUp2MS support is research to better understand the role that Clostridium Perfringens plays in Multiple Sclerosis. For that reason, we are pleased to share with you with his permission this article that KAREEM RASHID RUMAH published on April 9, 2023, on his SubStack.
In addition to the article below, you can listen to Dr. Rumah discuss Clostridium Perfringens and Multiple Sclerosis research in this 2:25-minute video.
Why Clostridium perfringens epsilon toxin mechanistically fits as an initial trigger for new multiple sclerosis lesion formation, while conventional autoimmunity leaves many unanswered questions.
A deep literature and conceptual review.
Clostridium Perfringens and Multiple Sclerosis Research Preface
Put simply, Clostridium perfringens epsilon toxin (ETX) directly binds to and damages the two tissues adversely affected by each new MS relapse; the blood-brain barrier (BBB) microvasculature and the central nervous system (CNS) myelin sheath. ETX binds to MS-related tissues via its receptor, myelin, and lymphocyte protein (MAL). Although ETX-encoding C. perfringens are not thought to typically colonize the human intestine, recent studies have identified these bacteria in more than half of patients sampled. Moreover, ETX effectively substitutes for Bordetella pertussis toxin, which is needed to disrupt the BBB when triggering the experimental autoimmune encephalomyelitis (EAE) rodent model of MS. ETX-EAE produces CNS lesions with a regional distribution that is more reminiscent of human MS than lesions produced by traditional pertussis-EAE. In this review, I will explore these important findings. However, I will also provide supporting ancillary evidence and suggestive context that will further implicate ETX as a bone fide environmental trigger for multiple sclerosis.
The Conventional Autoimmune Paradigm.
Although its fundamental cause remains poorly understood, MS is widely considered to be an autoimmune disease. The prevailing view, which has been relatively unquestioned in the modern scientific era, is that immune dysregulation drives new CNS lesion formation. Myelin-reactive blood lymphocytes adhere to the luminal surface of BBB endothelial cells, breaching the BBB as they enter the CNS. T cells secrete proinflammatory cytokines. B cells secrete myelin reactive antibodies and circulating monocytes differentiate into macrophages that strip myelin off the axons.1 Proponents of the autoimmune hypothesis will point to the heavy accumulation of peripheral inflammatory cells that reliably populate MS lesions. However, it is critical to note that the overwhelming majority of histologically analyzed lesions have been harvested from individuals who died of other causes, but happened to have had MS. Therefore, these lesions may be months, to years, to decades old, prior to cellular characterization.2 The autoimmune paradigm also rests on the fact that many MS treatments ostensibly target immune mediators. Indeed, corticosteroids, type I interferons, T lymphocyte blockade, and more recently, B lymphocyte blockade, comprise the lion’s share of current MS therapeutics.3,4 Although each of these anti-inflammatory interventions has led to a reduction in relapse rates and CNS lesion burden, we are still left searching for approaches that can halt the disease outright, as MS often progresses to permanent disability, regardless of treatment, for reasons that still remain unclear.3,4
The genetics of MS also point in an immune direction. Indeed, genome-wide association studies (GWAS) have identified HLA-DRB1*1501, interleukin 7 receptor-alpha (IL7RA) and interleukin 2 receptor-alpha (IL2RA) as being the most strongly MS-associated loci; HLA-DRB1* 1501 being the strongest. The association of an MHC II haplotype with MS may, at face value, seem to support the autoimmune hypothesis, as this haplotype is also associated with stereotyped autoimmune diseases such as Goodpasture syndrome,5 Systemic Lupus Erythematosus (SLE)6 and Sjogren’s syndrome7 for which autoantigens are known. However, it should also be noted that the HLA-DRB1*1501 haplotype is also associated with diseases of clear infectious origin, e.g., cervical cancer (human papilloma virus).8 Additionally, infectious triggers for autoimmune disease such SLE are still fertile areas of investigation. Therefore, distinguishing a disease as autoimmune or infectious in nature, based on immunophenotyping, may not be so clear-cut as the immune system’s primary function is to combat invasive foreign pathogens. An immunophenotype could just as easily influence a specific host-pathogen interaction as it could a particular autoimmune process.
The Leading Infectious Candidate: Epstein-Barr Virus.
While the focus of MS research has largely been immune-related, it is also well-accepted that an environmental trigger exists. Identical twin studies reveal an MS concordance rate that is only 38%, proving that while genetics are certainly important in MS, acquired factors are also necessary.9 Furthermore, migration studies point to a critical period (prior to the age of 15) during which the majority of MS risk is already established.10 While many environmentally acquired infectious agents have been considered, Epstein-Barr Virus (EBV) has, by far, been the most investigated. A recently published 2022 study tracked 35 EBV seronegative individuals, who subsequently developed MS, and compared their EBV seroconversion rates to that of 107 EBV seronegative individuals who remained disease-free over a 20-year period. As EBV seronegativity is rare in the human population (~6%), this study required that over 10 million healthy young adults be followed in order to successfully identify and track the small percentage who were initially seronegative. Researchers found an EBV seroconversion rate of 97% in the MS cohort compared to 57% in healthy individuals, with only 1 out of 35 MS patients failing to seroconvert. Importantly, researchers were also able to correlate the timing of EBV seroconversion to a rise in serum neurofilament, a marker of CNS damage and thus MS disease activity. EBV seroconversion preceded the rise of serum neurofilament and the authors interpreted this as evidence of EBV causing MS. 11 While a provocative result, it is clear that EBV exposure is not sufficient to cause MS as ~94% of the US population is EBV seropositive, while the highest estimate for the prevalence of MS in the US is ~0.3% according to the National Multiple Sclerosis Society.12
In line with the widely held autoimmune view of MS, a molecular mimicry mechanism for how EBV might trigger the disease has recently been proposed.13 Epitope mapping of antibodies secreted by clonally expanded B cells, harvested from MS cerebrospinal fluid (CSF), showed reactivity against a linear epitope within the Epstein Barr Nuclear Antigen protein (aa386-405). Exposure to this viral peptide was then shown to augment a traditional, active immunization model of experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. The authors propose that the linear EBNA1 peptide cross-reacts with a phosphorylated, intracellular, C-terminal epitope of the mammalian GlialCAM protein (aa370-389), which is expressed by astrocytes and by the oligodendrocyte-myelin unit. Importantly, rodent vaccination with EBNA1 aa386-405 alone was insufficient to trigger rodent EAE. Indeed, vaccination with the myelin antigen, proteolipid protein (PLP aa139-151) remained necessary for inducing EAE; the EBNA1 peptide being more of an aggravating factor than a sufficient trigger.13 Once again, an environmentally acquired agent that is sufficient to trigger the BBB breakdown and demyelination that is characteristic of MS remains elusive.
The Histopathology of Newly Forming Lesions.
While the root cause of MS still evades us, advances in MS histopathology may hold the key to unlocking its elusive secrets. Detailing the cellular changes that occur soon after the MS lesion begins to unfold gives us the best chance of revealing its most fundamental nature. Analogous to a crime scene, the forensics team would ideally start their discovery workup as close to the time of the incident as possible, in hopes of avoiding crime scene contamination by epiphenomena, and to avoid the degradation of crucial evidence over time. Along these lines, seminal work by Barnett & Prineas gives us the most detailed insight into the histopathology of the newly forming MS lesion to date. In an analysis of 10 lesions, collected hours to days after the rare deaths of 7 patients with fatal MS lesions (i.e., brainstem lesions; the earliest lesion being 17 hours old), Barnett & Prineas concluded that newly forming lesions arise in the absence of an inflammatory infiltrate. Instead, there was evidence of primary oligodendrocyte degeneration (apoptosis), BBB breakdown, and early microglial activation. In their opinion, some local change, to which oligodendrocytes are uniquely susceptible, is responsible for lesion formation.2 They offer the following timeline: within hours of lesion initiation, oligodendrocytes throughout the affected tissue appear apoptotic, myelin sheaths stain positively for activated complement while immunoreactivity for select myelin proteins that are closest to the axon (CNPase and Myelin Associated Glycoprotein, MAG) is diminished, and ramified microglia with thickened processes appear in increased numbers. T cells, early-activated macrophages, and myelin phagocytes are rare or absent in the apoptotic zone but are present elsewhere in the lesion. After 1 or 2 days, oligodendrocytes disappear, most presumably phagocytosed by the now amoeboid microglia present in the tissue. The tissue appears vacuolated because of the presence of widespread intramyelinic edema, which is the usual accompaniment of oligodendrocyte death. The third and most protracted stage involves fragmentation and uptake of vacuolated and smudged (vesiculated) myelin sheaths by macrophages in the presence of infiltrating T cells and macrophages.2
Surprisingly, Barnett & Prineas were not the first to describe fields of apoptotic oligodendrocytes and early microglial activation in the absence of an inflammatory infiltrate. In 1952, Adams & Kubik described a lesion, which they estimated to be 48 hours old. In this newly forming lesion, they observed unstained or poorly stained, but still intact myelin, and pyknotic nuclei, which they attributed to degenerating oligodendrocytes. They went on to postulate that the process that damages the myelin, at the same time destroys oligodendrocytes and causes a microglial reaction. Most importantly, there was no perivascular infiltration in this early lesion.14
While Barnett & Prineas admit that their study provides no direct evidence for what might be the cause of oligodendrocyte apoptosis, they point out that one unexplained finding, which may relate to the genesis of a new lesion, is the occasional occurrence of perivascular cuffs of mononuclear cells adjacent to normal-appearing periventricular white matter, at the corticomedullary junction and close to the pial surface; all areas where new MS lesions tend to form.2 Furthermore, Barnett & Prineas report serum protein leakage and the accumulation of perivenular monocytes in the absence of oligodendrocyte apoptosis or demyelination. These data suggest that insult to the endothelium and a subsequent innate immune response may be the earliest of changes in acute MS lesions.15 The provocative 2004 Barnett-Prineas study was not without controversy, however, as there was a contemporaneous and competing view/interpretation of MS histopathology.
In an analysis of 51 biopsies and 32 autopsy specimens, Lucchinetti & Lassmann concluded that a profound heterogeneity exists between patients with respect to the inflammatory response, oligodendrocyte survival, and patterns of demyelination. However, they observed a striking homogeneity among lesions within a given patient. In their opinion, MS may represent a common name for different pathologic entities that unify on the special vulnerability of CNS myelin to various immune and toxic mediators.16 The heterogeneous phenotypes observed in MS patients were classified into four distinctions.
- Pattern I: macrophage-associated demyelination.
- Pattern II: antibody/complement-associated demyelination. Pattern III: distal dying-back oligodendrogliopathy.
- Pattern IV: primary oligodendrocyte degeneration.
Although each pattern involves an inflammatory response composed mainly of T lymphocytes and macrophages, differences in plaque geography, extent and pattern of oligodendrocyte pathology, immunoglobulin deposition, complement activation, and myelin protein loss were observed. In patterns I and II, macrophages and T cells predominate in well-demarcated plaques that surround veins and venules; only pattern II lesions demonstrate local precipitation of immunoglobulin and activated complement in regions of active myelin breakdown. The expression of all myelin proteins is similarly reduced. Pattern III lesions also contain an inflammatory infiltrate, composed of macrophages, activated microglia, and T cells, however, the lesions are ill-defined and seldom surround vessels. There is no evidence of immunoglobulin deposition or complement activation, and MAG is selectively lost compared to other myelin proteins. MAG is a myelin protein localized to the most distal extension of the oligodendrocyte cell body (the periaxonal region), and early loss of this protein is believed to reflect a dying-back phenomenon in the oligodendrocyte, which precedes the apoptosis found to be prevalent in pattern III. Pattern IV lesions also contain T cells and macrophages but no preferential MAG loss, immunoglobulin deposition, or complement activation is observed. Instead, there is evidence of non-apoptotic oligodendrocyte death, the mechanisms of which are unclear. In summary, inter-individual rather than intra-individual differences in lesion heterogeneity, with respect to demyelination, is a characteristic feature of MS.17
The argument arises between these two groups when Barnett & Prineas conclude that pre-inflammatory oligodendrocyte changes represent the initial process for all MS lesions and that inter-individual lesion heterogeneity does not exist. Furthermore, Barnett & Prineas argue that the Lucchinetti-Lassmann findings represent different temporal stages in lesion evolution. Some years later, a third party seemingly resolved the dispute. Breij et al. analyzed 131 lesions from 39 patients with long-standing MS. The aim of the study was to determine if inter-individual differences persist in patients who are temporally farther away from their first symptoms. They found a homogenous pattern of demyelination in active lesions in patients with long-standing MS. The authors suggest that the immunopathological appearance of active demyelinating lesions in established MS is uniform and that the heterogeneity of demyelinating lesions in earlier phases of lesion formation may disappear over time, as different pathways converge into one general mechanism of demyelination.18 Therefore, it seems that Barnett & Prineas may have carried the day.
Tissue Outliers: Retinal Vasculitis and Macular Edema.
Other striking histopathological anomalies exist that do not align with the widely accepted anti-myelin autoimmune paradigm; the most salient being involvement of the retinal vasculature. The retina resembles the brain and spinal cord in that it consists of neurons, astroglia (muller cells and astrocytes), and vasculature that restricts the free flow of solutes from the blood into its parenchyma, the blood-retinal barrier (BRB). Most importantly, the retina lacks oligodendrocytes, the myelin-forming cells of the CNS, and thus lacks myelin. Therefore, inflammation of the retinal vasculature cannot be secondary to an anti-myelin, autoimmune demyelinating process. Despite this lack of myelin, many researchers have observed inflammatory scarring of the retinal microvasculature. This phenomenon remains unexplained. Ter Braal and Herwaarden first reported MS-associated retinal phlebitis in 1933.19 Since their initial observation, two types of inflammatory scar or “sheathing” have been described, active and inactive. Active venous sheathing consists of infiltrates that disappear over a period of months to as long as two years. Inactive venous sheathing consists of sharp, well-defined, permanent lines along veins; a presumed sequelae of chronic, active phlebitis. Inactive sheathing causes the venular walls to become thick and laminated with collagen. This occurrence has been the subject of a number of clinical studies, and the frequency of venous sheathing has been estimated to be between 9% and 36%. Furthermore, a multi-study analysis of venous sheathing in MS predicts a frequency of 11.5% and an average of 3.6 episodes during the course of the disease.19
In addition to venous sheathing of the retinal veins, ocular coherence tomography (OCT) studies have identified retinal abnormalities in MS patients even though the retina lacks myelin. Investigators have identified an increased thickness of the inner nuclear layer (INL) of the retina, which correlates with increased disease activity and contrast-enhancing MRI lesions. In the same study, they also found evidence of microcystic macular oedema (MMO), suggesting a breakdown of the blood-retinal barrier (BRB), which is analogous to the blood-brain barrier in the brain.20 The authors mention that BRB breakdown occurs in approximately 20% of MS patients and occurs concurrently with BBB breakdown during active disease.21
Vascular inflammation in the absence of myelin, as observed in retinal vasculitis, raises three important questions. 1. What causes the vascular injury observed in MS patients? 2. Why is the CNS vasculature targeted while the peripheral vasculature is spared? 3. Is the demyelination that typifies MS lesions caused by the vascular insult or is it a separate process?
Tissue Outliers: Red Blood Cell Macrocytosis and Osmotic Fragility.
In continuance with this vascular theme, another surprising abnormality has been reliably documented in MS patients, which the anti-myelin autoimmunity paradigm does little to explain. Although typically considered a disease confined to the CNS, MS has frequently been shown to cause hematologic abnormalities. Indeed, there have been numerous reports of red blood cell (RBC) abnormalities during, and up to one week prior to the onset of neurologic symptoms.22-27 During active RRMS, circulating RBCs are larger than normal (macrocytosis) and they break down more easily (increased osmotic fragility), which makes hemolysis more likely to occur. Interestingly, Lewin et al. have recently shed additional light on MS-related RBC abnormalities by showing that free hemoglobin, presumably released after hemolysis, correlates with iron deposition along CNS blood vessels. Iron deposition may lead to neuronal toxicity, axonal loss, and the progression from relapsing-remitting MS (RRMS) to secondary-progressive MS (SPMS).28 Despite the possible relevance to how MS fundamentally progresses, these hematologic abnormalities also remain unexplained.
A Blood-Borne Toxin May Fit the Bill.
The question remains, what environmental factor(s) is capable of triggering; 1. pre-inflammatory CNS lesions, 2. retinal vasculitis and breakdown of the blood-retinal barrier, and 3. RBC macrocytosis and increased osmotic fragility? It may be helpful to examine theories of old when techniques were much simpler and arguably more mechanistically agnostic. Because the primary role of blood is to transport molecules and cells throughout the body, the idea that a soluble, blood-borne, noxious agent may trigger MS readily comes to mind. Predictably, the circulating toxin theory of MS is not a new one. Indeed, two of the most influential MS researchers of the early 20th century, James Walker Dawson and Otto Marburg, supported the hematogenous toxin theory. Both Dawson and Marburg were struck by the reproducibly close proximity of MS plaques to the ventricular system and the equally reproducible presence of a vein or venule at the center of a lesion. Dawson also commented on the remarkable symmetry of periventricular plaques in MS. He explained this distribution on a vascular basis and concluded that MS was due to a specific morbid agent, probably a soluble toxin, which is conveyed to the nervous system by the blood channel.29,30 However, this idea precedes even Dawson. After a detailed histopathological investigation, documented in his groundbreaking thesis, Dawson quotes Byrom Bramwell’s theory that “the sclerotic lesions are the result of some irritant which is distributed through the nerve centers by the bloodvessels.”29 Similarly, Marburg speculated that the causative agent might be an enzyme or immuno-agent that diffuses from the blood or CSF into the brain.31
Although the soluble toxin theory’s popularity peaked in the early 20th century, one line of investigation has re-invigorated this past theory. The advent of more advanced myelin stains and MRI imaging techniques have made clear that MS lesions are not confined to the subcortical white matter. Indeed, modern research has shown that cortical demyelination exists, and this demyelination occurs in a very interesting pattern. Cortical lesions typically emanate from the subpial surface (the area of the brain that is in direct contact with the outer CSF). Additionally, these lesions typically occur as long strips, suggestive of a diffuse process, in contrast to the focal characteristics seen in subcortical white matter lesions with a venular or capillary focus.32 Dr. Richard Rudick and Dr. Bruce Trapp maintain that a soluble and diffusible agent may be at play (personal communication); an inference that revisits the conjecture of their 20th-century neuropathologist predecessors, Bramwell, Dawson, and Marburg. In support of this view, Lassmann et al. found that cortical lesions predominantly occur in deep indentations of the cortical ribbon and in cortical sulci. This is consistent with a soluble mediator, as CSF flow is more restricted in these areas than at the outer cortical surface.33 However, it should be noted, that Trapp and Lassmann believe this soluble mediator to be derived from inflammatory cells residing in the surrounding meninges.32,33 While antibodies may immediately come to mind as candidate soluble and diffusible immune mediators, it is worth revisiting the seminal histopathological analysis of Barnett & Prineas, which dispels the notion that immunoglobulin deposition plays a role in the formation of new MS lesions.2
The Case for a Gut Bacterium and its Soluble Neurotoxin
MS Clusters and Outbreaks.
MS outbreaks provide a valuable opportunity to possibly identify a triggering agent. There have been multiple reports of unusually high MS incidences and identifying a common theme between these clusters may be the key to identifying said trigger.
Key West:
In 1984, Dr. William Sheremata, a neurologist from The University of Miami, drew attention to the paradoxically high incidence of MS in Key West; the southernmost tip of the United States of America. He determined that of the 26,000 Key West residents, 37 of them had MS (140 per 100,000), which does not fit the expected low prevalence rate for its respective latitude, as MS is typically more common in northern latitudes.10 Dr. Sheremata speculated that the exposure may be related to the fact that Key West did not have an adequate sewage treatment plant and often endured exposure to unpotable water because of sewage contamination.34 Interestingly, nine of the 37 patients were nurses, who had at some time in their careers worked in the same community hospital.
Mansfield, MA:
14 MS cases were identified in the small town of Mansfield, Massachusetts (population 10,000). Remarkably, eight of these patients all lived within the same block between 1932 and 1936. All eight of these patients lived on the town’s water supply, which was heavily contaminated at the time. In August 1932, the Department of Public Health urged Mansfield to consider a sewerage system and disposal plan to eradicate excessive sewage bacteria found in all waterways in the center of the community. A pond located in the thickly settled area of town where the patients lived was highly contaminated. Eastman writes, “One can only assume that multiple episodes of exposure to a common contaminated water supply occurred during the four-year period.”35
The Faroe Islands:
This outbreak, identified by John F. Kurtzke, is perhaps the most well-known and the most carefully investigated. The Faroe Islands are a group of 18 Danish islands in the North Atlantic Ocean, situated between Norway and Iceland. According to Kurtzke, there were no native Faroese suffering from MS in the 20th century before July 1943, when the first of 21 cases occurred. From April 1940 until September 1945, British troops occupied the Faroe Islands during World War II. Because the presence of British forces coincided with the MS cases not only in time but also in space, Kurtzke postulated that they brought to the Faroes an infectious agent responsible for triggering MS. Kurtzke goes on to identify a coincident increase in acute gastrointestinal diseases during the British occupation and proposes that the MS agent may be an enteric pathogen, spread by fecal-oral transmission.36
Evidence of Gut Dysbiosis in MS Patients.
Increasingly, the gut microbiota and gut-brain axis communication are gaining more and more attention in MS research. Most recently, Ntranos et al. reported that gut bacterial metabolites (phenols and indoles) could be detected in the plasma and CSF of MS patients, indicating breach of the gut epithelial barrier.37 Moreover, there have been numerous studies indicating MS gut dysbiosis, with particular interest in the Firmicutes phylum and Clostridial genus.38-40 Of note, Clostridial dysbiosis may extend beyond MS when considering CNS demyelinating disease more generally, as patients suffering from neuromyelitis optica (a sister disease to MS) also display Clostridial overabundance in their GI tracts.41,42 Prior to microbiome studies that have been made possible by advancements in 16rRNA sequencing and metagenomics, gastroenterologists have long noted an association between MS and Crohn’s disease.43-46 The recent pivot of MS research toward the gut may be of critical importance to identifying the acquired factors that epidemiological studies point to as being necessary for triggering the disease.
Evidence of Gut Histopathology in MS Patients.
A provocative histological study reported subtle and unexplained alterations to the gut epithelium of MS patients. Lange and Shiner examined MS jejunal tissue by both light and electron microscopy. Intriguingly, gut epithelia from 6 of 8 patients displayed fine structural abnormalities such as increases in enterocyte and intraepithelial T lymphocyte lysosome numbers. Gut macrophages also contained large amounts of membrane-bound, electron-dense material in 5 of 8 patients, which the authors hypothesize to be derived from absorbed antigenic material or may represent phagocytosed lipid material.47 Another study by Rodrigo et al. showed that an increased number of MS patients displayed celiac disease-like changes in duodenal biopsy tissue.48
An Introduction to Clostridium perfringens types B and D and Epsilon Toxin (ETX).
Intrigued by the idea that MS may be associated with the concentration of global sheep populations, Dr. Timothy Murrell identified historically aberrant MS outbreaks and proposed that these epidemics stemmed from human contact with sheep.49 On this point, Murrell goes on to describe a rather unusual MS cluster. In 1947, four of seven researchers developed signs and symptoms of MS. They were studying swayback, which is a neurological disease of lambs caused by copper deficiency. Another researcher joined the group later in the study, making a final group of eight men. The chance of four or more out of eight men developing MS is about one in a billion. Of interest, MS has not occurred in workers studying swayback disease elsewhere.50 Murrell proceeded to delineate pathogens that may be transmitted from sheep to humans. He provocatively offered C. perfringens (C. p) type D as a possible infectious agent, as sheep are the natural reservoir for this bacterium. Humans typically carry C. p type A, which does not carry an ETX-encoding plasmid.51 Murrell notes that ETX causes MS-like symptoms upon entering the animal’s bloodstream such as blindness, ataxia, opisthotonos (a form of spastic paralysis) and CNS derangement due to malacia (softening) in brain areas with a distribution similar to MS; periventricular lesions perhaps being the most provocative, as they are commonly observed in both ETX-intoxicated animals and MS patients.49 Murrell concludes by suggesting that ETX-mediated BBB breakdown may open the human CNS to a demyelinating neurotropic virus resulting in MS. However, the 2008 finding that ETX specifically binds to myelin once it gains access to the CNS may obviate the need for a neurotropic virus to cause demyelination.52
There are many other properties of ETX that make it an attractive causative agent for MS. ETX-mediated veterinary disease bears the name focal symmetrical encephalomalacia (FSE), and if one examines the meaning of this clinical term, similarities between FSE and MS start to emerge. The characteristic subcortical white matter MS lesion is indeed focal in nature, matching the pathologic description of FSE. MS lesions are often symmetrical, especially lesions that form close to the lateral ventricles.53 Finally, encephalomalacia literally means “brain softening,” while sclerosis means “scar” or “hardening.” Although these two terms seem to oppose each other, sclerosis is indeed a misnomer when it comes to MS. One must consider that MS lesions harden over time. In reality, fresh MS lesions, which are in the processes of forming during the active disease state, are soft. The earliest of MS investigators Carswell, Cruveillhier and Dawson noted that fresh lesions were softer than the normal brain substance. Therefore, in terms of active disease, “encephalomalacia” more accurately describes MS than “sclerosis.”54
The experimental injection of rodents with ETX also shows many provocative parallels to the MS disease state. ETX not only binds specifically to brain microvessels but also has a penchant for vasculature residing in the myelinated regions of the brain.55,56 Additionally, intoxicated mice often develop periventricular lesions, similar to the lesion distribution seen in MS and display a form of spastic paralysis evinced by neck retroflexion.57 Similarly, MS patients often suffer from spastic paralysis, typically of the lower limbs. In this way, ETX intoxication may better reflect MS than the current EAE model. EAE mice develop a flaccid paralysis rather than the spastic paralysis that occurs in MS.58,59 Strikingly, intraperitoneal injections of rats with the non-activated ETX precursor protein, which is 1000X less active, results in the formation of focal ovoid lesions within the corpus callosum, in which the long axis of the ovoid is oriented perpendicular to the surface of the lateral ventricle.60 Dr. Timothy Vartanian astutely noted that these ETX-induced lesions resemble the flame-like lesions that radiate from the lateral ventricles in MS, termed Dawson’s fingers. Dawson first described this specific lesion morphology, and the radiographic equivalent is all but pathognomonic for clinically definite RRMS.29 To date, no MS animal model reproduces this highly specific lesion morphology.
The fact that ETX can disrupt the BBB, bind to myelin, and recapitulate Dawson’s finger lesion morphology and the spastic paralysis seen in MS are all provocative findings. However, if we look more closely at the mechanism by which ETX damages cells and compare this to what Barnett & Prineas observed in newly forming MS lesions, more similarities become clear. ETX forms heptameric pores in the cell membrane and allows the free flow of ions, water, and hydrophilic solutes (up to 1kDa) across the cell membrane. This osmotic imbalance causes cellular swelling, membrane blebbing, and cellular damage. ETX toxicity also leads to ATP depletion, AMP-activated protein kinase stimulation, mitochondrial membrane permeabilization, and mitochondrial-nuclear translocation of apoptosis-inducing factor, which is a potent caspase 3-independent cell death mechanism characterized by a marked reduction in nuclear size and nuclear pyknosis.61 Barnett & Prineas reported swelling of the oligodendrocyte cell body and the myelin sheath. Moreover, they found a reduction in nuclear size, observed nuclear pyknosis and caspase 3-independent cell death,2 all of which are reminiscent of the type of cell death described for ETX-mediated cytotoxicity.
All MS-Related Tissues Express the ETX Receptor, MAL, and Display ETX Toxicity.
Above, I have pointed to CNS vasculature (BBB and BRB microvessels), the oligodendrocyte-myelin unit, and circulating RBCs as being tissues that display evidence of damage during the active phase of RRMS. Of important note, each of these tissues have been shown to 1. bind ETX,52,62-66 2. be sensitive to ETX toxicity62-66 and, 3. express the putative receptor for ETX, myelin, and lymphocyte protein (MAL).67-70 As the name would suggest, MAL is also expressed by T lymphocytes,71 which raises an interesting question: what effect might circulating ETX have on the human immune system? While this is provocative question in the context of immune dysregulation and MS, the more important point is that primary action of ETX on retinal microvessels and on circulating RBCs might explain the “tissue outliers” discussed previously. Moreover, primary action of ETX on the BBB and on the oligodendrocyte-myelin unit could paint a histopathological picture reminiscent of what Barnett-Prineas and Adams-Kubik described in their analyses of newly forming MS lesions, between 17 and 48 hours past their initial onset.
Evidence of MS seroreactivity against ETX.
Although studies on human ETX exposure are sorely lacking, anti-ETX seroreactivity can be found in as many as 43% of MS patients and in as many as 16% of healthy individuals.72 These seroreactivity rates are worth highlighting, as conventional wisdom would suggest that ETX-secreting C. p strains are confined to the ruminant microbiota and that these strains do not inhabit the human gastrointestinal tract as commensals or as pathogens.
A Recap of the ETX-MS Hypothesis.
In this article, I have laid out a case for how and why RRMS may be triggered by a neurotropic bacterial toxin, emanating from the human GI tract. I point to mechanistic plausibility for ETX by showing that the primary tissues damaged during each new MS lesion, i.e., BBB vasculature and the myelin-oligodendrocyte unit, both express the putative ETX receptor, MAL, which is both necessary and sufficient for ETX binding and toxicity.67,68 I also suggest that primary damage to these tissues, by ETX, would paint a neuropathological picture similar to what has been documented by Barnett-Prineas and Adams-Kubik; two groups that have embarked on the rare study of newly forming MS lesions, in which no peripheral inflammatory infiltrate was observed.2,14 A throughline of MS clusters and outbreaks seems to be a communicable agent, likely harbored by contaminated water sources and/or spread by fecal-oral transmission.34-36 More recent studies have pointed to gut dysbiosis in MS patients, particularly relating to the Firmicutes phylum and the Clostridial genus,38-40 and even evidence of gut-barrier permeability, as bacterial small molecules (indoles and phenols) have been identified in MS sera and CSF.37 Additionally, ETX-specific serological data points to a ~3-fold increase in ETX exposure in MS patients vs. healthy individuals.72 Most intriguing, are the MS phenomena that the standard autoimmune paradigm fails to explain, i.e., retinal vasculitis/macular edema and macrocytosis/increased osmotic fragility of circulating RBCs. Strikingly, both the retinal vasculature and circulating RBCs bind ETX and display toxicity.19-28 Finally, C. p is a sporulating bacterium51 that can cycle between periods of dormancy vs. germination, exponential growth, and concomitant ETX secretion. Such a life cycle may offer mechanistic insight into the relapsing-remitting nature of RRMS.
New and Exciting Evidence Supporting the Role of ETX in Triggering MS.
In early 2023, Ma et al. published seminal work interrogating the gut microbiomes of MS patients for the presence and abundance of ETX-encoding C. p strains and hypothesized that C. p type B/D would be more prevalent and more abundant in the MS microbiota than that of healthy controls. This study found that; 1. ETX-encoding C. p strains are ~5 times more prevalent in the MS fecal microbiota (61%) than in that of healthy controls (13%); 2. ETX strains comprise ~35% of total C. p carriage in MS patients compared to ~0.002% in asymptomatic, healthy control carriers; and 3. ETX effectively replaces Bordetella pertussis toxin as the BBB disrupting agent in the rodent EAE model of MS, and causes experimental lesions with a regional distribution that more closely approximates human MS than the tradition pertussis toxin-triggered EAE model.73
Caveats to the ETX-MS Hypothesis.
Although a promising hypothesis, several caveats must be addressed when considering ETX as a possible MS trigger. First, in addition to binding CNS myelin, ETX has been shown to bind to peripheral nerve myelin when incubated with sciatic nerve tissue slices,52 however, MS is a disease that is restricted to the CNS. Radiolabeled ETX, injected into mice, only targets the CNS and not the PNS.74 I propose that ETX fails to access PNS myelin because of the dense ensheathing matrix that surrounds peripheral nerves (the perineurium and endoneurium).75 Second, one might expect GI discomfort and intestinal lesions in MS if caused by ETX present in the gut lumen. However, histological abnormalities are minor, inconsistent, and often completely undetectable in ETX-intoxicated sheep. Diarrhea is also very uncommon in sheep ETX-enterotoxaemia. The same is not true for intoxicated goats that often suffer from hemorrhagic enterocolitis and diarrhea.76,77 Therefore, even between ruminants, there is great species variation in the phenotype of ETX-enterotoxaemia. However, as mentioned previously, subtle small bowel changes have been observed in MS patients; varying degrees of villus atrophy, inflammatory cell infiltration, and thickening of connective tissue were described in jejunal biopsies from 12 randomly chosen MS patients.47 Third, enterotoxaemia in sheep, goats and less frequently cattle, often results in severe, fatal disease. MS attacks, while debilitating, are rarely fatal. Why this difference in disease severity? Attempts to develop a small rodent oral inoculation model for C. p enterotoxaemia may help address this question. When inoculated with toxinogenic C. p type D, mice remain unaffected unless the anus is sealed to halt intestinal transit.78 The authors propose that there may be more stasis in the ruminant gut, which allows for increased toxin accumulation and systemic absorption. Additionally, they propose that the ruminant animal has a larger gut absorptive surface area: body weight ratio than the mouse, thus making enterotoxaemia more likely in the ruminant.78 Furthermore, when toxinogenic C. p strains were orally administered to germ-free guinea pigs, all animals showed signs of intoxication. However, when this experiment was repeated in conventional guinea pigs with normal gut flora, none of the animals showed symptoms of intoxication.79 Therefore, a normal gut flora can protect against enterotoxaemia and may suggest that a critical biomass of toxinogenic C. p is necessary for CNS disease to occur. This may be critically important in animals for which C. p types B and D are not considered natural symbionts, i.e., humans and rodents. Much like guinea pigs, simple exposure to toxinogenic C. p may not be sufficient to cause disease, i.e., asymptomatic carriage may be possible if the enterotoxaemic-bacterial load threshold is not reached. It should be noted that germ-free animals inoculated with C. p type A, which is commensal to humans, remained unharmed. In contrast, each toxinogenic C. p strain resulted in a fatality rate of 100%.79 Fourth, ETX intoxication often bears the name pulpy kidney disease, suggesting damage to the kidneys, and there is no evidence of kidney damage in MS. However, pulpy kidney disease is a misnomer. The pathological changes in the kidneys of intoxicated sheep have been shown to be a post-mortem change characterized by rapid autolysis of the renal tubules. Kidneys that are inspected shortly after death show minimal, if any signs of damage.61 In contrast to what the name “pulpy kidney” disease may suggest, the kidneys have been shown to be critical for ETX detoxification. Nephrectomy of ETX-intoxicated mice greatly increases morbidity, mortality and considerably decreases the toxin LD50. Therefore, the kidneys are actually key players in ETX host defense.74 Finally, the putative ETX receptor, MAL, is expressed by various epithelial tissues, i.e., the pancreas, lung, prostate, thyroid and stomach, none of which display damage in MS. However, it is important to note that these tissues form lumens, and the epithelium will not be exposed to blood product under normal conditions. Therefore, blood-borne ETX will not encounter the MAL expressed by these organs.80
Clostridium Perfringens and Multiple Sclerosis Acknowledgements
I’d first like to thank Dr. Richard Daneman of The University of California San Diego for his edits to the above article. I’d also like to thank Dr. Timothy Vartanian of Weill Cornell Medical College for his keen observation that ETX triggers Dawson’s Finger-like lesions in experimentally exposed rodents and for our many discussions about the nature of MS over these many years. Finally, I’d like to highlight Dr. Jock Murray’s tremendous book entitled, “Multiple Sclerosis: The History of a Disease,” as it was crucial for sourcing many of the historical references about early MS investigators and for the notable MS clusters and outbreaks.
Clostridium Perfringens and Multiple Sclerosis Research References
1. Frohman, E. M., M. K. Racke, et al. (2006). “Multiple sclerosis–the plaque and its pathogenesis.” The New England journal of medicine 354(9): 942-955.
2. Barnett, M. H. and J. W. Prineas (2004). “Relapsing and remitting multiple sclerosis: Pathology of the newly forming lesion.” Annals of Neurology 55(4): 458-468.
3. Conway D, Cohen JA. (2010) “Combination therapy in multiple sclerosis.” Lancet Neurol. Mar;9(3):299-308. doi: 10.1016/S1474-4422(10)70007-7.
4. https://www.merckmanuals.com/professional/neurologic-disorders/demyelinating-disorders/multiple-sclerosis-ms
5. Phelps, R. G. and A. J. Rees (1999). “The HLA complex in Goodpasture’s disease: a model for analyzing susceptibility to autoimmunity.” Kidney international 56(5): 1638-1653.
6. Takeuchi, F., K. Nakano, et al. (2005). “Genetic contribution of the tumour necrosis factor (TNF) B + 252*2/2 genotype, but not the TNFa,b microsatellite alleles, to systemic lupus erythematosus in Japanese patients.” International journal of immunogenetics 32(3): 173-178.
7. Manoussakis, M. N., C. Georgopoulou, et al. (2004). “Sjogren’s syndrome associated with systemic lupus erythematosus: clinical and laboratory profiles and comparison with primary Sjogren’s syndrome.” Arthritis and rheumatism 50(3): 882-891.
8. Apple, R. J., H. A. Erlich, et al. (1994). “HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity.” Nature genetics 6(2): 157-162.
9. Sadovnick, A. D., A. Dircks, et al. (1999). “Genetic counselling in multiple sclerosis: risks to sibs and children of affected individuals.” Clin Genet 56(2): 118-122.
10. Marrie, R. A. (2004). “Environmental risk factors in multiple sclerosis aetiology.” Lancet neurology 3(12): 709-718.
11. Bjornevik, K. et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science 375, 296-301, doi:10.1126/science.abj8222 (2022).
13. Lanz, T. V. et al. Clonally Expanded B Cells in Multiple Sclerosis Bind EBV EBNA1 and GlialCAM. Nature, doi:10.1038/s41586-022-04432-7 (2022).
14. Adams R. D., Kubik C. S. “The morbid anatomy of the demyelinative disease.” Am J Med. 1952 May;12(5): 510-46.
15. Prineas, J. W. and J. D. Parratt (2012). “Oligodendrocytes and the early multiple sclerosis lesion.” Annals of neurology 72(1): 18-31.
16. Lucchinetti, C., W. Bruck, et al. (2000). “Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination.” Annals of Neurology 47(6): 707-717.
17. Lucchinetti, C. F., J. Parisi, et al. (2005). “The pathology of multiple sclerosis.” Neurologic clinics 23(1): 77-105, vi.
18. Breij, E. C., B. P. Brink, et al. (2008). “Homogeneity of active demyelinating lesions in established multiple sclerosis.” Annals of neurology 63(1): 16-25.
19. Kerrison, J. B., T. Flynn, et al. (1994). “Retinal pathologic changes in multiple sclerosis.” Retina 14(5): 445-451.
20. Runkle, E. A. and D. A. Antonetti (2011). “The blood-retinal barrier: structure and functional significance.” Methods in molecular biology 686: 133-148.
21. Saidha, S., E. S. Sotirchos, et al. (2012). “Microcystic macular oedema, thickness of the inner nuclear layer of the retina, and disease characteristics in multiple sclerosis: a retrospective study.” Lancet neurology 11(11): 963-972.
22. Plum, C. M., and Fog, T. (1959). Studies in multiple sclerosis. Acta psychiat. scand., 34, Suppl. No. 128.
23. LASZLO S. [OSMOTIC FRAGILITY OF THE ERYTHROCYTES IN MULTIPLE SCLEROSIS]. Acta Neurol Psychiatr Belg. 1964 May;64:529-33.
24. Caspary EA, Sewell F, Field EJ. Red blood cell fragility in multiple sclerosis. Br Med J. 1967 Jun 3;2(5552):610-1.
25. Prineas J. Red blood cell size in multiple sclerosis. Acta Neurol Scand. 1968;44(1):81-90.
26. Crellin RF, Bottiglieri T, Reynolds EH. Multiple sclerosis and macrocytosis. Lancet. 1989 Nov 11;2(8672):1157.
27. Crellin RF, Bottiglieri T, Reynolds EH. Multiple sclerosis and macrocytosis. Acta Neurol Scand. 1990 May;81(5):388-91.
28. Lewin A, Hamilton S, Witkover A, Langford P, Nicholas R, Chataway J, et al. Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis.Wellcome Open Res. 2016 Nov 15;1:10. doi:10.12688/wellcomeopenres.9967.2.
29. Dawson, J. W. (1916). ” The histology of disseminated sclerosis.” Trans R Soc Edinburgh 50: 517-740.
30. McAlpine, D. (1946). “The problem of disseminated sclerosis.” Brain 69(4): 233-250.
31. Adams, C. W., R. N. Poston, et al. (1985). “Inflammatory vasculitis in multiple sclerosis.” J Neurol Sci 69(3): 269-283.
32. Rudick, R. A. and B. D. Trapp (2009). “Gray-matter injury in multiple sclerosis.” The New England journal of medicine 361(15): 1505-1506.
33. Lassmann, H. (2011). “Review: the architecture of inflammatory demyelinating lesions: implications for studies on pathogenesis.” Neuropathology and applied neurobiology 37(7): 698-710.
34. Sheremata, W. A., D. C. Poskanzer, et al. (1985). “Unusual occurrence on a tropical island of multiple sclerosis.” Lancet 2(8455): 618.
35. Eastman, R., J. Sheridan, et al. (1973). “Multiple sclerosis clustering in a small Massachusetts community, with possible common exposure 23 years before onset.” The New England journal of medicine 289(15): 793-794.
36. Wallin, M. T., A. Heltberg, et al. (2009). “Multiple sclerosis in the Faroe Islands. 8. Notifiable diseases.” Acta Neurologica Scandinavica.
37. Ntranos A, Park HJ, Wentling M, Tolstikov V, Amatruda M, Inbar B, Kim-Schulze S, Frazier C, Button J, Kiebish MA, Lublin F, Edwards K, Casaccia P. Bacterial neurotoxic metabolites in multiple sclerosis cerebrospinal fluid and plasma. Brain. 2021 Dec 11:awab320. doi: 10.1093/brain/awab320.
38. Ventura RE, Iizumi T, Battaglia T, Liu M, Perez-Perez GI, Herbert J, Blaser MJ. Gut microbiome of treatment-na.ve MS patients of different ethnicities early in disease course.Sci Rep. 2019 Nov 8;9(1):16396. doi: 10.1038/s41598-019-52894-z.
39. Kozhieva M, Naumova N, Alikina T, Boyko A, Vlassov V, Kabilov MR. The Core of Gut Life: Firmicutes Profile in Patients with Relapsing-Remitting Multiple Sclerosis. Life (Basel). 2021 Jan 14;11(1):55. doi: 10.3390/life11010055.
40. Cekanaviciute E, Pr.bstel AK, Thomann A, Runia TF, Casaccia P, Katz Sand I, Crabtree E, Singh S, Morrissey J, Barba P, Gomez R, Knight R, Mazmanian S, Graves J, Cree BAC, Zamvil SS, Baranzini SE. Multiple Sclerosis-Associated Changes in the Composition and Immune Functions of Spore-Forming Bacteria.mSystems. 2018 Nov 6;3(6):e00083-18. doi: 10.1128/mSystems.00083-18. eCollection 2018 Nov-Dec.
41. Cree BA, Spencer CM, Varrin-Doyer M, Baranzini SE, Zamvil SS. Gut microbiome analysis in neuromyelitis optica reveals overabundance of Clostridium perfringens.Ann Neurol. 2016 Sep;80(3):443-7. doi: 10.1002/ana.24718. Epub 2016 Aug 4.
42. Zamvil SS, Spencer CM, Baranzini SE, Cree BAC. The Gut Microbiome in Neuromyelitis Optica. Neurotherapeutics. 2018 Jan;15(1):92-101. doi: 10.1007/s13311-017-0594-z.
43. Green C, Elliott L, Beaudoin C, Bernstein CN. A population-based ecologic study of inflammatory bowel disease: searching for etiologic clues.Am J Epidemiol. 2006 Oct 1;164(7):615-23; discussion 624-8. doi: 10.1093/aje/kwj260. Epub 2006 Aug 18.
44. Minuk GY, Lewkonia RM. Possible familial association of multiple sclerosis and inflammatory bowel disease.N Engl J Med. 1986 Feb 27;314(9):586. doi: 10.1056/NEJM198602273140921.
45. Purrmann J, Arendt G, Cleveland S, Borchard F, Fürst W, Gemsa R, Bertrams J, Hengels KJ. Association of Crohn’s disease and multiple sclerosis. Is there a common background? J Clin Gastroenterol. 1992 Jan;14(1):43-6. doi: 10.1097/00004836-199201000-00011.
46. Yang Y, Musco H, Simpson-Yap S, Zhu Z, Wang Y, Lin X, Zhang J, Taylor B, Gratten J, Zhou Y. Investigating the shared genetic architecture between multiple sclerosis and inflammatory bowel diseases. Nat Commun. 2021 Sep 24;12(1):5641. doi: 10.1038/s41467-021-25768-0.
47. Lange LS, Shiner M. Small-bowel abnormalities in multiple sclerosis. Lancet. 1976 Dec 18;2(7999):1319-22. doi: 10.1016/s0140-6736(76)91972-3.
48. Rodrigo L, Hern.ndez-Lahoz C, Fuentes D, Alvarez N, L.pez-V.zquez A, Gonz.lez S. Prevalence of celiac disease in multiple sclerosis. BMC Neurol. 2011 Mar 7;11:31. doi: 10.1186/1471-2377-11-31.
49. Murrell TG, O’Donoghue PJ, Ellis T. A review of the sheep-multiple sclerosis connection. Med Hypotheses. 1986 Jan;19(1):27-39. doi: 10.1016/0306-9877(86)90134-9.
50. Dean, G., E. I. McDougall, et al. (1985). “Multiple sclerosis in research workers studying swayback in lambs: an updated report.” Journal of neurology, neurosurgery, and psychiatry 48(9): 859-865.
51. Carman, R. J., S. Sayeed, et al. (2008). “Clostridium perfringens toxin genotypes in the feces of healthy North Americans.” Anaerobe 14(2): 102-108.
52. Dorca-Arevalo, J., M. Martin-Satue, et al. (2012). “Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system.” Veterinary Microbiology 157(1-2): 179-189.
53. McAlpine, D. (1973). “Multiple sclerosis: a review.” Br Med J 2(5861): 292-295.
54. Murray, T. J. (2005). Multiple Sclerosis: The History of a Disease, Demos Health.
55. Finnie, J. W. (1984). “Histopathological changes in the brain of mice given Clostridium perfringens type D epsilon toxin.” J Comp Pathol 94(3): 363-370.
56. Finnie, J. W. (1984). “Ultrastructural changes in the brain of mice given Clostridium perfringens type D epsilon toxin.” J Comp Pathol 94(3): 445-452.
57. Morgan, K. T. and B. G. Kelly (1974). “Ultrastructural study of brain lesions produced in mice by the administration of Clostridium welchii type D toxin.” J Comp Pathol 84(2): 181-191.
58. Stromnes, I. M. and J. M. Goverman (2006). “Active induction of experimental allergic encephalomyelitis.” Nature Protocols 1(4): 1810-1819.
59. Stromnes, I. M. and J. M. Goverman (2006). “Passive induction of experimental allergic encephalomyelitis.” Nature Protocols 1(4): 1952-1960.
60. Zhu, C., M. N. Ghabriel, et al. (2001). “Clostridium perfringens prototoxin induced alteration of endothelial barrier antigen (EBA) immunoreactivity at the blood-brain barrier (BBB).” Experimental neurology 169(1): 72-82.
61. Popoff, M. R. (2011). “Epsilon toxin: a fascinating pore-forming toxin.” The FEBS journal 278(23): 4602-4615.
62. Finnie J.W., Manavis J., Casson R. J., Chidlow G. Retinal microvascular damage and vasogenic edema produced by Clostridium perfringens type D epsilon toxin in rats. J Vet Diagn Invest. 2014 May;26(3):470-472.
63. Mander K.A., Finnie J.W. Loss of Endothelial Barrier A ntigen Immunoreactivity in Rat Retinal Microvessels is Correlated with Clostridium perfrignens type D Epsilon Toxin-induced Damage to the Blood-Retinal Barrier. J Comp Pathol. 2018 Jan;158:51-55
64. Finnie J.W., Navarro M.A., Uzal F.A. Pathogenesis and diagnostic features of brain and ophthalmic damage produced by Clostridium perfringens type D epsilon toxin. J Vet Diagn Invest. 2020 Mar;32(2):282-286.
65. Wioland L, Dupont JL, Doussau F, Gaillard S, Heid F, Isope P, Pauillac S, Popoff MR, Bossu JL, Poulain B. Epsilon toxin from Clostridium perfringens acts on oligodendrocytes without forming pores, and causes demyelination. Cell Microbiol. 2015 Mar;17(3):369-88. doi: 10.1111/cmi.12373. Epub 2014 Oct 31.
66. Gao J., Wenwen X., Huang J. et al. Research articleHemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors. Virulence. 2018;9(1):1601-1614
67. Linden JR, Flores C, Schmidt EF, Uzal FA, Michel AO, Valenzuela M, Dobrow S, Vartanian T. Clostridium perfringens epsilon toxin induces blood brain barrier permeability via caveolae-dependent transcytosis and requires expression of MAL.PLoS Pathog. 2019 Nov 8;15(11):e1008014. doi:10.1371/journal.ppat.1008014. eCollection 2019 Nov.
68. Linden JR, Ma Y, Zhao B, Harris JM, Rumah KR, Schaeren-Wiemers N, Vartanian T. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination. MBio. 2015 Jun 16;6(3):e02513. doi: 10.1128/mBio.02513-14.
69. Geng Z., Huang J., Kang L., et al. Clostridium perfringens epsilon toxin binds to erythrocyte MAL receptors and triggers phosphatidylserine exposure. J Cell Mol Med 2020 Jul;24(13):7341-7352.
70. Bryk AH, Wiśniewski JR. 2017. Quantitative Analysis of Human Red Blood Cell Proteome. J Proteome Res 16(8):2752-2761. doi: 10.1021/acs.jproteome.7b00025. Epub 2017 Jul 24.
71. Alonso, M. A. and S. M. Weissman (1987). “cDNA cloning and sequence of MAL, a hydrophobic protein associated with human T-cell differentiation.” Proceedings of the National Academy of Sciences of the United States of America 84(7): 1997-2001.
72. Wagley, S. et al. Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis. Mult Scler 25, 653-660, doi:10.1177/1352458518767327 (2019).
73. Ma Y, Sannino D, et al. Epsilon toxin-producing Clostridium perfringens colonize the MS gut and epsilon toxin overcomes immune privilege. J Clin Invest. 2023 Feb 28:e163239. doi: 10.1172/JCI163239. Online ahead of print.
74. Tamai, E., T. Ishida, et al. (2003). “Accumulation of Clostridium perfringens Epsilon-Toxin in the Mouse Kidney and Its Possible Biological Significance.” Infection and Immunity 71(9): 5371-5375.
75. Stassart R.M., Woodhoo A. Axo-glial interaction in the injured PNS. Dev Neurobiol. 2021 Jul;81(5): 490-506.
76. Blackwell, T. E., D. G. Butler, et al. (1991). “Differences in signs and lesions in sheep and goats with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D.” American journal of veterinary research 52(7): 1147-1152.
77. Fernandez Miyakawa, M. E. and F. A. Uzal (2003). “The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep.” Veterinary research communications 27(3): 231-241.
78. Fernandez-Miyakawa, M. E., S. Sayeed, et al. (2007). “Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection.” Infection and immunity 75(9): 4282-4288.
79. Horton, R. E., D. L. Madden, et al. (1970). “Pathogenicity of Clostridium perfringens for germ-free guinea pigs after oral ingestion.” Applied microbiology 19(2): 314-316.
80. Marazuela, M., A. Acevedo, et al. (2003). “Expression of MAL, an integral protein component of the machinery for raft-mediated pical transport, in human epithelia.” J Histochem Cytochem 51(5): 665-674.