![. Frequency of various adverse reactions to MMR before and after January 2003 [US FDA, VAERS]. The P-values were computed according to a χ 2 goodness-of-fit test.](https://www.researchgate.net/profile/Anthony-Samsel/publication/316601847/figure/tbl1/AS:613852207149057@1523365179075/Frequency-of-various-adverse-reactions-to-MMR-before-and-after-January-2003-US-FDA_Q320.jpg)
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© 2017 Collegium Basilea & AMSI
doi: 10.4024/25SA16A.jbpc.17.01
Journal of Biological Physics and Chemistry 17 (2017) 8–32
Received 16 November 2016; accepted 15 March 2017 8
25SA16A
________________________________________________________________________________________________________
1. INTRODUCTION
At first glance, multiple sclerosis (MS) and autism appear
to have little in common, aside from the fact that both are
neurological diseases. Autism is a condition with prenatal
or early childhood onset, characterized by repetitive
behaviours, impaired social interaction and cognitive
impairment. The male:female ratio for autism is 4:1, while
multiple sclerosis is twice as common in women as in
men; its first symptoms usually begin in early adulthood to
involve impaired lower limb mobility, although in later
stages it affects both mental and physical capabilities. Both
conditions are, however, associated with inflammatory
autoimmune features [1, 2], and both diseases are viewed
as having an environmental and a genetic component [3–6].
A study comparing a population of 658 MS patients
with the general population found an association between
MS and increased rates of asthma, inflammatory bowel
disease (IBD), type 1 diabetes mellitus, pernicious
anaemia and autoimmune thyroid disease [7], all of which
have also been linked to autism [8–11]. These conditions
are all considered to be autoimmune diseases, which can
be triggered through molecular mimicry, where an
antibody responding to a foreign protein that resembles a
native protein becomes sensitized to the native protein as
well [12]. A paper by Shoenfeld and Aron-Maor in 2000
developed the argument that both autism and MS may be
examples of an autoimmune reaction via mimicry
following exposure to an antigenic stimulus, possibly from
an infection or through vaccination [13]. They further
propose specifically that myelin basic protein (MBP) and
other proteins constituting the myelin sheath are attacked
by the immune system in both autism and MS. This has
been recognized by many others in autism [14, 15] and MS
[16–20]. In 1982, Weizman et al. reported a cell-mediated
autoimmune response to human MBP in 76% of the
autistic children studied [16]. Immune sensitization to the
myelin sheath proteins could arise either through mimicry
as a consequence of exposure of the immune system to a
foreign antigen with a similar peptide sequence that is
* Corresponding author. E-mail: seneff@csail.mit.edu
Glyphosate pathways to modern diseases VI: Prions, amyloidoses and autoimmune
neurological diseases
Anthony Samsel1 and Stephanie Seneff
2, *
1Samsel Environmental and Public Health Services, Deerfield, NH 03037, USA
2Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA 02139, USA
Usage of the herbicide glyphosate on core crops in the USA has increased exponentially
over the past two decades, in step with the exponential increase in autoimmune diseases
including autism, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, coeliac
disease, neuromyelitis optica and many others. In this paper we explain how glyphosate,
acting as a non-coding amino acid analogue of glycine, could erroneously be integrated with
or incorporated into protein synthesis in place of glycine, producing a defective product that
resists proteolysis. Whether produced by a microbe or present in a food source, such a peptide
could lead to autoimmune disease through molecular mimicry. We discuss similarities in other
naturally produced disease-causing amino acid analogues, such as the herbicide glufosinate
and the insecticide L-canavanine, and provide multiple examples of glycine-containing short
peptides linked to autoimmune disease, particularly with respect to multiple sclerosis. Most
disturbing is the presence of glyphosate in many popular vaccines including the measles,
mumps and rubella (MMR) vaccine, which we have verified here for the first time.
Contamination may come through bovine protein, bovine calf serum, bovine casein, egg
protein and/or gelatin. Gelatin sourced from the skin and bones of pigs and cattle given
glyphosate-contaminated feed contains the herbicide. Collagen, the principal component of
gelatin, contains very high levels of glycine, as do the digestive enzymes: pepsin, trypsin and
lipase. The live measles virus could produce glyphosate-containing haemagglutinin, which
might induce an autoimmune attack on myelin basic protein, commonly observed in autism.
Regulatory agencies urgently need to reconsider the risks associated with the indiscriminate
use of glyphosate to control weeds.
Keywords: autism, autoimmune disease, collagen, glycine, glyphosate, multiple sclerosis,
protein misfolding, vaccines
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 9
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
resistant to clearance, or because the proteins
themselves have been altered in some way that renders
them defective, exposed and/or resistant to proteolysis.
Unlike DNA synthesis, protein synthesis is highly
prone to error [21, 22]. It appears that biological systems
have adopted a strategy of allowing coding errors to
survive during active synthesis, but use protein misfolding
as a criterion to mark a defective peptide for degradation
and recycling through ubiquitination. It is estimated that
15% of average-length proteins will have at least one
misincorporated amino acid. Typically, 10–15% of random
substitutions disrupt protein function, mostly because of
misfolding [22]. Such destabilization causes protein–
protein aggregation, and can lead to multiple neurological
diseases and amyloidoses. Drummond et al. propose that
early-forming toxic oligomers of amyloidogenic proteins
are enriched with missense errors [22].
Glyphosate is the active ingredient in the pervasive
herbicide Roundup and in many other formulations of
herbicides used to control weeds on agricultural, residential
and public land worldwide. A recent study based in
Germany involving 399 urine samples from adults not
involved in agricultural work revealed glyphosate residues
above the detection limit in the urine of 32% of the subjects,
and residues of AMPA, a metabolite, in 40% [23]. In a
paper published in 2014, Swanson et al. showed a
remarkable correlation between the rising rate of
glyphosate usage on corn (maize) and soy crops in the
USA and an alarming rise in a number of different chronic
diseases [24]. Additional strong correlations for other
conditions and diseases are provided in two follow-on
papers [25, 26]. While correlation does not necessarily
mean causation, causation becomes much more likely if a
plausible mechanism can be found. Swanson et al. found a
remarkable 0.98 correlation coefficient between the rise in
autism rates in the USA and the use of glyphosate on crops
(P-value ≤ 9.6 × 10–6). The correlation for multiple
sclerosis was not as high, but still highly significant at 0.83
(P-value ≤ 1.1 × 10–5). IBD had a correlation coefficient of
0.94 (P-value ≤ 7.1 × 10–8) (see Table 1 for other diseases).
IBD, especially among children, is an emerging
global epidemic [27] that is linked to autism [28, 29].
Impairment of intestinal barrier function is a core feature
of IBD [30]. Increased intestinal permeability promotes
infiltration of unmetabolized peptides into the lymph
system and general circulation. This provides an
opportunity for an immune antigenic response, which by
molecular mimicry can lead to an attack on crucial
proteins in the brain and spinal column. Disturbances of
collagen texture are a major factor leading to the onset of
diverticular disease and IBD along with the disturbed
wound-healing mechanisms seen in the pathogenesis of
anastomatic leakage following large bowel surgery [31].
In a recent paper [32], we suggested that
glyphosate, a non-coding amino acid analogue of glycine,
could substitute for glycine in error during protein
synthesis. Such misincorporation and disruption of
proteostasis could explain the strong correlations
observed between glyphosate usage and multiple modern
diseases. In this paper, we show that this could be one
of the most important mechanisms by which glyphosate
could induce multiple autoimmune diseases.
A prime site for initiation of the disease process is
the colon, where misfolded collagen, resistant to
degradation, could lead to an autoimmune disease and,
subsequently, a leaky gut. Autoantibodies against type
VII collagen have been detected in up to 68% of IBD
patients [33]. Glycine is the most common amino acid in
collagen, making up one fourth of the residues in the
protein. Proline is also a very common component of
collagen and, as we discuss later in this paper, proline
resists hydrolysis. Incomplete collagen degradation by
matrix metalloproteinases in the gut could lead to the
accumulation of short pro–gly–pro peptides that are
resistant to proteolysis. These could then induce the
infiltration of neutrophils or the activation of resident
immune cells to induce an inflammatory response [34].
An unpublished study conducted by Monsanto and
submitted to the US Environmental Protection Agency
(EPA) traced the accumulation of radiolabeled glyphosate
in various tissues of rats following low-dose oral
administration (10 mg/kg body weight) [35]. By far the
highest accumulation was found in the bones (Table 11 in
[36]). Radioactive levels in the colon were 4–6 times as
high as those in the stomach and small intestine.
The production of novel non-coding amino acids by
plants and microbes wards off predators. The toxicity of
these products may be due to the fact that they replace
coding analogues during protein synthesis. Examples
include: azetidine-2-carboxylic acid (Aze), a proline
analogue [37, 38]; glufosinate, a glutamate analogue that
is also a popular herbicide [39]; β-N-methylamino-L-alanine
Disease Correlation
coeff icient (R) P-value
Autism (p revale nce) 0.98 9.6 × 10
–
6
MS (deat hs) 0.83 1.1 × 10
–
5
IBD 0.94 7. 1 × 10
–
8
A
n
aemi a 0. 90 1.8 × 10
–
4
Diabetes (prevalence) 0.97 9.2 × 10
–
9
Thyroid canc er (incid ence) 0.99 7. 6 × 10
–
9
Table 1. Correlations between time trends in several diseases
and conditions recorded by the US Centers for Disease Control
(CDC) with glyphosate usage on corn (maize) and soy crops
reported by the USDA. Data reproduced from [23] and [25].
10 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
(BMAA), an analogue of serine [40]; and L-canavanine,
a natural analogue of L-arginine that is exploited as an
insecticide [41, 42].
A remarkable true-life story involving a 119-day
Alaskan wilderness experiment conducted by Christopher
McCandless was recounted in the book Into the Wild by
Jon Krakauer (later made into a popular movie) [43].
McCandless was thought to have died in the wilderness
from starvation; however, Krakauer always suspected a
toxin in the seeds of the wild potato, Hedysarum alpinum,
which formed a staple of his diet in his last month of life.
Krakauer had originally suspected a poisonous alkaloid
but, through later research, was able to identify a
significant level of L-canavanine in the wild potato seeds
and published a paper on this analysis with several other
authors in 2016 [42].
A key factor in L-canavanine’s toxicity is its ability to
insinuate itself into peptides in place of L-arginine. L-
canavanine can be assimilated into essentially any protein
to create aberrant canavanyl proteins that can disrupt many
fundamentally important biochemical reactions across a
broad spectrum of organisms [41, 44]. L-canavanine is
exploited in agriculture as a potent insecticide against the
tobacco hornworm [45], although the tobacco budworm
has developed tolerance with a unique enzyme,
canavanine hydrolase, which can quickly metabolize it
[46]. Larvae exposed to L-canavanine incorporate it into
the protein lysozyme, resulting in a 48% loss in catalytic
activity [41]. Furthermore, diptericins B and C of
Protoformia terranovae, but not diptericin A, are
negatively impacted by L-canavanine. The distinction is
that diptericin A has histidine at position 38 instead of the
L-arginine found in the other two diptericins. Presciently,
with respect to glyphosate, Rosenthal wrote: “These
insect studies support the view that the biological effects
of canavanine result from its incorporation into a protein,
resulting in an alteration in protein conformation that
leads ultimately to impairment of protein function” [41].
2. SHIKIMATE PATHWAY INHIBITION REVISITED
The shikimate pathway enzyme, 5-enolpyruvylshikimate-
3-phosphate synthase (EPSPS) is believed to be the main
target of glyphosate’s toxicity to plants [47]. A 1991
paper by Padgette et al. describes studies to gain insight
into the mechanism by which glyphosate disrupts EPSPS
[47]. Surprisingly, it is not understood exactly how
glyphosate binds to the active site.
The microbes Klebsiella pneumoniae, Escherichia
coli [47, 48] and Agrobacterium sp. strain CP4 [48, 49]
have all evolved to produce versions of EPSPS that are
glyphosate-resistant. The CP4 variant has been widely
exploited by importing it into genetically modified
glyphosate-resistant crops [48]. Insight can be gained by
investigating the alterations to the peptide sequence that
afforded resistance. All three mutations involved replacing
a glycine residue at the active site with alanine [47, 48].
In the case of E. coli, the mutated enzyme is about 72
times less efficient than the wild-type enzyme, but 69
times more efficient in the presence of glyphosate.
Changing the DNA code from glycine to alanine
completely disables glyphosate’s inhibiting effects on the
enzyme [48].
Substitution of gly-96 at the active site in E. coli by
serine leads to a version of the enzyme that is unable to
bind PEP, most likely due to steric hindrance. The authors
speculated that the hydroxymethyl group of serine
displaces the phosphate of PEP and functions as a
nucleophile. In fact, this mutated enzyme achieves a kind
of reverse reaction, breaking EPSP down into shikimate-
3-phosphate and pyruvate via hydrolysis.
We propose that substitution of gly-96 (gly-100 in the
CP4 variant) by glyphosate during protein synthesis could
explain its disruption of the enzyme’s function. One can
expect that the highly reactive and bulky glyphosate
molecule, if substituted for gly-96, would behave more
like serine than alanine. An additional disruptive factor is
glyphosate’s chelation of manganese, which would
disrupt the catalytic action of EPSPS. A cell containing
both wild-type and glyphosate-substituted forms of the
enzyme would arguably circuitously convert PEP to
pyruvate via EPSP without producing ATP from ADP;
i.e., would waste the energy in the phosphate bond, as
shown in Fig. 1, and end up with excess pyruvate and a
deficiency in EPSP.
Figure 1. Diagram of the hypothetical pathway by which
glyphosate substitution for glycine in EPSPS could result in
the synthesis of pyruvate from PEP without generating ATP;
i.e., wasting the energy in the phosphate group, as discussed
in the text.
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 11
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JBPC Vol. 17 (2017)
3. GLYPHOSATE AS A GLYCINE ANALOGUE
While glyphosate’s main mechanism of toxicity to plants
is considered to be disruption of the shikimate pathway, it
is also likely that it disrupts other biological pathways
where glycine is either a substrate or a ligand, due to the
fact that it is a glycine analogue. It has been proposed
that, through glycine mimicry, glyphosate’s rôle as a ligand
to NMDA receptors in the brain could explain its known
ability to activate NMDA receptors and cause neuronal
damage [49, 50]. In [51], acute exposure of rat
hippocampal slices to Roundup (0.00005–0.1%) for 30
minutes caused oxidative stress and neuronal cell death,
which was attributed to NMDA receptor activation.
Glyphosate also interferes with the synthesis of porphyrin,
a precursor to haem, by disrupting the first step in the
pathway where glycine is substrate [52].
N-substituted glycine “peptoids” are an attractive
class of synthetic molecules that can be constructed by
linking component N-substituted glycines at sequential
nitrogen–carbon bonds; they are directly analogous to the
linking of amino acids into peptides [53]. Glyphosate is of
course an N-substituted glycine, where the nitrogen side
chain is a methyl phosphonyl group. Part of the attraction
of peptoids is that they are highly resistant to proteolysis,
just as is the amino acid proline, in which the carbon side
chain circles back and binds to the peptide nitrogen.
Impaired ability to break down proline-rich gliadin has been
proposed as a contributing factor in coeliac disease and
gluten intolerance [54]. This can explain why common
cereals with high proline contents are especially problematic
to gluten-sensitive individuals [55, 56].
Glyphosate is probably particularly problematic
when it substitutes for N-terminal glycines in proteins
where these glycines are highly conserved and play a
significant rôle. Several proteins rely on an N-terminal
glycine for anchoring to the plasma membrane (e.g.,
endothelial nitric oxide synthase (eNOS) [57]) or to the
cytoskeleton (e.g., Kelch-like ECH-associated protein 1
(KEAP1) [58]). Protein N-myristoylation and prenylation
depend on an amide bond to the N-terminal glycine residue
[59]. For example, myristoylated G proteins involved in
many signaling mechanisms depend on an N-terminal
glycine residue [59]. This would be disrupted if the
nitrogen atom has a side chain through glyphosate
substitution for the terminal glycine.
N-nitrosoamino acids form a reasonable model for
N-nitrosoglyphosate, a carcinogenic derivative of
glyphosate that was of concern to the EPA during
Monsanto’s early studies. N-nitrosoproline is particularly
relevant because proline, like glyphosate, has an extra
carbon atom bound to the nitrogen atom. With respect to
non-coding amino acids, and especially the incorporation
of N-nitrosoamino acids into peptides and proteins,
R.C. Massey remarked: “In addition to their presence as
free N-nitrosoamino acids, species such as N-
nitrosoproline (NPRO) and N-nitroso-4-hydroxyproline
(HONPRO) may exist in a peptide- or protein-bound
form as a result of N-nitrosation of an N-terminal imino
acid residue” [62]. Tricker et al. [63] and Kubacki et al.
[64] devised high performance liquid chromatography–
thermal energy analyser (HPLC–TEA) techniques for
analysis of multiple dipeptides with a nitrosylated N-
terminal, including N-nitrosoprolylalanine (NPROALA), N-
nitrosoprolyl-4-hydroxyproline (NPROHOPRO) and N-
nitrosoprolylglycine (NPROGLY) [63, 64]. Tricker notes
that the average recoveries for NPROALA, NPROHOPRO
and NPROGLY, 200 μg of which was added to cured
meat, were between 69 and 88%. Tricker also used the
method to analyse the nitroso-tripeptide N-nitrosoprolylgly-
cylglycine [65].
Nitrosamines of glyphosate (N-phosphonomethylgly-
cine), its salts and esters include: N-nitrosoglyphosate
(NNG) (Monsanto CP 76976), N-nitrosoiminodiacetic acid
(NNIDA), N-nitrosoglyphosate sodium salt (NNGNa), N-
nitrosoglyphosate isopropylamine ester (NNGIPA), N-
nitrosoglyphosate potassium salt (NNGK), the metabolite
N-nitrosoAMPA (NNAMPA), the metabolites N-nitrosodi-
methyl amine (NDMA) and N-nitrosarcosine (NSAR),
which occur in glyphosate products or may be generated
in vivo or in soils and waterways. N-nitroso compounds
derived from secondary amines are considered carcinogenic.
Monsanto glyphosate documents reveal analysis
and quantification of five nitrosamines of concern [61].
Out of six lots of Roundup analysed for NNG, four lots
contained NNG residues of 0.61 to 0.78 ppm and two lots
had residues from 0.22 to 0.40 ppm NNG. Analysis of six
lots of Monsanto Rodeo revealed NNG residues in the
range 0.13–0.49 ppm.
Recently, a powerful metatranscriptome study on
bacterial gene expression following glyphosate treatment
was conducted on microbes growing within the
rhizosphere of glyphosate-tolerant corn [66]. RNA
transcript abundance was compared between control
and glyphosate-treated samples in order to characterize
which protein genes were upregulated or downregulated.
While they found many changes in gene expression, most
striking to us was the upregulation of genes involved in
both protein synthesis and protein hydrolysis. The
ribosomal proteins L16p (L10e) and Firmicutes ribosomal
L7Ae family proteins involved in the synthesis of the
ribosomal large subunit increased 1.4- and two-fold,
respectively, and the small subunit ribosomal protein S11p
(S14e) increased 1.5-fold. Upregulation of genes involved
in protein degradation was even more dramatic. For
12 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
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JBPC Vol. 17 (2017)
example, transcripts for a proteasome β 2 subunit (EC
3.4.25.1) increased 4.3-fold and aminopeptidase YpdF
increased threefold. An explanation could be an increase
in the number of proteins that fail to fold properly due to
glyphosate substitution for glycine in the protein. These
authors also suggested a potential shift towards an
increase in glyphosate-tolerant bacteria, a point that will
become important later in this paper.
These results are corroborated by a study on pea
plants grown in hydroponic culture, which revealed that
glyphosate induced a significant increase in two major
systems for proteolytic degradation: the ubiquitin-26 S
proteasome system and papain-like cysteine proteases
[67]. It also increased the total free amino acid content
and decreased the soluble protein in the root system.
4. GLYPHOSATE-CONTAMINATED COLLAGEN AND
PROTEOLYSIS RESISTANCE
We mentioned in the Introduction the gly–pro–gly peptide
sequence that is common in collagen and linked to
autoimmune disease. There are several enzymes in
multiple organisms that are devoted to the proteolysis of
peptide sequences containing proline, particularly the
gly–pro sequence. These include enzymes that detach a
terminal proline, enzymes that detach a dipeptide sequence
where the second residue is a proline molecule and the
first one is often glycine, and enzymes that break apart
the X–pro dipeptide to release two free amino acids, one
of which is proline. Certain pathogens have special
modified versions of these enzymes, and there are genetic
diseases related to pathologies in these enzymes.
Substitution of glyphosate for glycine in this sequence is
likely to cause extra stress to the enzymes that break down
these sequences, potentially leading to autoimmune disease.
Prolyl aminopeptidase is an enzyme that detaches a
terminal proline residue from a peptide. The enzyme is
expressed predominantly by pathogenic bacteria in the
gut, in particular Serratia marcescens, a common
pathogen in the gut as well as in the urinary tract; it is
often multiply antibiotic-resistant and is a serious threat in
hospital-acquired infection [34]. This enzyme is
especially important to the pathogens for degrading
collagen, providing amino acids as fuel. It is conceivable
that the pathogens are able to degrade glyphosate-
contaminated peptides terminating in proline whereas the
human form of the enzyme is not. It is intriguing that the
S. marcescens version of prolyl aminopeptidase is unusual
in having extra space at the active site [34], which could
potentially accommodate the larger glyphosate molecule
adjacent to the terminal proline residue. This might also
contribute to glyphosate’s observed effect on the gut
microbiome: excessive growth of pathogens.
Multiple strains of the toxic mould Aspergillus
secrete an X–prolyl dipeptidyl aminopeptidase (X-PDAP)
that is important for digesting collagen because it can
separate out an X–pro pair to bypass the difficult step of
breaking the X–pro bond. Research has shown that this
enzyme is essential for hydrolysing proline-containing
peptides [69, 70]. It is likely that it becomes even more
essential when X is glyphosate, as the peptoid sequence
glyphosate–proline is likely almost impossible to break.
Since gly–pro is a very common sequence in collagen,
glyphosate–pro is likely to impede the breakdown of collagen
fragments, which may then encourage Aspergillus
infection in both plants and animals. Glyphosate has been
shown to increase the growth rate of Aspergillus [71].
The most disturbing question is, what happens in the
absence of pathogens that can effectively clear collagen
peptides contaminated with glyphosate? As we will see
later in this paper, antibodies to collagen are linked to
antibodies to vaccines. A genetic defect in the enzyme
prolidase, which can break apart the very common gly–pro
dipeptide to release the individual amino acids, leads to a
severe disease with mental deficiencies and multiple skin
lesions [72]. Intriguingly, a common plant pathogen,
Xanthomonas campestris, which causes blight on
multiple plant species has a unique variant of prolidase
with two mutations, a substitution of tyrosine for gly-385
and valine for tyr-387, two highly conserved residues in
the peptide sequence [73]. Is it possible that swapping out
glycine affords protection from glyphosate substitution
for this residue? We hypothesize that peptides derived
from multiple proline and glyphosate-contaminated
proteins, which are highly resistant to proteolysis, are
causing an autoimmmune epidemic that is an important
contributor to autism and other autoimmune disorders.
5. BMAA AND ALS IN GUAM
β-N-methylamino-L-alanine (BMAA) is another noncoding
amino acid and an analogue of serine [40]. BMAA is
synthesized by cyanobacteria, the microbes responsible
for the toxic algal blooms that occur in lakes experiencing
an accumulation of nitrogen and phosphate nutrients
following hot, rainy weather [74]. An in vitro study by
Dunlop et al. in 2013 demonstrated that BMAA can be
misincorporated into human proteins, causing protein
misfolding that could lead to neurological diseases [40].
BMAA has, in fact, been linked to several
neurodegenerative diseases, including Parkinson’s,
Alzheimer’s and amyotrophic lateral sclerosis (ALS)
[75]. A 2013 study linked an ALS cluster in Chesapeake
Bay to consumption of BMAA-contaminated crabs [76].
A study in France investigated an ALS cluster near a
lagoon that supplied oysters and mussels to the local
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 13
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JBPC Vol. 17 (2017)
population. The authors demonstrated that the shellfish
were contaminated with BMAA, but also remarked that
there was intensive chemical-based agriculture in the
region [77]. Interestingly, cyanobacteria have been found
to be remarkably resistant to glyphosate [78, 79], and this
could contribute to the recent record-setting algal blooms
in the Great Lakes region, where glyphosate is
extensively used on genetically modified (GM) Roundup-
Ready crops [80].
One likely molecule that could be adversely affected
by BMAA is the glutamate transporter, whose defective
expression has been linked to ALS [81]. Glutamate
excitotoxicity in motor neurons is associated with ALS,
and this could be caused by an impaired glutamate
transport system. Ordinarily, astrocytes quickly clear
glutamate from the synapse, following its release by
neurons, and the transporter is essential for this
clearance. A conserved serine-rich motif in the glutamate
transporter forms a reëntrant loop, similar to a structure
found in many ion channels [82]. This loop is crucial for
the enzyme’s proper function, and would be disrupted by
substitution of BMAA for serine.
An interesting detective story has evolved around an
epidemic of a complex neurological condition termed
amyotrophic lateral sclerosis–Parkinsonism dementia
complex (ALS–PDC), which reached epidemic
proportions during a short interval after World War II
among the native Chamorro people on the small island of
Guam in the South Pacific. At the peak of the epidemic,
the natives had a hundredfold increased risk to ALS and
Parkinson’s disease compared to the risk in the general
human population.
A plausible explanation for this epidemic relates to a
popular native food source: seeds from the cycad trees
[83–85]. Cycad seeds contain BMAA, likely derived
from associated cyanobacteria. However, what is
especially interesting is that the BMAA becomes
concentrated in the skin of fruit bats that feed on the
cycad seeds. Fruit bats were a popular delicacy among
the natives, who ate every part of them, including the
skin. Increased access to firearms from the USA during
the war may have made it easier to kill the bats, on which
the natives then feasted, ultimately leading to the natives’
near-extinction through the accumulation of BMAA in
their brains [86]. Meanwhile the near-extermination of
the bats through the hunting removed the presumed
source of the epidemic [83].
However, the warfare also led to the accumulation
of many toxic chemicals in the soil, which could have
encouraged the proliferation of cyanobacteria, which are
especially resilient in the face of stressors. The bats’
demise was undoubtedly hastened by the accumulation of
excess BMAA in their tissues. A measurement of the
amount of BMAA in three dried specimens of fruit bats
from Guam taken from a museum in Berkeley found
concentrations between 1200 and 7500 μg/g, which
indicates up to hundredfold bioamplification over the level
in the seeds of the cycad tree [87].
There have been inconsistent results in measuring
the levels of BMAA in different tissue samples, but this
has been explained recently by the realization that any
BMAA incorporated into proteins may be missed in
analysis without sufficient proteolysis. Ince et al. wrote:
“When the insoluble, protein-containing fraction following
TCA (trichloroacetic acid) extraction is further
hydrolysed to release BMAA from protein, there is a
further pool of protein-bound BMAA that is present in a
ratio of between 60:1 and 120:1 compared with the pool
of free BMAA” [84, p. 348]. We believe that this point
has great significance when it comes to glyphosate: we
highly suspect that different methodologies used to
measure glyphosate contamination in any situation where
there is a significant protein-bound component may yield
different results depending on the degree to which protein
hydrolysis is carried out.
6. GLYPHOSATE CONTAMINATION IN COLLAGEN,
ENZYMES, GELATIN AND VACCINES
Gelatin is commonly used as an excipient stabilizer in
vaccines, particularly the live virus vaccines. Gelatin is
derived from animal skin and bone, especially of pigs and
cattle; they may be fed glyphosate-contaminated forages,
including GM Roundup-Ready corn and soy feed, which
are sometimes supplemented with GM Roundup-Ready
beet pulp. Gelatin is mainly derived by partial hydrolysis
from the collagen in skin and bone. 26% of the amino
acids in collagen are glycine; proline and hydroxyproline
together make up 18% [88]; and glutamate constitutes
6%. All three of these components are problematic. The
proline could be substituted by Aze from the sugar beet,
the glycine could be substituted by residual glyphosate in
the feed, and glutamate is a neurotransmitter but known
to be neurotoxic at high concentrations; it works together
with glycine to excite NMDA receptors in the brain. The
vaccine virus may incorporate some of the noncoding
amino acids into its own proteins to produce versions of
them that resist proteolysis and induce autoimmunity
through molecular mimicry.
One of us (Samsel) analysed a number of animal
protein products for glyphosate. These included the
bones of pigs, cows, horses’ hooves, bees and bee
products, collagen and gelatin products, vitamins, protein
powders, enzymes and vaccines. Results are shown in
Tables 2 and 3. Both high performance liquid
14 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
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JBPC Vol. 17 (2017)
chromatography with tandem mass spectrometry
(HPLC–MSMS) and enzyme-linked immunosorbent
assay (ELISA) methods were utilized. It has been shown
that both HPLC and ELISA are comparable in terms of
accuracy and precision for detection and quantification of
glyphosate in water-based analysis and including
Nanopure, tap and river waters. Water-based solvents for
glyphosate demonstrate a detection limit of 0.6 ng/mL
and a linear functional range of 1–25 ng/mL [200].
However, HPLC was not able to achieve detection
below 5 ppb;1 hence, in cases including water-based
vaccines, analysis using numerous sample runs was
made including using two independent labs to test the
same samples.
1Parts per (US) billion. To put this into perspective, 1 ppb = 1 μg/kg, and 1 μg of glyphosate (N-phosphonomethylglycine)
contains 3.561 × 1012 molecules of the substance, each one of which could integrate with a protein.
Protein substrate Type Test date Glyphosate
residue (ppb)1
GELATI N JELL-O ORANGE # 07 JAN 2018 DB02
02:36 29 Ju ly 2016 9.00
GELATI N POWER-MAX PROTEIN POWDER
ADVANC ED NUTRITION 29 Ju ly 2016 14.94
GELATI N DISNEY GUMMIES VI TAMINS 9 Augu st 2 016 8 .27
GELATI N FLIN TSTONES GUMMI ES VITAMINS 9 August 2016 5.32
ORAGEL CHILDREN’S ORAGEL 7.5%
BENZOCAINE FORM ULA 26 Se ptember 2016 2.81
Table 2. Residues of glyphosate found in animal-based products that were reported to the US Food and Drug
Administration (FDA) by Samsel Environmental & Public Health Services. The limit of detection for glyphosate
using hot water extraction is 0.075 parts per billion (ppb).1
HPLC–MSMS was also later used, where the
method detection limit (MDL) permitted, for additional
confirmation and quantification of glyphosate in digestive
enzymes and collagens. Spiked sample recoveries were
done for all samples tested. Freshly prepared glyphosate
standard solutions were run as controls and results were
calculated based on a standard curve.
In 1989, Monsanto researchers conducted an experi-
ment on exposure of bluegill sunfish to 14C-radiolabeled
glyphosate [89]. One of us (Samsel) obtained the
(unpublished) report from the EPA through the Freedom
of Information Act. The researchers had found that, with
EDTA extraction, the amount of radiolabel in tissue
samples was much higher than the amount of detected
glyphosate. They decided to apply a digestive enzyme,
proteinase K, and discovered that this “caused a
substantial improvement in extractability”. It brought the
yield from 17–20% in the case of EDTA to 57–70%
following digestion with proteinase K. They summed up
as follows: “Proteinase K hydrolyses proteins to amino
acids and small oligopeptides, suggesting that a significant
portion of the 14C activity residing in the bluegill sunfish
tissue was tightly associated with or incorporated into
protein” (present authors’ emphasis). In this context it is
important to recall that a 60- to 120-fold higher detection
level of BMAA was obtained following protein
hydrolysis of contaminated proteins [84].
Since Monsanto found bioaccumulation of
glyphosate in all animal tissues, with the highest levels in
the bones and marrow [35, 36], one would expect that all
tissues derived from animals fed a diet containing
glyphosate residues and used for food by people around
the globe would be contaminated. Knowing that the
bioaccumulation of glyphosate would be evident in the
vast majority of animals raised for market and fed a
contaminated diet, as well as their products; and
suspecting the possibility of contamination of even the
digestive enzymes derived from these animals, one of us
(Samsel) decided to analyse random samples.
Results from various gelatin-based products, along
with the results for several different vaccines (discussed
later) were reported to the FDA by Samsel
Environmental & Public Health Services in August 2016.
Table 2 shows results for glyphosate residues found in
these gelatin-based products. The highest level found in a
gelatin sample was almost 15 ppb.1
Having found glyphosate in animal gelatins,
analysing the collagen at the source was a logical next
step. Tissues from pork and cattle obtained from a local
supermarket, commercially available collagen sourced
from industrially-raised swine and oxen, as well as the
purified digestive enzymes pepsin, lipase and trypsin,
derived from pigs, were selected for evaluation. Three
methods of laboratory analysis were used to determine if
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 15
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JBPC Vol. 17 (2017)
glyphosate was present in porcine pepsin and in the
glycine-rich collagen from the tissues of pigs and cattle,
protein sources that are regularly consumed by
Americans. The results are given in Table 3.
Glyphosate integration with enzymes is a serious
consideration, as glyphosate may serve as an enzyme
inhibitor like other phosphonates [90–92]. Inhibition and
immobilization of enzymes may occur via three basic
categories: covalent linkage; adsorption on a carrier; or
entrapment within macromolecules [93].
Inhibition of enzymes may be reversible or
irreversible. Types of reversible enzyme inhibition include
competitive, noncompetitive and uncompetitive.
Irreversible inhibitors covalently bond to the functional
groups of the active site, thus permanently inactivating
catalytic activity. Irreversible inhibition includes two
types: group-specific inhibition and “suicide” inhibition.
The importance of fully functional digestive enzymes
cannot be understated. They are essential for metabolic
function, as they convert food into nutrients and other
molecules that are then available to cells for tissue and
organ growth, maintenance and repair. The precursor
trypsinogen, produced in the pancreas, is enzymatically
transformed into the serine protease trypsin. Trypsin
catalyses the hydrolysis of proteins into peptides and
provides substrates for further enzymatic hydrolysis for
protein absorption.
Pepsin, a primary protease of digestion, is also
responsible for the metabolism of dietary protein.
Pepsin’s cleavage of peptide bonds is responsible for the
availability of the aromatic amino acids phenylalanine,
tyrosine and tryptophan. It is also responsible for the
cleavage and release of several other amino acids,
including valine, glycine, histamine, glutamine, alanine
and leucine.
Lipase participates in cell signaling, inflammation
and metabolism. Pancreatic lipase is the catalyst for the
hydrolysis of dietary lipids, which include fats, oils,
cholesterol esters and triglycerides [94]. Triglyceride
triester is metabolized for utilization as glucose and
three fatty acids. Glyphosate integration into and inhibition
of lipase could induce excessive bioaccumulation of
fatty material in the blood vessels, gut, liver, spleen and
other organs, as well as mimic lysosomal acid lipase
deficiency. It would also allow for an increase in
triglycerides in the blood, leading to numerous disease
cascades, including malabsorption, fatty liver disease,
jaundice, failure to thrive in infants, calcification of the
adrenal gland, anaemia, hypercholesterolaemia, biliary
dysfunction, decreased HDL, increased LDL, blood
clots, fat-enlarged hepatocytes and liver fibrosis and
failure. Samsel found that radiolabeled glyphosate was
not detectable by HPLC–MSMS in samples of lipase
deliberately spiked for analysis, suggesting that
glyphosate may irreversibly inhibit lipase. On the other
hand, pepsin and trypsin had good spike recoveries,
demonstrating reversibility as glyphosate was released
from the protein.
Protein substrate (Method) Type Glyphosate residue (ppb)
Bone (ELISA) Bovi ne leg 11.56
Bone
m
arrow (ELISA ) Bovi ne leg mar row 4.2 2
Bone (ELISA) Porcine foot 9.81
Skin (ELISA) Porcine 0.325
Gela tin (ELISA) Bovi ne, Sigma Aldrich , gel streng th
225 Ty pe B
2.04
Collagen (ELISA) Bovine I & III 120.18
Collagen (GC-MS) Bovine I & III 130 µg/kg
Col l ag en (HPL
C
-MSMS) Bovine I & III 95 µg/kg
Pepsin (EL ISA) Purified porcine enzyme < 40.00
Pepsin (GC-MS) Purified
p
orc in e en zy me 430 µg /kg
Pepsin (HPL
C
-MSMS) Purified
p
orc in e en zy me 290 µg /kg
Trypsin ( ELISA) Purifie d
p
orc in e en zy me 61. 9 9
Lipase (ELISA) Pu rified
p
orc in e en zy me 24. 4 3
Bee
b
read (HPLC-MSMS) Bee
b
rea
d
2300 µg/kg
Bees (HPL
C
-MSMS) Api s me llif era < 10 µg/kg trace
Honey & comb (HPLC-MSMS) Hone
y
< 10 µg/kg trace
a The trace amount found in the bee substrates appeared as a small peak, which directly corresponded to glyphosate, complete
with retention time and molecular features confirming contamination using HPLC–MSMS.
Table 3. Integration of glyphosate residues in various proteins, assessed using three testing methods.a
16 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
Table 3 shows results for various bovine and porcine
products, including enzymes, bone, bone marrow, skin,
collagen and gelatin. Acid hydrolysis was used on the
bovine and porcine skin, bones and marrow, which were
shaken and digested with 0.15 M hydrochloric acid for
24 h. The analysis methods were ELISA, gas chroma-
tography–mass spectrometry (GC–MS) and HPLC–
MSMS. All of the tested products were contaminated,
with the highest level detected being 430 µg/kg in porcine
pepsin (via GC–MS).
Additional evidence of glyphosate accumulation was
found by Samsel in 2015 in the bodies of dead bees, bee
bread and honey from bee hives suspected of colony
collapse disorder (CCD), and these are also shown in
the table. Colony collapse disorder (CCD) is an ever-
increasing problem threatening pollination of crops
globally. It may share a similar aetiology to that of
Alzheimer’s disease with regard to learning and memory
within the bee’s brain. Integration of glyphosate with the
structural proteins and enzymes of the bee may affect
protein folding and function. Additionally, glyphosate may
also affect the digestive enzymes and bacterial
homeostasis within the digestive system, which in turn
may affect the quality of the honey produced. Glyphosate
in bees may become part of their chitin, which has a
structural function, in their bodies, analogous to
glyphosate becoming part of the collagens of humans and
other animals.
The results in Table 3 show ubiquitous contamination
of the bee and bee products. Honey is derived from
nectar and is the source of carbohydrates in the bee diet,
whereas pollen turned into bee bread supplies the fats
and proteins. Royal jelly, made from the secretions of the
glands found in the hypopharynx of the worker bees, is fed
to the queen and developing larvae [96].
Results for nineteen different vaccines, from five
manufacturers, are shown in Table 4. Some vaccines do
not contain live viruses and do not involve gelatin in their
preparation, but many involve the use of eggs, bovine calf
serum, fetal bovine serum or bovine proteins [95].
Engerix Hepatitis B vaccine is manufactured through a
novel procedure, which involves culturing genetically
engineered Saccharomyces cerevisiae yeast cells that
carry the surface antigen gene of the hepatitis B virus.
The procedures result in a product that can contain up to
5% yeast proteins, which could be a source of glyphosate
if the yeast is grown on broths or media that utilize
glyphosate-contaminated nutrient sources such as animal
or plant proteins.
Vaccines that tested negative for glyphosate included
Merck’s Hep-B vaccine, most of the pneumococcal
vaccines and the sterile diluent included as a control.
Gelatin is not listed as an ingredient in any of these
vaccines, nor is bovine serum. In contrast, all of the
vaccines that listed gelatin as an excipient tested positive
for glyphosate, and nearly all of them also included bovine
serum (including Varicella, MMR-II, MMRV and Zoster).
It is significant that MMR-II consistently contained
the highest levels of glyphosate, significantly more than
any of the other vaccines. This vaccine uses up to 12%
hydrolysed gelatin as an excipient–stabilizer; as well as
foetal bovine serum albumin, human serum albumin and
residual chick embryo; all of which are contaminated by
glyphosate during animal production.
7. EVIDENCE FOR A ROLE FOR COLLAGEN IN VACCINE
ADVERSE REACTIONS
Post-vaccination allergic reactions to MMR and varicella
vaccines have been linked to the gelatin excipient, and
confirmed through observation of induced gelatin-
specific IgE antibodies [97–100]. 24 out of 26 children
with allergic reactions to vaccines (e.g., anaphylactic
shock) had anti-gelatin IgE ranging from 1.2 to 250 μg/mL.
Seven were allergic to gelatin-containing foods. A pool of
26 control children all tested negative for anti-gelatin IgE
[99]. A study from 2009 that looked at gelatin sensitivity in
children who were sensitive to cows’ milk, beef and/or
pork as determined by IgE antibody levels [101] found
that 16% of beef-sensitized children and 38% of pork-
sensitized children had IgE antibodies to beef- or pork-
derived gelatins that were cross-reactive with each other.
In a published case study, a 2-month-old baby
developed Kawasaki disease one day after receiving its
first dose of Infanrix (DTaP-IPV-Hib) and Prevenar, a
pneumococcal conjugate vaccine [102]. Kawasaki
disease is an acute, multisystemic vasculitis whose
occurrence very early in life is extremely rare. Extensive
tests for the presence of infection with multiple bacteria
and viruses were all negative. We suggest that glyphosate
contamination in one or both of the vaccines may have
contributed to the vasculitis through glyphosate uptake
into common proteins such as collagen in the vasculature
to induce the autoimmune reaction.
Kelso (1993) reported the case of a 17-year-old girl
who experienced anaphylaxis within minutes of receiving
an MMR vaccine [98]. The girl described the event as
“kind of like what happens when I eat Jell-O2”. Further
testing found gelatin to be the component of the vaccine
2Jell-O is a proprietary brand of gelatin-based desserts, popular in the USA, and manufactured by Kraft Foods, part of the Kraft
Heinz Company, headquartered in Chicago.
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 17
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
to which the girl was allergic. The connexion may be to
misfolded proteins, which include the collagens and
associated partially hydrolysed gelatins. Indeed, both
Jell-O and vaccines have been contaminated by
glyphosate, as we reported in the previous section.
Puppies immunized with the rabies vaccine and a
multivalent canine vaccine were compared to unvaccinated
Table 4. Glyphosate levels in vaccines determined by ELISA reported to the US CDC, NIH, FDA and UN WHO of the Americas in
September 2016 by Samsel Environmental & Public Health Services.a
Vaccine undiluted Manufacturer Lot number
Exp da te
Test date
Lab #
Glyphosate residue
(ppb)
% Recovery in
s
p
iked sample
DTaP ADACEL SANOFI PASTEUR
N
DC
58160 -820-4 3
3- 30-2 018
7-15-2016
LAB #1
0.1 09 82%
DTaP SANOFI PASTEUR C5041 8A
9- 2-20 18
5-11-2016
LAB #1
< 0.075 81%
DTaP ADACEL SANOFI PASTEUR
N
DC 58 160-82 0-4 3
3- 30-2 018
7-12-2016
LAB #2
N
D -
HEPATITIS-B MERC
K
LO16427
4- 13-2 017
5-11-2016
LAB #1
< 0.075 97%
HEPATITIS
EN GERIX -B
GLAXOSMI TH-
KLINE
NDC 58160-820-43
6- 1-20 18
7-15-2016
LAB #1
0.3 37 73%
INFLUENZA
FLUZONE QUAD
SANOFI PASTEUR 6762
6- 30-2 016
7-15-2016
LAB #1
0.1 70 95%
INFLUENZA
N
OVARTIS 1573 3P
05/2 016
5-11-2016
LAB #1
0.2 27 106%
Pneumococcal
PNEUMOVA X 23
MERC
K
700281601
5- 18-2 017
9-19-2016
LAB #1
0.1 12 118%
MMR II MERC
K
70021 51400
9- 9-20 17
7-15-2016
LAB #1
3.7 40 -
MMR II MERCK 009545
3- 19-2 017
5-11-2016
LAB #1
2.9 63 -
MMR II MERCK 7002151400
9- 9-20 17
9-19-2016
LAB #1
3.1 54 -
MMR II MERCK 7002151400
9- 9-20 17
7-12-2016
LAB #2
2.90 -
MMRV PROQUAD MERC
K
70023 05700
9- 12-2 017
9-19-2016
LAB #1
0.6 59 103%
MMRV PROQUAD MERC
K
70023 05700
9- 12-2 017
7-15–2016
LAB #1
0.5 12 86%
MRV PROQ UAD MERC
K
70023 05700
9- 12-2 017
7-12-2016
LAB #2
0.43 -
Pneumococcal
PNEUMOVA X 23
MERCK 70 028160 1
5- 18-2 017
7-15-2016
LAB #1
< 0.075 77%
Pneumococcal
PREVNAR 13
WYETH 73332
07/2 017
5-11-2016
LAB #1
< 0.075 82%
Pneumococcal
PNEUMOVA X 23
MERCK 70 026816 01
5- 18-2 017
7-12-2016
LAB #2
ND -
STERILE DILUENT MERCK, SHARP
& DOHM
E
LO 40058
5- 11-2 018
7-15-2016
LAB #1
< 0.075 97%
VARI CELLA
VARIVAX
MERC
K
70020 25000
2- 8-20 18
7-15–2016
LAB #1
0.5 56 84%
MVARICELLA
VARIVAX
MERC
K
70020 25000
2- 8-20 18
7-12-2016
LAB #2
0.41 -
ZO STER
ZOSTAVAX
MERCK 70 025024 01
6- 1-20 17
9-19-2016
LAB #1
0.6 20 95%
ZOSTER
ZOSTAVAX
MERCK 70 026024 01
6- 1-20 17
7-15-2016
LAB #1
0.5 58 98%
ZOSTER
ZOSTAVAX
MERCK 70 026024 01
6- 1-20 17
7-12-2016
LAB #2
0.42 -
a Limits of detection for glyphosate in vaccines in parts per billion (ppb):1 0.075 (LAB #1); 0.15 (LAB #2).
control puppies [103]. The vaccinated puppies, but not the
unvaccinated ones, developed autoantibodies to their own
collagen. A follow-up study where either just the rabies
vaccine or just the multivalent vaccine was administered
produced a similar result. The authors suggested that this
could explain issues of joint pain that are currently common
among dogs, particularly as they age.
18 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
8. MULTIPLE SCLEROSIS (MS)
8.1 Sugar beet and MS
The world obtains 30% of its sugar supply from beet
sugar. While sugar cane is grown in tropical regions,
sugar beet requires a temperate climate. The highest
incidences of MS worldwide are in the USA, Canada and
western Europe [5], where most of the beet sugar is
produced. MS rates are higher in the northern states of
the USA compared to the south, corresponding to the
distribution of sugar beet cultivation. MS rates in Canada
are highest in the Alberta prairie region, at the centre of
the Canadian sugar beet industry [104]. Studies on
migrants have shown that those who move from a low-
risk to a high-risk area tend to adopt high-risk only if they
migrated during childhood [105]. This implicates local
environmental factors acting before adolescence.
Tokachi province in Japan hosts only 0.3% of the
population, but produces 45% of the sugar beet consumed
in Japan [37]; this province has the highest rate of MS
among all Asian populations [106].
A fascinating proposition how sugar beet could
cause MS implicates a unique noncoding amino acid that
is produced by sugar beet, namely Aze. Both proline and
Aze have a unique structure for an amino acid: the side
chain loops back round to connect up to the nitrogen
atom. In the case of Aze, there are only 3 carbons in the
ring instead of the 4 carbons in proline (Fig. 2). It has
been shown experimentally that Aze can be inserted by
mistake into proteins in place of proline [38].
Myelin basic protein (MBP) is an essential protein
for maintaining the myelin sheath, and it interacts with
actin, tubulin, calmodulin and SH3 domains [107]. It
assembles actin filaments and microtubules, binds actin
filaments and SH3 domains to membrane surfaces, and
participates in signal transduction in oligodendrocytes and
myelin. A central proline-rich region in MBP is
functionally significant [108–110] and, in particular, is a
binding site for Fyn-SH3, a key regulatory protein [111].
Proline substitutions of the SH3 ligand decrease its
affinity for the Fyn-SH3 domain [108]. Fyn is localized to
the cytoplasmic leaflet of the oligodendrocyte plasma
membrane, where it participates in numerous signaling
pathways during development of the central nervous
system [112, 113]. Phosphorylation at a polyproline
structure in the Fyn-binding region of MBP affects its
structure.
A study using recombinant murine MBP inserted into
E. coli strains demonstrated conclusively that Aze makes
its way into MBP, substituting for up to three of the
eleven possible proline sites. Molecular modeling of a
proline-rich region of the recombinant MBP illustrated
that misincorporation of Aze at any site would cause a
severe bend in the polypeptide chain, and that multiple
Aze substitutions would completely disrupt the structure
of MBP [114, 115].
A possible concern regarding Aze is that over 90%
of the sugar beet grown in the USA and Canada is
genetically engineered to resist glyphosate. Therefore,
the crops are exposed to significant amounts of
glyphosate. The electronic Code of Federal Regulations
e-CFR 180.364 Glyphosate; Tolerances for Residues,
allows up to 25 ppm residue of glyphosate in dried sugar
beet pulp. In 1999, Monsanto realized that its GM sugar
beet crop well exceeded the upper limit established by
the EPA for glyphosate residues. They requested, and
were granted, a 125-fold increase in the upper residue
limit for dried beet pulp (from 0.2 to 25 ppm). At the same
time, the upper limit for fresh beet was increased fiftyfold
to 10 ppm.
Glyphosate has been shown to increase the risk of
root rot in sugar beet, caused by fungi [116]. Aze has
been demonstrated to have antifungal activity [117].
Plants tend to increase synthesis of toxins under stress
conditions, and it is plausible that an increased potential
for root rot would result in increased synthesis of Aze.
This is especially likely given that plants increase proline
synthesis under a variety of different stress conditions
[118]. However, to our knowledge, whether glyphosate
causes an increase in either proline or Aze synthesis in
sugar beet has not been investigated.
Consumption of milk worldwide is strongly
correlated with MS risk (Spearman’s correlation test =
0.836; P < 0.001) [119]. For the past several decades,
cows’ feed has been supplemented with either beet
Figure 2. Molecular structures of the coding amino acids
proline, L-arginine, glycine and glutamic acid; and their
respective noncoding analogues Aze, L-canavanine,
glyphosate and glufosinate.
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 19
______________________________________________________________________________________________________
JBPC Vol. 17 (2017)
molasses or sugar beet pulp, left as a residue after the
sugar has been extracted [120]. Aze has been
experimentally found in three sugar beet by-products that
are fed to farm animals: sugar beet molasses, and both
shredded and pelleted sugar beet pulp [38]. Casein is
relatively enriched in proline [121]. If cows are exposed
to Aze from the sugar beet, it will likely get inserted by
mistake into casein, causing it to resist proteolysis.
MBP’s critical proline-rich sequence is vulnerable to
misincorporation of Aze. The characteristic plaques of
MS show loss of MBP within lesions in axon sheaths
[107]. It is unclear whether this autoimmune reaction
would arise through molecular mimicry from antibodies to
unmetabolized peptides from casein or as a direct result
of improperly folded MBP due to Aze insertion.
Glyphosate, an analogue of glycine, can be expected
to be found in all tissues, including the milk of all mammals
consuming glyphosate residues in the diet. Radiolabeled
glyphosate studies conducted with lactating goats found
13C and 14C residues of glyphosate (N-phosphono-
methylglycine), N-acetylglyphosate and other radiola-
beled metabolites in milk. Monsanto found daily average
14C residue levels from 19 to 86 ppb, with levels falling
after five days of depuration to 6 ppb prior to sacrifice for
organ examination. Results disseminated by Monsanto
indicate that lactating animals (goats) fed a diet
containing glyphosate and AMPA can be expected to
have measured residue levels in edible tissues and milk
[122]. In 2007 Dupont, in a similar study, examined the
metabolism of N-acetylglyphosate in lactating goats.
Detectable residues of N-acetylglyphosate, glyphosate
and AMPA were detected in milk and other tissues. Milk,
liver and kidney each contained 0.03% of the
administered dose. Individual daily radiolabeled residues
in the milk ranged from 0.030 to 0.036 μg/g [123].
Lactobacillus plays an important rôle in
metabolizing casein in the human gut. A detailed study of
the prolyl aminopeptidase from Lactobacillus revealed
that it is a member of the class of α/β hydrolases.
Multiple sequence alignment has revealed three distinct
highly conserved regions in this family and all three
contain at least two highly conserved glycines [124] that
would be vulnerable to displacement by glyphosate. The
motif gly-x-ser-x-gly-gly characterizes the domain sur-
rounding the catalytic serine residue of prolyl
oligopeptidases in general. The glycine residues in this
motif contribute to the correct positioning of the catalytic
serine with respect to its substrate. A second glycine-rich
domain appears essential to activity, as it likely
corresponds to the oxyanion hole. The function of the
third highly conserved glycine-rich domain, with the motif
asp-x-x-gly-x-gly-x-ser, remains unknown. Lactobacillus
spp. are also highly dependent on manganese to protect
them from oxidative damage, hence glyphosate’s
preferential chelation of manganese likely harms
Lactobacillus [125].
An examination of collagen in the jugular veins of
MS patients undergoing surgical reconstruction revealed
an abnormal collagen structure, characterized by thin,
loosely packed type III fibres [126]. Collagen is rich in
proline. If too many of the prolines in procollagen are
displaced by Aze, the polypeptide does not fold into a
stable triple-helical conformation, which is a prerequisite
for normal secretion of procollagen [127]. This reduces
the release of procollagen and the misfolded molecules
are subjected to proteolysis for recycling. resulting in the
useless expenditure of energy for building and degrading
procollagen molecules. Those that are released can be
expected to produce defective collagen matrices. Collagen
is even more highly enriched in glycine than in proline, as
its core structure consists of a triple peptide repeat, where
glycine is always the third residue of the triplet, and proline
and hydroxproline often occupy the other two positions
[128]. Glyphosate substitution for glycine in structural
proteins; i.e., collagen, elastin, fibronectin and laminin;
would contribute to disrupted folding as well as defective
strength and elasticity.
Conserved prolines also play a crucial rôle in ion
channel gating, the regulation of hypoxia-inducible factor
(HIF) and embroygenesis; in fact, substituting Aze for
proline is a technique used to test whether a particular
proline residue is critical to the protein’s proper
functioning [37].
8.2 Rôle of Acinetobacter and Pseudomonas
aeruginosa in MS
A series of papers by Ebringer et al. have suggested
an important rôle for the Gram-negative bacteria
Acinetobacter and Pseudomonas aeruginosa in MS
[129–131] as well as a proposed link to prion diseases.
Their most recent paper in Medical Hypotheses
presents the evidence to support this idea from multiple
dimensions [130]. First, MS patients were shown to have
elevated levels of antibodies to these two microbes but
not to the common gut microbe E. coli [132, 116]. They
have autoantibodies to MBP and myelin oligodendrocyte
glycoprotein (MOG) [131]. MS patients are also prone to
sinusitis and Acinetobacter is one of the most common
microbes found in nasal sinuses. Ebringer et al. also
proposed that the increased prevalence of sinusitis in
colder climates may explain the geographical distribution
of MS in more northerly latitudes [130]. P. aeruginosa
causes upper respiratory infections and it is among the
microbes that have developed multiple antibiotic
20 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
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JBPC Vol. 17 (2017)
resistance in recent years, presenting a huge problem in
hospital infection [133]. Acinetobacter has also become
resistant to multiple antibiotics [134].
The number of microbial species that can metabolize
glyphosate is quite small. A 1996 study showed that
Acinetobacter is able to fully metabolize both glyphosate
and AMPA and utilize these molecules as a source of
phosphorus [135]. A study of agricultural soil heavily
polluted with glyphosate identified only three species
capable of degrading glyphosate when exposed at a level
of 1000 ppm: Pseudomonas putida, P. aeruginosa and
Acetobacter faecalis [136]. Another study on marine
species identified Pseudomonas as being among the rare
microbial species that can utilize the phosphonate in
glyphosate as a source of phosphorus [137]. It can be
predicted that Pseudomonas and Acinetobacter species
in the nasal or digestive tracts would have a substantial
advantage over other microbes if they can degrade
glyphosate. On the other hand, they would also be heavily
exposed if they actively take it up, and it would not be
unreasonable to assume that some of the glyphosate
might end up in their synthesized proteins by mistake in
place of glycine. Both Pseudomonas aeruginosa and
Acinetobacter strains have recently become a serious
problem in hospitals, and a public health issue, due to their
multiple-antibiotic resistance [138]. Glyphosate has been
shown to induce generic antibiotic resistance in other
microbial species, including E. coli and Salmonella,
through the induction of a generic capability to export
toxic chemicals through efflux pumps [139].
A PEP transferase enzyme synthesized by
Acinetobacter calcaceticus has sequence homology
with a bovine prion sequence, and antibodies against
synthetic peptides containing the structurally related
sequences were found to be significantly elevated in
cattle with bovine spongiform encephalopathy (BSE)
compared to negative controls [140]. Ebringer et al.
(2005) [129] link MS to BSE, also known as “mad cow
disease”, and to the related human disease, Creutzfeldt–
Jakob disease (CJD). Cows suffering from BSE manifest
hindquarters paralysis early after onset, similar to the
mobility issues afflicting MS patients at onset. Ebringer
et al. found elevated levels of antibodies to both
Acinetobacter and Pseudomonas, along with autoanti-
bodies to both white and grey matter components, in
BSE-affected animals, as is also the case for MS [129].
Of particular note are the molecular similarities they
identified between certain peptides found in these two
microbes and peptides in MOG and MBP that are known
to be allergenic. Strikingly, all three of the microbial
sequences they identified and all three of their human
protein analogues contain conserved glycines (Table 5).
a Note that all six peptides have a glycine residue.
Table 5. Amino acid sequences of three peptides from Acinetobacter and Pseudomonas and the
corresponding human peptides from MBP that they mimic.a
Microbe Acinetobacter Acinetobacter Pseudomonas
Protein 3-OACT-A 4-CMLD Gamma-CMLD
Peptide Le
u
-Tyr -Arg-Ala-G l
y
-Lys Ser-Arg-Phe-Ala-Ty
r
-Gl
y
Th
r
-Ar
g
-His-A la-Ty
r
-Gly
MBP Le
u
-Tyr-Arg -Asp-Gly-Lys Se
r
-Ar
g
-Phe-Ser -Ty
r
-Gly Ser-Arg-Phe-Ser-Tyr-Gl
y
MOG is strongly implicated in the disease pathology
of MS; autoantibodies recognizing MOG have been
found in the CNS of MS patients [141]. One of the major
encephalitogenic peptides in MOG is the sequence from
residue 92 to residue 106, which contains a highly
conserved glycine near its centre [142].
Both diabetes and MS are associated with abnormal T-
cell immunity to proteins found in cow’s milk [143]. In a
study conducted in dairy cows by Monsanto in 1973, 14C-
radiolabeled glyphosate was studied in the distribution of
residues in milk, urine, faeces and other tissues of the
lactating cow. Glyphosate contamination of milk ranged
from 9 to 15 ppb with the highest accumulation in the kidney
and rumen fluid (201 ppb and 109 ppb, respectively) [201].
An epitope of bovine serine albumin found in milk that is
linked to MS but not to diabetes is BSA193. It shows
structural homology with exon 2 of MBP through the peptide
sequence GLCHMYK. Note that the first peptide in this
sequence is glycine. Exon 2 is a target peptide in both MS
autoimmunity and in experimental autoimmune encephalitis
(EAE), an animal model of MS [144–146]. Exon 2 of MBP
is implicated in remyelination [144]. Its expression is largely
restricted to the developing brain and to areas of myelin
reconstruction, notably MS lesions [147].
The gly-ser-gly-lys tetrapeptide is highly conserved
among MBPs from multiple species [148]. The serine
in this sequence is the site of attachment of
polyphosphoinositide. The highly conserved nature of this
sequence suggests that the phospholipidation of MBP is
important biologically. Substitution of glyphosate for either
of the glycines would likely disrupt this modification.
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 21
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JBPC Vol. 17 (2017)
9. MMR VACCINE AND AUTISM
In this section, we make a case for a direct link between
the measles, mumps, and rubella (MMR) vaccine and
autism, via autoantibody induction through molecular
mimicry. In a paper provocatively titled, “Peptide cross-
reactivity: the original sin of vaccines”, Kanduc makes
the point that massive cross-reactivity between antigens
in vaccines and similar sequences in human proteins
makes it almost inevitable that vaccines lead to
autoimmune disease through molecular mimicry [149].
Reported post-vaccination autoimmune diseases include
systemic lupus erythematosus, rheumatoid arthritis,
inflammatory myopathies, multiple sclerosis, Guillain–
Barré syndrome and vasculitis [150].
It is becoming increasingly acknowledged that
autism may be an autoimmune disease. Family members
of autistic children have a significant increased risk to
other known autoimmune diseases such as
hypothyroidism, rheumatic fever and multiple sclerosis
[151]. Several studies on both humans and monkeys have
revealed a potential link between maternal antibodies
directed against specific foetal brain proteins and a future
autism diagnosis in the foetus [152–155]. Furthermore, it
has already been demonstrated that vaccines are capable
of inducing autoimmune antibodies against proteins in the
brain. The narcolepsy epidemic in Europe following an
aggressive immunization campaign against the H1N1 ’flu
virus was eventually conclusively resolved as being
attributed to autoimmune reactions to the hypocretin
receptor through molecular mimicry from a peptide in the
surface-exposed region of the influenza nucleoprotein A
that was present in the H1N1 vaccine [156] (hypocretin
is an important regulator of sleep).
Much controversy surrounds the concept that the
MMR vaccine may be contributing to the autism
epidemic in the USA and elsewhere. In an immune-
compromised child, the live measles virus from the
vaccine is capable of infecting the brain and sustaining a
chronic measles infection, resulting in loss of neurons,
eosinophilic intranuclear inclusions and gliosis, a condition
termed “subacute measles encephalitis”. This can result
in a seizure disorder and developmental delay in language
and motor skills (as was clearly observed in a case study
involving an HIV-positive 2-year-old boy [157]).
Singh et al. have published a series of papers over
the past two decades [14, 158–160] proposing that there
is a subpopulation among the autism community who can
be characterized as suffering from “autoimmune autistic
disorder” [14]. The 1998 study by Singh et al. found that
90% of measles-IgG-positive autistic sera were also
positive for anti-MBP antibodies, supporting the hypothesis
that a virus-induced autoimmune response may be
causal in autism [158]. A follow-on serologic study of
antibodies to viruses associated with autism published in
2003 revealed a statistically significantly elevated level of
measles antibody in children with autism compared to
their siblings (P = 0.0001) or to unrelated children (P =
0.003), but not with antibodies to mumps or rubella [159].
In a later study, 60% of 125 autistic children had
significantly elevated levels of antibodies to measles
haemagglutinin unique to the MMR strain of the virus,
compared to the 92 control children [160]. Over 90% of
the children who had elevated antibody levels also tested
positive for MBP autoantibodies. It was suggested that
this could be linked to virus-induced autoimmunity
through mimicry.
In fact, there is a sequence homology of 78%
between a peptide sequence from MBP
(EISFKLGQEGRDSRSGTP) and one found in a measles
virus protein, MP3 (EISDNLGQEGRASTSGTP) [161,
Table 2, p. 7]. Three of the matches between these two
sequences are glycines. Measles virus-neutralizing
antibodies are mainly directed to haemagglutinin,
implying that it is essential for acquired immunity from
the vaccine [162]; yet over-production, particularly if
the virus penetrates the blood–brain barrier, runs the
risk of inducing an autoimmune response to the myelin
sheath. In fact, high measles antibody titres have been
previously linked to MS [163].
Gonzalez-Granow et al. found high titres of autoanti-
bodies in both the IgG and IgA classes specific to MBP in the
serum of patients with autism [15]. The IgA antibodies in
particular were shown to act as serine proteinases to
degrade MBP in vitro. They also induced a decrease in
long-term potentiation in perfused rat hippocampi.
Reduced long-term potentiation in the hippocampus is a
feature of autism, as has been clearly demonstrated in
studies using mouse models of autism [164].
Dr Andrew Wakefield was the first to reveal a
possible connexion between MMR and autism. His
controversial Lancet paper, published in 1998 and then
later retracted, proposed that this vaccine caused an
acute reaction in children with gut dysbiosis (abdominal
pain, diarrhoea, food intolerances, bloating etc.) [9]. The
paper reported on a group of 12 children who had
experienced developmental delay following an MMR
vaccine and who were diagnosed with autism. These
children suffered from rash, fever, delirium and seizures
following the vaccination with MMR. He and several
colleagues later published additional papers elaborating
the hypothesis that dysbiosis in the gut, combined with
impaired protein hydrolysis, leads to autoimmune lesions
in the duodenum that are associated with extensive colonic
lymphoid hyperplasia. The release of undigested peptides
22 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
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JBPC Vol. 17 (2017)
into the vasculature across a leaky gut barrier and,
ultimately, from the vasculature across a leaky blood–brain
barrier, could induce encephalopathy [165–167].
In an epidemiological study from 1998, encepha-
lopathy was clearly demonstrated as an acute reaction to
measles vaccine, where 48 cases were found following
vaccination, with no cases identified after administration
of either monovalent mumps or rubella [168]. Among
these 48 children, eight died, and the remainder experienced
mental regression, chronic seizures, movement disorders
and sensory deficits in the subsequent months.
The FDA’s vaccine adverse event reporting system
(VAERS) database is a valuable tool for uncovering
trends in vaccine adverse reactions. Our earlier studies on
VAERS comparing MMR with an age-matched, equal-
sized distribution of all other vaccines showed a
significant association of MMR with autism (P < 0.007)
[169]. This was puzzling, because MMR has never
contained either aluminium or mercury, the two prime
candidates for the kind of neurological damage that might
lead to autism [170–174]. Strong associations also appeared
with fever and rash. In that paper, we proposed that the
adverse reaction might be caused by the acetaminophen
administered to the child to try to curb the seizures.
Since glyphosate usage on crops has gone up
dramatically since the GM Roundup Ready crops were
first introduced in 1996, we decided it would be
worthwhile to compare the early data on MMR in
VAERS with the later data. We defined a cutoff date on
1 January 2003, such that the events where MMR was
included as an administered vaccine could be separated
into “early” and “late”, based on whether they were before
or after that date. Each dataset represented a 13-year
interval. We found 10
639 events in the early set and
19
447 events in the late set; thus, the raw number of
events nearly doubled in the later years.
We also tabulated the frequency of different adverse
reactions in the two sets, and used a standard statistical
analysis to compute the significance of any differences
observed: we randomly down-sampled both sets as
needed such that there was an identical total count and an
identical distribution over age in the two datasets. Results
were surprising: many symptoms associated with atopy or
with an allergic reaction were significantly higher in the
later set, and “hospitalization” was highly significantly
overrepresented in the later set [Table 6]. Other
overrepresented symptoms included seizures, dyspnea,
hyperventilation, asthma, eczema, autism, hives,
anaphylatic [shock], and irregular heart rate. Interestingly,
the early set had more frequent occurrences of joint pain
and arthritis, suggesting that the toxic elements in the
vaccine impacted the joints rather than the brain.
Table 6. Frequency of various adverse reactions to MMR before and after January 2003 [US FDA, VAERS]. The P-values
were computed according to a χ2 goodness-of-fit test.
More common
b
efore 2003
Reaction Count < 2003 Count ≥ 2003 P-value
Arthritis 52 18 0.045
Joi nt pain 175 75 0.012
More common after 2002
Reaction Count < 2003 Count ≥2003 P-value
Hospital 132 423 0.00041
Seizures 314 534 0.0 055
Dyspnea 139 279 0.0086
Hives 444 654 0. 011
Anaphyl acti c 28 91 0.017
Eczema 10 47 0.028
Autism 105 1 84 0. 031
Hyperventilatio
n
1 8 57 0.035
Genera l infection 77 136 0.044
Asth ma 22 58 0. 046
Immunogl obu lin G 0 17 0.048
Ear infection 32 72 0.048
Hear t r ate irr egular 11 39 0. 049
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 23
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JBPC Vol. 17 (2017)
To our knowledge, there have been no significant
changes to the formulation of MMR since its
introduction. The explanation for the significant changes
in adverse reactions must, therefore, lie in external
factors, one of which is likely to be glyphosate. We
suggest that both chronic exposure to glyphosate from
food, water and air and direct exposure to glyphosate
residues in the vaccine are relevant factors. A child with
a disrupted gut microbiome due to chronic glyphosate
exposure will also suffer from a leaky blood–brain barrier,
and this will lead to a much greater possibility of measles
antigenic proteins entering the brain and causing
anaphylaxis and seizures.
The measles virus is a member of the family of
paramyxoviruses, which have two highly-conserved
glycine residues at positions 3 and 7 in the hydrophobic
fusion peptide (FP) region of the viral fusion-mediating
glycoproteins [175]. This FP region is the most highly
conserved region of the glycoproteins, and it plays a
critical rôle in destabilizing the membrane of the host cell
to gain entry. Substitutions of other amino acids for either
the G3A or G7A glycines caused increases in both cell–
cell fusion and the reactivity of the protein to antibodies,
leading to both a higher infection rate and increased
chances for an autoimmune reaction. Glyphosate
substitution is likely to do the same, as well as leading to a
form of the protein that would resist proteolysis.
The FPs of both the influenza virus and human
immunodeficiency virus (HIV) gp41 contain numerous
glycine residues at regular intervals, with glycine overall
making up 29 and 26%, respectively, of the total peptide
sequence [175]. Optic neuritis, an immune-mediated
demyelinating injury of the optic nerve, has been
recognized as a side effect of the influenza vaccine that
can lead to blindness [176].
10. OTHER AUTOIMMUNE DISEASES
10.1 Neuromyelitis optica and aquiporin
Neuromyelitis optica is a rare severe inflammatory
demyelinating disorder of the central nervous system,
which is related to multiple sclerosis but distinctly
different and manifested mainly by paralysis and optic
nerve damage [177, 178]. It has been conclusively
demonstrated that this condition is caused by an
autoimmune reaction to aquaporin-4, which is highly
expressed in the astrocyte membrane [177, 178].
Aquaporins are important membrane proteins,
which can transport water molecules through pores into
the cell while excluding protons [179]. They are highly
expressed by astrocytes, one of whose rôles is to
mediate water flow among the vasculature, the
cerebrospinal fluid and the lymph system [178]. Thus,
aquaporins are implicated in brain oedema [180]. Plants
produce aquaporins as well, and mimicry between plant
and human aquaporins has been proposed as a
mechanism for the development of an autoimmune
sensitivity to this protein [181]. Plants considered to
show aquaporin mimicry notably include corn and soy as
well as tomato, tobacco and spinach [182].
Autoimmune sensitivity to aquaporin has also been
found in association with MS [182]. Vojdani et al. found
significant elevations in antibodies against both human
and plant aquaporin 4, in addition to antibodies against
MB, MOG and S100 calcium-binding protein B (S100B)
in patients suffering from MS.
Among the aquaporins, aquaporin-6 is unique in that
it operates as an anion channel instead of as a water
channel. Analysis of the peptide sequence in comparison
to other aquaporins reveals that aquaporin-6 has an
asparagine substituted in place of a glycine at residue
60. This one small difference completely changes the
way the molecule behaves in the membrane. A glycine
at this position is conserved among all the other
aquaporins. Furthermore, aquaporins are constructed of
α-helices, and there are three sites where the helices
cross. Highly conserved glycine residues are found at all
three sites [57, 183].
Aquaporin is also found in bacteria, although
homology with human aquaporin is only about 20%. The
bacterial aquaporin is a 27 kDa trypsin-resistant protein
called aquaporin-Z, which was originally described in E.
coli [184]. Sequence analysis conducted by Ren et al.
[185] revealed four regions where homology was
considerably stronger (90%, 60%, 50% and 45%
respectively). They convincingly showed cross immunore-
activity between the human and bacterial versions of the
protein. Antibodies to aquaporin Z bind to astrocytes,
activate complement, and cause death.
Ren et al. [185] identified all the residues where the
bacterial and human peptides were identical (Fig. 1 in
[185]). A tally of counts reveals that glycine was by far
the most common among these matched residues,
representing 14 of the total 66 matches. The second most
common amino acid was lysine with 8 matches. Alanine,
isoleucine and valine had 7, 5 and 4 matches respectively,
and all other amino acids had less than four.
Thus, it appears that glyphosate-substituted trypsin-
resistant aquaporin from both gut microbes and from GM
glyphosate-resistant corn and soy foods are plausible
sources of antigens that could induce neuromyelitis optica
and contribute to the disease process in MS through
misincorporation.
24 A. Samsel and S. Seneff Glyphosate and autoimmune diseases
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JBPC Vol. 17 (2017)
10.2 Type 1 diabetes
Type 1 diabetes is considered a genetic disease, but its
incidence has been increasing by 3–4% worldwide every
year in the recent past [186, 168]. Although an environ-
mental component is highly suspected, environmental
factors have not yet been identified. An increased
incidence of type 1 diabetes is associated with both MS
[187] and autism [188]. The disease is characterized by
an autoimmune reaction to various proteins expressed in
the pancreatic islet cells. Specifically, antibodies against
glutamic acid decarboxylase (GAD65) are often found
[189]. Cross-reactivity with proteins from foods and
microbes in the gut are both possibilities.
One microbe that may be inducing antibody
production through mimicry is Mycobacterium avium
paratuberculosis (MAP). Blast analysis revealed 75%
homology between a previously identified antigenic region
of GAD65 [190] and a MAP heat-shock protein (HSP65)
[189]. The specific 16-residue matched sequence in
HSP65 centrally contains a pair of glycines which could be
substituted by glyphosate to cause resistance to
proteolysis. This microbe has been linked to numerous
other human diseases including ulcerative colitis, irritable
bowel syndrome, sarcoidosis, Hashimoto’s thyroiditis, MS
and autism [188]. With respect to MS and autism, cross-
reactivity between HSP65 and MBP through mimicry may
provide the link.
Patients with type-1 diabetes commonly have an
antibody reaction to bovine serum albumin, a component
of cows’ milk [191]. The hypothesized explanation is an
autoimmune reaction to a beta-cell specific surface
protein through mimicry.
Insulin-derived amyloidosis is a condition that can
develop following long-term insulin therapy, whereby an
“insulin ball” develops at the site of injection. This hard
mass has been analysed and found to contain
accumulations of insulin fibrils reminiscent of amyloid
β-plaque in the Alzheimer’s brain. Insulin amyloidosis is
more common for animal (cows and pigs)-derived than
human-derived insulin products. Nowadays, cows and
pigs are chronically exposed to glyphosate in their feed.
The rôle of glycine residues in proteins may indeed be to
protect from aggregation into amyloid fibrils [192].
Substitution of glyphosate for any of these conserved
glycines would therefore tend to promote amyloidosis.
Glutamic acid and glycine are by far the largest
component amino acids of bovine proinsulin and make up
25% of the amino acid residues in the molecule [193].
The same is true for human insulin, which differs very
little from the animal versions. The herbicide glufosinate
is a natural noncoding amino acid analogue of glutamic
acid (Fig. 2). Substitution of either glufosinate for
glutamic acid or glyphosate for glycine in insulin is likely to
impair its function, and may also lead to amyloidosis.
The widespread appearance of glyphosate-resistant
weeds among the glyphosate-resistant crops has forced
some farmers to turn to glufosinate as the herbicide of
choice [194]. Glufosinate-tolerant corn and soybean have
been available on the US market since their approval by
the USDA in 1995 and 1996, respectively. A tri-resistant
form of soybean tolerant of glyphosate, glufosinate, and
2,4-D was approved by the FDA in September 2014.
Dual resistance to glufosinate and glyphosate in corn was
approved in November 2015.
10.3 Coeliac disease
Coeliac disease and, more generally, gluten intolerance,
have reached epidemic proportions in the USA in the past
decade [195]. Wheat grown there is being routinely
sprayed with glyphosate for staging and desiccation just
before harvest. This practice clears the field of weeds
prior to harvest and planting of the next crop, but
increases the amount of residual glyphosate in the grain.
The practice has been increasing in popularity in step
with the increase in gluten intolerance. Glyphosate is
systemic in the plant and enters the seed as the plant dies,
hence eventually ending up in wheat-based foods.
Proline residues make up 20% of the first 100 amino
acids of both α- and γ-gliadins [54]. Related proteins from
rye and barley are also unusually proline-rich [56]. As we
implied earlier, proline is inaccessible to most digestive
proteases because the bond between the peptide nitrogen
atom and the side group complicates hydrolytic attack.
As a consequence, specialized prolyl aminopeptidases
detach the amino-terminal proline from a peptide. These
enzymes depend on manganese as a catalyst, and
manganese is one of the metals most dramatically
affected by glyphosate chelation [125]. Unhydrolysed
gliadin peptides bind to HLA-DQ molecules (receptors on
antigen-presenting cells) and trigger pathogenic T-cell
responses [196]. Genetic variants of HLA-DQ are linked
to both coeliac disease and type 1 diabetes [197, 198].
Analysis of the X-ray crystal structure of a human
cytosolic prolyl aminopeptidase worked out in 2008
revealed that it is a dimer with a dependency on two
manganese ions as the catalytic centres [199]. The full
sequence of the catalytic domains of six prolyl peptidases
from both human and microbial species is shown in Fig. 6
in ref. 199. Six of the twenty sites of fully conserved
residues across all species were glycine residues, three
were histidine, two were tyrosine and two were proline.
The remaining seven were seven different amino acids.
Glyphosate and autoimmune diseases A. Samsel and S. Seneff 25
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JBPC Vol. 17 (2017)
11. CONCLUSION
In this paper, we have shown that widespread
misincorporation of glyphosate for glycine during protein
synthesis could explain the aetiology of multiple
autoimmune diseases that are currently increasing in
incidence in the USA. Misincorporation is plausible by
analogy with multiple known toxins produced by
organisms in defence against pathogens, including Aze,
BMAA, L-canavanine and glufosinate, which work in a
similar manner. We have shown that proteins from foods
such as milk, wheat and sugar beet, as well as peptides
derived from microbes resident in the gut or nasal tract or
introduced iatrogenically through vaccination, are all
potential causes of autoimmune disease induced through
molecular mimicry. It is highly significant that two
microbes linked to MS through molecular mimicry are
among the very few microbes that can fully metabolize
glyphosate. Using the VAERS database, we have shown
that severe adverse reactions to the MMR vaccine have
increased significantly over the past decade in step with
the increased use of glyphosate. Glyphosate in MMR
may originate from growth of the live virus on culture
materials derived from glyphosate-exposed animals and/
or from gelatin used as an excipient stabilizer. We have
confirmed the presence of glyphosate contamination in
MMR and in many other vaccines where the live virus is
cultured in eggs, bovine protein or gelatin, or where
animal products are used as an excipient component.
Notably, some vaccines prepared without live culture on
gelatin were free of glyphosate contamination.
Substitution of glyphosate for glycine during protein
synthesis could yield a peptide that resists proteolysis,
making it more likely to induce an immune response.
Furthermore, enzymes involved in proteolysis are likely
to be disrupted due to their confirmed contamination with
glyphosate. A non-exhaustive list of possible diseases
that can be attributed to this mechanism include autism,
multiple sclerosis, type 1 diabetes, coeliac disease,
inflammatory bowel disease and neuromyelitis optica.
ACKNOWLEDGMENT
This research is supported in part by Quanta Computers,
Taiwan, under the auspices of the Qmulus program.
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