BIOMEDICAL
The Science Behind
Treatment Strategies:
Underlying physiological
conditions, biomarkers,
and the Defeat Autism
Now! approach
BY ELIZABETH MUMPER, MD
Dr. Mumper is the Medical Director of the Autism
Research Institute and founder of the RIMLAND Center.
I
n the September issue of The Autism File,
I wrote about vicious cycles in autism and
how rewarding it can be to restore those
cycles to better function. This month, I will
share some of the scientific literature behind
our concepts of how the gut, brain, and
immune system are interconnected and how
all are influenced by metabolic factors.
Intestinal factors in autism
Let’s start with the role of the gut. We
coexist with a hundred trillion bacteria that
live in our gut. These bacteria outnumber
our own body cells ten to one! At birth, our
guts are sterile, but quickly are colonized
with our mother’s gut, vaginal, respiratory,
and skin flora, as well as germs in the
environment. During the first two years of
life, our colonies of gut bacteria become
established and play a major role in
modulating our immune systems. At least
70% of our immune defenses are in our gut,
so early disruption in the establishment of
gut flora or early inflammation can have
far-reaching consequences for our ability
to fight infections and avoid allergies. Over
time, our bodies develop oral tolerance to
ingested foodstuffs (so our immune systems
don’t attack the food we eat) yet develop
appropriate immune responses to pathogenic
bacteria.
Dysbiosis exists when our good flora
are outnumbered by the overgrowth of
microorganisms (that may even be of low
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virulence) that induce pathology by altering
the immune response or nutritional status
of the host. Dendritic cells in the intestine
are exposed to gut flora and ingested
microorganisms and can be activated
to induce T-cell responses and produce
chemokines, which act as messengers that
inflammation is present and other immune
cells need to mobilize to help. Many
clinicians utilizing the Defeat Autism Now!
approach recommend probiotics early on in
the treatment of children with autism:
“…Probiotics possess the ability to modulate
dendritic cell surface phenotype and
cytokine release in granulocyte-macrophage
colony-stimulating factor-stimulated bone
marrow-derived dendritic cells. Regulation
of dendritic cell cytokines by probiotics may
contribute to the benefit of these molecules
in treatment of intestinal diseases.”
(Drakes, 2004) Fedorak enumerated several
mechanisms of action for probiotics,
including “(1) competitive exclusion,
whereby probiotics compete with microbial
pathogens for a limited number of receptors
present on the surface epithelium; (2)
immunomodulation and/or stimulation of an
immune response of gut-associated lymphoid
and epithelial cells; (3) antimicrobial activity
and suppression of pathogen growth; (4)
enhancement of barrier function; and (5)
induction of T cell apoptosis in the mucosal
immune compartment.” (Fedorak, 2004).
Let’s examine some evidence that shows
that the gut flora in children with autism
is different from that of neurotypical
children. Finegold and colleagues have
performed elegant studies demonstrating
that both clostridial colony counts and the
number of clostridia species were higher in
the stools of children with autism than in
controls. Children with autism had significant
numbers of non-spore-forming anaerobes
and microaerophilic bacteria in gastric and
duodenal specimens, which were strikingly
absent in control children. “These studies
demonstrate significant alterations in the
upper and lower intestinal flora of children
with late-onset autism and may provide
insights into the nature of this disorder.”
(Finegold, 2002).
Children with autism are demonstrated to
have high rates of gastrointestinal disease.
A study at the University of Maryland
demonstrated by endoscopy that of 36
children with autism, 69.4% had reflux
esophagitis, 41.6% had chronic gastritis, and
66.6% had chronic duodenal inflammation
(Horvath, 1999). Further studies showed
chemical messenger and white blood cell
(in this case, lymphocytes) evidence of
inflammation, with significant increases in
CD3(+) and CD3(+)CD8(+) intraepithelial
lymphocytes and CD3(+) lamina propria
lymphocytes in children with autism
compared to developmentally normal non-
inflamed control children (p<0.01). CD3(+)
CD4(+) IEL (intraepithelial lymphocytes) and
ISSUE 30 2009
LP (lamina propria) CD19(+) B cells were
dramatically increased in affected children
compared to both non-inflamed and inflamed
control groups, including inflammatory bowel
disease (p<0.01) (Ashwood, 2003). This
study demonstrated that some children with
autism have evidence of inflammation and
immunopathology anywhere from throat to
anus. These children deserve to have their
gastrointestinal problems investigated and
treated and not written off as “just part of
the autism.” Dr. Horvath went on to suggest
that “treatment of the digestive problems
may have positive effects on their behavior.”
(Horvath, 2002).
Other researchers demonstrated a
lymphocytic colitis (inflammation of the
intestine associated with lymphocytes,
a type of white blood cell) in autistic
children that was not as severe as classical
inflammatory bowel disease (such as Crohn’s
or ulcerative colitis). Mucosal gamma
delta cell density and basement membrane
thickness were significantly increased, more
than in patients with inflammatory bowel
disease. CD8(+) density and intraepithelial
lymphocyte numbers were higher than those
in the Crohn’s disease, lymphoid nodular
hyperplasia (LNH), and normal control
groups. Epithelial glycosaminoglycans were
disrupted and the authors concluded they
had found “increasing evidence for gut
epithelial dysfunction in autism.”
(Furlano, 2004).
Pathologic changes observed in intestines
of children with autism include: increased
size and number of lymphoid follicles,
particularly in the ileum (Wakefield, 2000;
Ullman, 2002); crypt cell proliferation
(Torrente, 2002); neutrophil and eosinophil
infiltration of the intestinal lumen (Krigsman,
personal communication); thickening of
Endoscopy
 Submucosal hemorrhages of
the colon
 
Lymphoid Nodular Hyperplasia
(LNH) Persistent
 
Esophagitis with friability and
cobblestoning of the mucosa
and blunting of vascular
markings
the basement membrane (Furlano, 2004);
ulceration of the epithelium (Balzola, 2005);
and decreased brush border digestive
enzyme function (Kushak, personal
communication). These pathology findings
are consistent with the establishment of a
chronic inflammatory condition. Evidence
of autoimmunity includes IgG deposition
on the “basolateral epithelial surface in
23/25 autistic children, co-localizing with
complement C1q.” (Torrente, 2002). We
will return to the issue of autoimmunity in
children with autism and their families later.
Food intolerance in autism
Can gut dysfunction affect brain function?
Dr. Hadjivassiliou investigated the frequency
of antigliadin antibodies and celiac disease
in neurological patients. Using ELISA
techniques, he assessed serum IgG and
IgA antigliadin antibodies in 53 patients
with neurological dysfunction of unknown
cause despite full investigation (25 ataxia,
20 peripheral neuropathy, 5 mononeuritis
multiplex, 4 myopathy, 3 motor neuropathy,
2 myelopathy) and 94 patients with a specific
neurological diagnosis. They found that
gluten sensitivity was common (30 of 53
of their cases) in patients with neurological
disease of unknown cause (Hadjivassiliou,
1996). One of our teenage patients has only
a few words, and does better neurologically
when he stays on a casein-free, gluten-free
diet. My staff can tell if he has gone off his
diet by his ataxic gait when he comes to our
office.
Most children with cow’s milk intolerance
have chronic diarrhea, but Iacano and
colleagues hypothesized that cow’s milk
intolerance could also cause painful perianal
lesions and, therefore, constipation. A
double blind placebo controlled crossover
study comparing cow’s milk to soy confirmed
their hypothesis and provided the treatment
option of discontinuing cow’s milk in children
with chronic constipation unresponsive
to laxatives (Iacano, 1998). It seems like
blasphemy to suggest that certain children
should be taken off milk, but many infants
with chronic diarrhea, constipation, allergies,
recurring otitis media, and children with
autism improve off dairy products. Children

Gut Disorders in Autism

Historical clues: Physical/Lab clues:
 Difficulty breastfeeding Food  Abnormal stools
 Peristent Colic Gut
sensitivities inflammation  Abnormal cytokine profile
 Gastro-esophageal reflux Malabsorption  Lymphonodular
 Infantile eczema hyperplasia of ileum
 Food sensitivities  Esophagitis
 Failure to thrive Abnormal  Gastritis
 Frequent antibiotics (abnormal flora) intestinal
permeability
 Abnormal posturing
 Self injurious behavior
 Poor sleep LNH

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 BIOMEDICAL
need to have adequate protein, vitamin D
and calcium when on a casein-free diet.
Marked improvement in behavioral symptoms
of patients with autism was noted after eight
weeks on an elimination diet without cow’s
milk and other foods to which the children
had positive skin tests. High levels of IgA
antibodies to casein, lactalbumin, and beta-
lactoglobulin and IgG and IgM antibodies to
casein were significantly higher in cases than
controls (Lucarelli, 1995).
The “hygiene hypothesis” suggests that
children who are not exposed to enough
germs in infancy are at risk for increased
allergy. Infants with a family history of atopic
allergy had a 100% higher prevalence of
allergies and asthma at two years old than
infants who received a Lactobacillus probiotic
(Hanaway, personal communication). One
way that maternal nutrition can affect
offspring is that mothers who have low levels
of omega-3 essential fatty acids and high
levels of omega-6 essential fatty acids have
infants more likely to develop problems with
oral tolerance and, therefore, more likely to
have food sensitivities.
The gut brain connection
There are several interesting animal
models that demonstrate the intimate
connections between the gut and the brain.
In one set of experiments, a metabolite
of gut flora (clostridia, which Finegold
has demonstrated in several studies to
be common in children who have autism)
induced neuroinflammation and oxidative
stress. MacFabe and colleagues infused
propionic acid into the ventricles of rats
and observed autistic behaviors such
as obsessive compulsiveness and social
withdrawal. These behaviors appeared
and disappeared as the propionic acid
infusion was turned on and off. Pathology
Immune Dysregulation in Autism
Clinical clues to immune dysregulation
 Allergic shiners
 Eczema
 Fungal skin infections
 Oral thrush
 Molluscum contagiosum
 Warts
Treatment of oxidative stress in autism, as in many chronic diseases, is absolutely crucial.
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analysis of the rat brains showed evidence
of neuroinflammation (MacFabe, 2008).
Welch and her colleagues showed that
experimentally induced inflammatory bowel
disease in an animal model was associated
with secondary neuropathological changes
and that those changes could be treated with
neuropeptides, such as secretin and oxytocin
(Welch, 2005).
Seizures in autism
Seizures are a common disorder in children
with autism; diagnosis requires a high index
of suspicion. Some inattentive or “brain fog”
behaviors may be due to absence seizures.
Diagnosis of seizure disorders in children
with autism may be missed on standard EEGs
and may require 24-hour EEGs. Seizures
may be a complicating manifestation of
neurologic dysfunction in children who
are slow to respond to nutriceuticals and
standard therapies. Anticonvulsants of
particular benefit in children with autism
include gabapentin (neurontin) and
lamotrigine (lamictal).
Neurontin may help with self-stimulatory
behaviors as well as seizures, and lamictal
has the advantage of blocking glutamate in
addition to stabilizing mood.
Neuroinflammation in autism
A watershed article that my colleagues at
the Autism Research Institute have come
to rely on heavily for our understanding of
brain dysfunction in autism was done by
Vargas and colleagues at Johns Hopkins.
They examined the brains of 11 autistic
patients aged 4–45 years and found
degeneration in the cerebral cortex, white
matter, and, particularly, the cerebellum.
Their elegant neuropathologic studies, using
cytokine profiles, immunocytochemistry,
and enzyme linked immunosorbent assays,
Chronic Viral TH1 to TH2
and fungal shift
infections
Increased
autoimmunity
and allergy
demonstrated neuroglial activation and
activation of the innate immune system (the
nonspecific first responders of our immune
systems), but not the adaptive immune
system (which creates antibodies targeted
specifically to the offending antigen). There
was marked activation of the microglia and
astroglia. Microglia are the immune cells
in the central nervous system that exist
throughout the brain in a resting state
under normal conditions. With an insult
to the brain, these resting cells assume an
ameboid shape and are able to move and act
as scavengers of the injured cells. Human
microglia have receptors for many cytokines,
including interleukins (IL-6, IL-8, IL-10,
IL-12), tumor necrosis factor alpha (TNF
alpha), macrophage chemotactic protein-1
(MIP-1), and monocyte chemotactic
protein (MCP-1). Activated microglia
express anti-inflammatory cytokines (such
as IL-10), immunomodulatory cytokines
(such as IL-5 and IL-12), and inflammatory
cytokines (such as IL-6 and tumor necrosis
factor alpha). In the Vargas study, the most
prevalent cytokines in brain tissue were
macrophage chemoattractant protein (MAP)
and tumor growth factor beta 1, both of
which are derived from neuroglia. Their
study confirmed Purkinje cell loss, as noted
on previous studies, and demonstrated a
microvasculitis (Vargas, 2005).
Autoimmunity in autism
Other scientists have demonstrated
autoantibodies to the brain in autism,
Landau-Kleffner variant, and other
neurologic disorders; anti-brain antibodies
were present in 27% of the children with
autism compared to 2% of control children
(Connolly, 1999). Families of children
with autism have a higher incidence of
autoimmune disease than do families
Evidence of Immune Dysregulation
 Increased IgE
 Autoimmunity markers
 Abnormal natural killer cell function
 IgA deficiency
 Lymphopenia
 T cell abnormalities
ISSUE 30 2009
of neurotypical children (Comi, 1999).
Activation of cellular immunity in children
with autism is supported by the observed
increase in urinary neopterin in autism and
suggests “involvement of autoimmunity
in the pathogenesis of autism.” (Messahel
1998).
The cholinergic nervous system
The cholinergic anti-inflammatory
pathway plays an important role in
neuroimmunomodulation. The efferent
vagus nerve plays an important role in
controlling inflammation through interaction
with nicotinic acetylcholine receptors
expressed on macrophages (Pavlov, 2003).
Galantamine and nicotine have been shown
to have a synergistic effect on microglial
activation (Gunta, 2004). Since chronic brain
inflammation is a final common pathway
in many neurodegenerative diseases,
and Vargas and colleagues demonstrated
microglial activation (which is central to
the process of chronic brain inflammation)
in the brains of patients with autism, novel
therapeutic strategies may be developed
to modulate microglial activation. As these
strategies are tested, it will be prudent
to respect the delicate balance between
enough inflammation to protect the host and
overactivity of cytokines in ways that lead to
chronic inflammation.
Anti-inflammatory strategies
Therapeutic strategies to address the
ongoing inflammation that, based on the
evidence, seems to be occurring in the brains
of children with autism include nonsteroidal
anti-inflammatories (NSAIDS), which carry
side effects such as abdominal pain and
diarrhea, and which may worsen existing
pathologies like gastritis and esophagitis.
Steroids may be effective, but the myriad
side effects when used long-term often
prohibit their use. Practitioners associated
with Defeat Autism Now! have used novel
agents such as pioglitazone (Actos),
minocycline, and spironolactone (aldactone).
Actos (pioglitazone) inhibits NF-
kappaB, which is involved in immune and
inflammatory responses, cellular growth,
and apoptosis. NF-kappaB proteins are
transcription factors persistently active in
chronic inflammation and neurodegenerative
diseases. One therapeutic approach to
the inflammatory-autoimmune conditions
in children with autism would be the use
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Dramatically, symptoms were reversed in each case by various
molecular interventions, even in older animals! These experiments
must be recognized as changing the paradigm that such conditions
are beyond intervention; potentially, the quality of life for the child
and family can be significantly improved.
of agents, such as Actos, that inhibit
nuclear factor kappa B (NF-KB) or anti-
leukotrienes. Boris and colleagues have
studied pioglitazone at doses of 30 mg/day
for children 10-20 kg, 45 mg/day for 20-30
kg, and 60 mg/day for children over 30
kg (Boris, 2007). A new black box warning
for Actos will warn that this class of drugs
increases a person’s risk of congestive heart
failure and fluid retention.
Minocycline is an antibiotic that also has
immunomodulating effects on microglia.
Anecdotal experience suggests improvements
in a subset of children with autism; this
therapy for neuroinflammation is currently
being evaluated in children with autism by
the NIH (National Institutes of Health).
Interferon gamma, testosterone, TNF
alpha, MCP 1, in addition to inflammation
and oxidative stress, have been elevated in
children with autism. Spironolactone has
been shown to decrease all of the above, so
it has been proposed as a potential hormonal
and immunologic intervention for a subset of
children with autism (Bradstreet, 2007).
Mitochondrial dysfunction in autism
Mitochondrial dysfunction is more common
than initially recognized in children with
autism; in addition to classic congenital
mitochondrial disorders associated with low
tone, it behooves us to consider acquired
mitochondropathies.
You may remember mitochondria
being described in high school biology
as the powerhouses or batteries of the
cell. Their primary role is to make ATP
(adenosine triphosphate) for energy,
which is used to build and maintain our
bodies. Mitochondria have their own DNA.
Mitochondria are important in regulating
apoptosis, which is programmed cell death,
a crucial process in living beings where cells
are constantly turning over. Mitochondria
in the liver help detoxify ammonia, which
can accumulate during the metabolism of
protein. Mitochondria also help produce
heme via the porphyrin pathway. Carnitine
levels in 100 children with autism showed
significantly low levels of carnitine, slightly
elevated lactate, and significantly high
levels of ammonia and alanine levels, all
of which are consistent with mitochondrial
dysfunction (Filipek, 2004). Potential
strategies for helping mitochondrial function
include: antioxidants, L-carnitine, CoQ10
(UBHQ), and alpha-ketoglutarate.
Genetics and autism
Autism has classically been considered a
genetic disease, with the implication that it
is therefore incurable. Typically coded as a
“static encephalopathy” by developmental
pediatricians, traditional therapies have
included speech, physical, and occupational
therapies and applied behavior analysis
(ABA). Three recent papers described
mouse models of developmental disorders,
specifically Fragile X (Hayashi, 2007),
Rett syndrome (Guy, 2007), and tuberous
sclerosis (Ehninger, 2008), all previously
considered beyond interventions that could
change the trajectory of those conditions.
Dramatically, symptoms were reversed in
each case by various molecular interventions,
even in older animals! These experiments
must be recognized as changing the
paradigm that such conditions are beyond
intervention; potentially, the quality of life
for the child and family can be significantly
improved.
Genetic susceptibilities play a role in
determining a child’s vulnerability to
environmental stressors. Single nucleotide
polymorphisms (SNPs) are variations in an
individual’s genetics affecting enzymes
and body metabolism that might make one
person prone to heart disease, hypertension,
and stroke and another person more prone
to cancer. SNPs of interest in autism
include: PON-1 (paraoxonase 1), which
makes children more susceptible to pesticide
toxicity; ALAD (aminolevulinate, delta-,
dehydratase), which increases risk of lead
toxicity; and GST M1-null (Glutathione
S-transferases), which increases a child’s
39
 BIOMEDICAL
Vicious Cycles - metabolic
The vital role of Methylation
and Transulfuration
When this cycle does not function,
downstream effects are huge:
 cannot make normal neurotransmitters
 cannot make creatine
 cannot control gene expression
 cannot make cell membranes
susceptibility to mercury and xenobiotic
toxicities. SNPs that are associated with
interference in proper methylation and
transulfuration biochemistry include:
MTHFR (methylene tetrahydrofolate
reductase), MTRR (methionine synthase
reductase), and adenosine deaminase
weakness. The HLA-DR4 allelle is associated
with increased allergies and poor handling
of heavy metals. Children with autism
are more likely than controls are to have
problems with the function of reduced
folate carrier (associated with entry of folate
into the cell where it can perform its jobs,
which include promoting proper neurologic
functions) and transcobalamin II (associated
with the transport of methylcobalamin
into the cell, where it performs a crucial
role in the re-methylation of methionine
from homocysteine) (James, 2006).
Glutathione is the body’s major intracellular
anti-oxidant, and it also has important
roles in detoxification, T cell regulation,
maintenance of the gut epithelium, and
mitochondrial function. Total reduced
glutathione levels (the reduced form is the
active form) were 46% lower in children with
autism compared to neurotypical controls,
and oxidized glutathione (the bad stuff)
was 72% higher in the children with autism.
Cysteine levels were 19% lower in autism
(James, 2004). Cysteine is the rate limiting
amino acid for glutathione synthesis.
Furthermore, the parents of children with
autism showed abnormal methylation
and transulfuration metabolism and DNA
hypomethylation, which puts those parents
at risk for diseases like heart disease and
cancer (James, 2008).
This recent research has opened doors
of opportunity for treating dysfunctional
metabolic pathways in children with autism
via nutriceutical means. No longer should
we only consider classic inherited inborn
errors of metabolism; managing children
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Increased Dysfunctional
oxidative enzymes
stress
Abnormal
Methylation
biochemistry
with autism in the new millennium requires
consideration of acquired and potentially
treatable metabolic dysfunctions.
Oxidative stress in autism
Oxidative stress is common in children with
autism, as demonstrated by increased lipid
peroxidation markers. Transferrin (which
binds iron) and ceruloplasmin (which binds
copper) are both reduced in children with
autism compared to their non-autistic
siblings. Low levels of these two antioxidant
proteins were strikingly correlated with
loss of previously acquired language in
the children with autism (Chauhan, 2004).
Environmental factors that act as pro-
oxidants include toxins, pollution, heavy
metals, viral infections, pathogenic bacteria,
thalidomide, and valproic acid. Endogenous
(in the body) pro-oxidants include
homocysteine, xanthine oxidase, and nitrous
oxide. Decreased antioxidant capabilities
in children with autism are related to low
levels of the major intracellular antioxidant,
glutathione, poor function of antioxidant
enzymes such as catalase and superoxide
dismutase, and low levels of the major
antioxidant proteins, transferrin and
ceruloplasmin.
Treatment of oxidative stress in autism,
as in many chronic diseases, is absolutely
crucial. Ongoing oxidative stress makes
metabolic abnormalities worse. Foods and
supplements are rich sources of antioxidants
and come in sufficient varieties so that
appropriate forms can be found for virtually
any child.
Biomarkers in autism
Clinicians caring for children with autism can
use various biomarkers to work up immune
function, evaluate oxidative stress, and
assess mitochondrial function. Of particular
value are neopterin, which signifies immune
activation leading to oxidation, and
biopterin, which signifies oxidation due
to inflammation. Isoprostane elevations
imply oxidized fatty acid membranes, and
8 OHG elevations imply oxidized RNA.
Low cysteine and glutathione are nearly
ubiquitous in children with autism, and
these reflect abnormalities in methylation
and transulfuration biochemistry. Elevations
in ammonia and lactic acid may be a result
of mitochondrial dysfunction, although the
differential diagnosis of each includes many
other potential causes. Low L-carnitine
levels are associated with mitochondrial
problems. Quantitative immunoglobulins
with IgG sub-classes may reveal immune
dysfunction, and assessment of natural killer
cell function may reveal impaired ability
to fight bacterial and fungal infections.
At the Autism Research Institute, we
believe that the use of biomarkers in the
evaluation of children with autism will
help us to categorize our patients into
different phenotypes and determine specific
therapies influenced by the metabolic and
inflammatory profiles of subgroups of
patients.
A call for urgent action
Clinicians, researchers, and parents who are
part of the Defeat Autism Now! initiative
have spoken of the autism epidemic for
over a decade now. Dr. Martha Herbert
has articulated the paradigm shift from
considering autism to be a brain disorder, to
seeing it as a disorder with many potential
etiologies that affect the brain and must
include environmental factors (Herbert,
2006). The mantra of the Autism Research
Institute is that autism is treatable. Our
children deserve nothing less than heroic
efforts on the part of their governments,
medical establishments, and educational
institutions. It is crucial that we apply the
currently available science in ways that
are designed to help the many families
affected by this catastrophe. It is critical
that we work within a paradigm that
acknowledges the reality that many of the
changes affecting the brains of children
with autism are metabolic or inflammatory
and, therefore, subject to intervention. It
is imperative that we use what we know
now to organize treatment-oriented
research initiatives to help as many children
as possible. Many children in the autism
spectrum have so much more potential than
they currently are able to achieve.
ISSUE 30 2009