Introduction
In the quest for the causative agent of MS, the principal search must be for something, which enables the blood brain barrier (BBB) to be breached in order to facilitate the further damage to the myelin of oligodentrocytes. ETX fits this requirement exactly and there is extensive research in the literature into this toxin and its mechanism of action as the cause of a well-documented disease, Enterotoxaemia, in sheep and goats.
ETX is known to bind to endothelial cells of brain capillary vessels before passing through the blood-brain barrier. ETX has also been shown in the laboratory to be capable of causing the initial damage not only to the BBB but also to oligodentrocytes (27), blood retina barrier, optic nerve and lymphocytes. It is also known to damage erythrocytes (red blood cells).
Another section of this website has detailed that the presence of one protein, myelin and lymphocyte (MAL), is expressed in all the cell lines that are damaged by MS. There is now a considerable body of evidence that MAL is necessary for the binding and activity of ETX. Recent research has shown that ETX can bind directly to MAL, which further validates the hypothesis that the expression of MAL on certain human cell lines is necessary for ETX to cause damage.
8Oct2019Pres2Summary of the Evidence to Date
The principal evidence of ETX in humans is set out below:
8Oct2019Pres1Western blotting analysis of serum from MS patients and controls has been carried out to identify if more MS patients test positive for antibodies to ETX compared to controls.
In K Rumah’s 2013 publication (18): “Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease”, evidence was provided of Western blotting of MS patients and controls. He found 10% of MS patients tested positive to ETX and 1% of controls.
The same (but more sensitive) method was used by MS Sciences and Exeter University and the results were published in the Multiple Sclerosis Journal (MSJ) (40) “Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis” in June 2018. A total of 250 people using Western blotting (half with some form of MS diagnosis and half without). The results showed that between 24% and 35% of people with MS had antibodies for ETX compared to 10% among the control group. This was significantly higher that the results from K Rumah.
More recently, as yet unpublished results from a pilot study of patients with neuromyelitis optica (NMO), which in the past has been implicated as a “sister disease” to MS as there are many commonalities, indicate that these patients have an even higher incidence of antibodies to ETX.
Patients with NMO are typically identified from the presence of antibodies to Aquaporin 4 (AQP4), which is a water channel transporter associated with Astrocytes, which are known to be damaged during an NMO episode. It is also reported that ETX is capable of damaging Astrocytes (12) and (30). This finding needs further research.
All these results are summarised in the the table below:
Pepscans show reactivity to Peptides of ETX in humans
Pepscan testing of MS patients and controls was also undertaken to identify whether more MS patients show positive to ETX. This was reported in the same MSJ article (40). Pepscans are short, linear peptides of the toxin tested for reactivity against serum in MS patients and controls.
This Pepscan analysis was conducted on 75 of the 250 people referred to above (57% with some form of MS diagnosis and 43% without). 33% of those people with MS reacted with at least one peptide of ETX (and most recognised several peptides), compared to 16% among the control group.
In summary, the results found that, of the 75 samples from people who were tested under two separate methods (Western blotting and Pepscan), a total of 43% of MS patients and 16% of controls were found to be positive by at least one assay. These are significant results in the search for the elusive trigger of MS.
ETX has been identified in certain Lymphocytes in humans with Multiple Sclerosis
At the Annual MS Conference in Berlin in October 2018, a poster from Dr J Linden was presented, which was entitled “Analysis of CD4+ Cells Reveal Increased Exposure of Multiple Sclerosis Patients to C. perfringens Epsilon Toxin” (43). This provided evidence of ETX in certain lymphocytes of 33%MS patients compared with 8% of controls.
It is known that many MS patients experience flu like symptoms before an episode and that white blood cells are damaged by MS. It is therefore speculated that these symptoms may be caused by ETX damage to MAL-producing white blood cells. It is further speculated that long term damage to certain of these white blood cells (in particular, the ones that help regulate the immune response in humans) may somehow lead to the auto-immune nature of MS and Secondary Progressive MS. These areas certainly merit further research.
C. perfringens types B and D bacteria have been cultured from faecal samples from MS patients
The first evidence of the C. perfringens type B bacteria in a newly diagnosed patient with MS was reported by Rumah et al (18) (2013). Eight months after initially testing positive for C Perfringens type B, she remained positive for toxinotype B upon repeat analysis (data not shown in the paper).
S Haigh exhibited a poster at the MS Conference in Berlin in October 2018 entitled “Intestinal Colonization by Epsilon Toxin-producing C. perfringens Strains is Associated with Multiple Sclerosis” (42). This showed that from 28 MS patients, C. perfringens (type B or D) were cultured from faecal samples from 6 patients (21%) compared with 0 (0%) controls.
The low value of immunoreactivity to ETX may be explained by the difficulty that mammals have sustaining humoral immunity to ETX. For example, when vaccinated with epsilon toxoid, only 50% of goats have protective anti-toxin titres at week nine. By week 30, at the time of the 3rd vaccination only 2% of the goats maintain protective titres. At week 32 (2 weeks after the 3rd vaccination), 100% have protective titres, but by week 56 only 11% show protective titres. Thus, in mammals exposed to epsilon toxin, seronegativity and seroreversion are common even when the toxin is administered with an adjuvant. We thus postulate that the values we obtained from healthy controls and MS subjects for ETX immunoreactivity are likely to underestimate the true incidence of ETX exposure.
Other Links between ETX and MS
There are a number of other biochemical signals, which are common outcomes in both ETX and MS. These common signals include published papers in relation to Ceramide (16 and 24), Neurofilaments, Glutamate and Sulphatides, for which there are various published papers. The results of a search of the literature are summarised below:
Ceramide 16 and 24
MS: “Evidence of increased levels of ceramide C16:0 and C24:0 in the cerebrospinal fluid from patients with multiple sclerosis. 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” (21) and (28).
ETX: “These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of neutral sphyngolmelinase (nSMase) caused by the toxin” (29).
Neurofilaments
The study of Neurofilamanent Light chains are a new “hot” area for MS disease identification and measurement of disease progression.
MS: “Serum Neurofilament light chain levels are not only significantly higher in MS patients versus controls, they correlate with focal lesion presence and activity in both the brain and the spinal cord….. (34) and (36).
ETX: “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 ”. Extract from article: “Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes” (33)
Glutamate
MS: “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…” (8) and (32).
ETX: Microdialysis revealed that epsilon toxin induced excessive glutamate release in the hippocampus. Article entitled: “Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus” (2), (37) and (17).
Sulphatides
Sulfatides are one of the main constituents of brain lipids and ceramides are released as complex sphingolipids in the brain are broken down. Lipids are fat-like substances widely found in blood and body tissues, including, in particular, cell walls. Sphingolipids are fatty compound derivatives, which occur chiefly in the cell membranes of the brain and nervous tissue. One would therefore expect to see that ETX causes elevated sulfatides.
MS: “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). ”. Extract from article: “Antibodies to sulfatide in cerebrospinal fluid of patients with multiple sclerosis” (3).
ETX: “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.” Article entitled: “Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide” (26).
Although this evidence is anecdotal and contributory to the link, it is not causative. In other words, if ETX is the trigger for MS, you would expect to find the presence of Ceramide (16 and 24), Neurofilaments, Glutamate and Sulphatides in patients. However, the presence of these biochemical markers could also indicate other neurological problems. What can be said is that if you did not find such biochemical evidence, then that would cast doubt on the MS/ETX hypothesis.
The Links between ETX and MAL
The main evidence for ETX causing MS symptoms comes from showing that the initial damage to the blood brain barrier (BBB), oligodentrocytes, blood retina barrier, optic nerve and lymphocytes can all be initiated by ETX in laboratory conditions. ETX, produced by C. perfringens types B and D is one of the most potent poisonous substances known. ETX binds to endothelial cells of brain capillary vessels before passing through the BBB.
The previous paragraphs have detailed that the presence of one protein, MAL, is associated with all the cells that are damaged by MS. There is now considerable evidence that MAL is necessary for binding and activity of ETX. References can be found as follows:
A) Rumah et al (23) published a paper in 2015 showing that MAL is required for binding and activity (cytotoxicity) of ETX.
B) Furthermore, Dr J Linden et al. (27) found evidence in 2015 that ETX specifically targets the myelin-forming cells of the CNS – mature oligodendrocytes – leading to cell death. She found ETX was highly specific for oligodendrocytes, as other cells of the CNS are unaffected and ETX-induced oligodendrocyte death results in demyelination and is dependent on the expression of MAL .
C) In a poster presentation, Dr J Linden also found that ETX binds to intestinal endothelial cells and confirmed ETX’s ability to bind to brain endothelial cells and cause BBB permeability. New evidence was provided that ETX binds to the retinal vasculature and causes blood retinal barrier (BRB) permeability. These observations are consistent with MS related retinal periphlebitis and macular oedema. In addition, ETX also binds to the blood vessels and myelin of the optic nerve, possibly explaining the high incidence of optic neuritis in MS patients.
D) A recent article published in July 2018 (39) provides evidence of a physical interaction between ETX and MAL, which was another missing piece of the jigsaw. A summary of the research follows:
“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 as a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, Myelin and Lymphocyte protein (MAL) has been identified as 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 through a direct Etx-MAL interaction. Experiments on lymphocytic cell lines reveal that MAL expressing T cells, but not B cells, are sensitive to Etx, and revealed the toxin as a molecular tool to distinguishing subpopulations of lymphocytes. The overall results open the door to investigate the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.”
E) A further experiment shows that MAL is critical for ETX to affect mice. There is a video of two mice, one a normal mouse given a lethal dose of ETX and the other a “knock-out MAL” transgenic mouse (i.e. one which has been bred without the MAL gene) and given 50 times the lethal dose of ETX. The knock-out MAL mouse survives while the normal mouse dies.
MAL is therefore known to be critical for ETX to form pores and damage cells. MAL is expressed in all the cell types that are damaged by MS. In conclusion, it is increasingly clear from the evidence that ETX can damage the exact cells, which are known to be damaged during a MS relapse and give rise to the early stage symptoms of MS.
Conclusion
The physical evidence set out above seems to indicate that the various detection techniques show evidence of ETX in between 25% to 50% in MS or NMO patients compared with 8% to 12% for controls. These are significant percentages but the question to ask is why are the results not 80% to 100%?
The cause of MS has proved to be so elusive (for at least 150 years), so it is not surprising that the evidence is in short supply for a number of reasons. Whilst the investigative techniques are not new, the detailed processes vary and can produce differing results – for instance Dr Murrell, who originally proposed the link between MS and sheep in 1986, could not find any evidence ETX in MS patients by the less sensitive ELISA test (1).
Another reason that identifying the evidence of ETX maybe difficult is that the humoral response could be poor or short-lived. It is not uncommon for mammals to have difficulty in sustaining humoral immunity to ETX. Whilst a response to ETX may be generated shortly after an exposure, bearing in mind that MS is typically a relapsing and remitting disease, which can come and go over many years, but after 6-12 months the response may have been lost or at a barely detectable level. It should be noted that in all of the antibody responses detailed above, many of the responses were weak and not one patient generated any “neutralising antibodies”.
For example, when vaccinated with epsilon toxoid, research shows that only 50% of goats have protective anti-toxin titres at week nine. By week 30, at the time of the 3rd vaccination only 2% of the goats maintain protective titres. At week 32 (2 weeks after the 3rd vaccination), 100% have protective titres, but by week 56 only 11% show protective titres [Link]. Thus, in mammals exposed to epsilon toxin, seronegativity and seroreversion are common even when the toxin is administered with an adjuvant. It is therefore postulated that the values obtained from healthy controls and MS subjects for ETX immunoreactivity are likely to underestimate the true incidence of ETX exposure.
Type B and D strains of C. perfringens are not recognised as part of the human commensal gut system but Type A is recognised as a human commensal (microbiome). Type A is not uncommon in the human microbiome and K. Rumah (18) found reduced prevalence of C. perfringens Type A in MS patients compared to healthy controls and found:
“….that people with MS are less likely to harbor C. perfringens type A when compared to controls. Soil studies have identified that the presence of C. perfringens type A is coincident with the absence of other toxinotypes, suggesting that toxinotype A may compete with other C. perfringens toxinotypes for resources. While the type A toxinotype may outcompete C. perfringens types B and D……”
From another section has been shown that MAL is critical for ETX to form pores and damage cells. There is also considerable evidence that MAL is expressed in all the cell types that are damaged by MS. In conclusion, it is increasingly clear from the evidence that ETX can damage the exact cells, which are known to be damaged during a MS relapse and give rise to the early stage symptoms of MS.