Download in Children with Autism.

The Role of Environmental
Factors in Autism
Aristo Vojdani, Ph.D., M.T.
Immunosciences Lab., Inc.
822 S Robertson Blvd, Ste 312
Los Angeles, California 90035
Tel: 310-657-1077
E-mail: [email protected]
Autism One
May 20-24, 2009
Chicago, Illinois
1
Understanding The Puzzle of Complex
Diseases
 Understanding mechanisms
of action responsible for the
development of complex
diseases including
gastrointestinal,
cardiovascular and
autoimmune diseases.
 These diseases cannot be
ascribed to mutation in a
single gene; rather they
arise from the combined
action of many genes,
environmental factors
and risk-conferring
behavior.
2
Autism’s Cause May Reside in Abnormalities at the Synapse
New genetic evidence is leading researchers to home in on the cleft
Separating neurons as the site where the disorder may originate.
Autism’s origin? Neuroligins and neurexins, proteins crucial for aligning and activating synapses, have now been implicated in autism, along with the Shank3 scaffolding
protein. An altered balance between excitatory synapses (left) and inhibitory (right)
could affect learning and memory during development.
Science, 317:190-191, 20073
N Engl J Med 2008;358
Background
Autism spectrum disorder is a heritable developmental disorder in which
chromosomal abnormalities are thought to play a role.
Conclusions
We have identified a novel, recurrent microdeletion and a reciprocal
microduplication that carry substantial susceptibility to autism and appear to
account for approximately 1% of cases. We did not identify other regions
with similar aggregations of large de novo mutations.
4
nervous system
5
Gastrointestinal microflora studies
in late-onset autism
Finegold S.M. et al., Clin Infect Dis, 2002 Sep 1;35(Suppl 1):S6-S16
Some cases of late-onset (regressive) autism may involve abnormal
flora because oral vancomycin, which is poorly absorbed, may lead to
significant improvement in these children. Fecal flora of children with
regressive autism was compared with that of control children, and
clostridial counts were higher. The number of clostridial species found
in the stools of children with autism was greater than in the stools of
control children. Children with autism had 9 species of Clostridium not
found in controls, whereas controls yielded only 3 species not found
in children with autism. In all, there were 25 different clostridial
species found. In gastric and duodenal specimens, the most striking
finding was total absence of non-spore-forming anaerobes and
microaerophilic bacteria from control children and significant numbers
of such bacteria from children with autism. 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.
6
Ileal-lymphoid-nodular
Ileal-lymphoid-nodularhyperplasia,
hyperplasia,nonnonspecific
specificcolitis,
colitis,and
andpervasive
pervasivedevelopmental
developmental
disorder
disorderin
inchildren
children
A.J.
A.J.Wakefield
Wakefieldetetal.,
al.,The
TheLancet,
Lancet,1998,
1998,351:
351:637-641
637-641
FINDINGS: Onset of behavioral symptoms was associated, by
FINDINGS: Onset of behavioral symptoms was associated, by
the parents, with measles, mumps, and rubella vaccination in
the parents, with measles, mumps, and rubella vaccination in
eight of the 12 children, with measles infection in one child, and
eight of the 12 children, with measles infection in one child, and
otitis media in another. All 12 children had intestinal
otitis media in another. All 12 children had intestinal
abnormalities, ranging from lymphoid nodular hyperplasia to
abnormalities, ranging from lymphoid nodular hyperplasia to
apthoid ulceration. Histology showed patchy chronic
apthoid ulceration. Histology showed patchy chronic
inflammation in the colon in 11 children and reactive ileal
inflammation in the colon in 11 children and reactivce ileal
lymphoid hyperplasia in seven, but no granulomas. Behavioral
lymphoid hyperplasia in seven, but no granulomas. Behavioral
disorders included autism (nine), disintegrative psychosis (one),
disorders included autism (nine), disintegrative psychosis (one),
and possible postviral or vaccinal encephalitis (two). Results
and possible postviral or vaccinal encephalitis (two). Results
showed low haemoglobin and a low serum IgA in four children.
showed low haemoglobin and a low serum IgA in four children.
7
Autistic Enterocolitis
Crohn’s Disease
8
Science, 2002, 298:1424-1427
 Activation-induced cytidine deaminase (AID) plays an essential role in
class switch recombination (CSR) and somatic hypermutation (SHM) of
immunoglobulin genes.
 Deficiency in AID results in the development of hyperplasia of isolated
lymphoid follicles (ILF) associated with a 100-fold expansion of
anaerobic flora in the small intestine.
 Reduction of bacterial flora by antibiotic treatment of AID-/- mice
abolished ILF hyperplasia as well as the germinal center enlargement
seen in secondary lymphoid tissues.
9
9
AID deficiency leads to hyperplasia of isolated
lymphoid follicles in the small intestine
Peyer’s patch
Isolated
lymphoid
follicles
A duodenal segment of the small intestine of AID-/- mouse showing
many protruding follicles and an enlarged Peyer’s patch.
10
2005
1111
Prenatal viral infection leads to pyramidal cell
atrophy and macrocephaly in adulthood:
implications for genesis of autism and
schizophrenia
Fatemi S. H., Earle J., Kamodia R., Kist D., Emamian E. S., Patterson, P.
H., Shi L., Sidewell R., Cell. Mol. Neurobiol., 2002, 22:25-33.
Maternal influenza infection causes marked
behavior and pharmacological changes in the
offspring
Shi L., Fatemi S. H., Sidewell R., Patterson, P. H., J. Neurosciences,
2003, 23:297.
12
Infectious
Agents
Child
with
Autism
Maternal
Exposure
to Infection
Abnormal
Brain
Development
Limin Shi, et al., The Journal of Neuroscience, 23(1):297-302 (2003)
Child with
Schizophrenia
13
14
IgG
Patient #1
1- B. b.
2- C2+C6
3- LFA
4 - OspA+OspC
5 - OspE
6 - VMP
7- B. afz.
8 - B. s. s.
9
8
7
130
IgM
9 - B. gar.
10 - Babe
11 - Bart
12 - Ehr
13 - Un Spir
14 - MBP
15 - Nf
16 - BBB
120
110
100
90
80
6
70
5
60
4
50
3
40
2
Normal 40.0
30
Normal 2.0
20
1
10
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
20
IgG
Patient #2
1- B. b.
2- C2+C6
3- LFA
4 - OspA+OspC
5 - OspE
6 - VMP
7- B. afz.
8 - B. s. s.
9
8
7
130
IgM
9 - B. gar.
10 - Babe
11 - Bart
12 - Ehr
13 - Un Spir
14 - MBP
15 - Nf
16 - BBB
120
110
100
90
80
6
70
5
60
4
50
3
40
2
Normal 40.0
30
Normal 2.0
20
1
10
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
21
Polyreactive myelin oligodendrocyte
glycoprotein antibodies: Implications for
systemic autoimmunity in progressive
experimental autoimmune encephalomyelitis
Peterson LK et al., J Neuroimmunol, 2007; 183:69-80
Two myelin oligodendrocyte glycoprotein (MOG92–106)
monoclonal antibodies (mAbs) were produced from an
A.SW mouse with progressive experimental autoimmune
encephalomyelitis. Polyreactivity/specificity of the mAbs
was demonstrated by ELISA. Functionality and a
potential role in pathogenesis of systemic autoimmunity
were demonstrated in vitro in a lymphocytotoxicity assay
and in vivo upon injection into naïve mice. Injection of
MOG mAb producing hybridomas into naïve mice
resulted in immunoglobulin deposition in kidneys and
liver.
17
nervous system
18
19
Immunogenicity of Metals by Interaction
with Amino Acids of Proteins
Haptens comprise organic compounds and metal ions, and bind to proteins
forming either covalent bonds (a) or coordination complexes (b).
a. Organic haptens forming covalent bonds bind to a single amino acid sidechain; the covalent binding of trinitrophenyl (TNP) to lysine is shown.
b. Metal complexes consist of a central metal ion and a set of atoms, ions or
small molecules, regarded as ligands. These ligands are aligned in a characteristic geometric form, e.g. a plane square or an octagon. The interactions
between a metal ion and ligands allow the electron-rich ligands to transfer part
of their electron density to the positively charged metal ion (coordination
bond) in order to increase complex stability; a square planar complex of nickel
with three histidines and one cysteine is shown.
c. Alternatively, certain reactive chemicals can irreversibly oxidize protein sidechains, such as those of cysteine and methionine; a methionine sulfoxide is
shown.
Hg
20
THE PROTOTYPIC Th2 AUTOIMMUNITY INDUCED BY MERCURY IS
DEPENDENT ON IFN- AND NOT Th1/Th2 IMBALANCE
Kono et al., Journal of Immunology 161: 234-240 (July 1998)
WILD-TYPE MOUSE
IL-4 GENE KNOCKOUT
IFN- GENE KNOCKOUT
INJECT WITH 40 g MERCURY CHLORIDE TWICE A WEEK FOR 2 WEEKS
SIGNIFICANT INCREASE
SIGNIFICANT INCREASE
SIGNIFICANT INCREASE
 IL-4
 No IL-4
 IL-4
 IgG1 Antibodies
 IgG1 Antibodies
 No IFN-
 ANA
 ANA
 Low ANA
 Immune Complexes
 Immune Complexes
 Low Immune Complexes
 Abnormal Histology
 Abnormal Histology
 Normal Histology
AUTOIMMUNE
DISEASE
AUTOIMMUNE
DISEASE
NO
AUTOIMMUNE
DISEASE
21
Analysis of the Autoantibody Response to
Fibrillarin in Human Disease and Murine
Models of Autoimmunity
Takeuchi et al., The Journal of Immunology, 1995, 154:961-971.
Fibrillarin, a component of the U3 RNP particle, is a
target for the spontaneously arising autoantibodies in
human scleroderma and a monoclonal autoantibody
(72B9) derived from the autoimmune mouse strain (NZB
x NZW) F1. Autoantibodies against fibrillarin can also
be induced in H-25 mice by treatment with mercuric
chloride (HgCl2).
Our results indicate that spontaneous human and
toxin-induced murine autoantibodies to fibrillarin share
common reactivity against this highly conserved
nucleolar protein.
22
Exposure to Methyl Mercury Results in
Serum Autoantibodies to Neurotypic and
Gliotypic Proteins
El-Fawal et al., Neurotoxicity 17 (1) 267-276, 1996.
 Environmental exposure to methyl mercury (MeHg)
continues to pose a threat to humans, making early
detection of neurotoxic effects a pressing concern.
 Exposure to 32 ppm MeHg resulted in decreased
(p<0.05) levels in the cortex at 14 days.
 Both levels of MeHg resulted in increased GFAP in
the cerebellum at 14 days.
 This study suggests that assays of autoantibodies
against nervous system proteins may provide a
means of assessing the early neurotoxic effects of
environmental MeHg exposure.
23
Detection of low-level environmental
chemical allergy by a long-term
sensitization method
Fukuyama et al. Toxicology Letters, 2008; vol. 180
Multiple chemical sensitivity (MCS) is characterized by various
signs, including neurological disorders and allergy. We are interested
in the allergenicity of MCS and the detection of low-level chemicalrelated hypersensitivity.
We used long term sensitization followed by low-dose challenge to
evaluate sensitization by well-known Th2 type sensitizers (trimellitic
anhydride (TMA) and toluene diisocyanate (TDI)) and a Th1 type
sensitizer (2,4-dinitrochlorobenzene (DNCG)).
After topically sensitizing BALB/c mice (9 x in 3 weeks) and
challenging them with TMA, TDI or DNCB, we assayed their auricular
lymph nodes for number of lymphocytes, surface antigen expression
of B cells, and local cytokine production, and measured antigenspecific serum IgE levels.
24
Detection of low-level environmental
chemical allergy by a long-term
sensitization method
Fukuyama et al. Toxicology Letters, 2008; vol. 180
TMA and TDI induced marked increases in levels of antigenspecific serum IgE and of Th2 cytokines (IL-4, IL-5, IL-10 and IL-13
produced by ex vivo restimulated lymph node cells.
DNCB induced a marked increase in Th1 cytokine (IL-2, IFN- and
TNF-a) levels, but antigen-specific serum IgE levels were not
elevated.
All chemicals induced significant increases in numbe of
lymphocytes and surface antigen expression of B cells.
Our mouse model enabled the identification and characterization of
chemical-related allergic reactions at low levels, this long-term
sensitization method would be useful for detecting environmental
chemaical-related hypersensitivity.
25
nervous system
26
27
The Humoral Response in the
Pathogenesis of Gluten Ataxia
Hadjivassiliou et al., Neurology 2002;58:1221-1226.
 Objective: To characterize humoral response
to cerebellum in patients with gluten ataxia.
 Background: Gluten ataxia is a common
neurologic manifestation of gluten sensitivity.
 Conclusions: Patients with gluten ataxia have
antibodies against Purkinje cells. Antigliadin
antibodies cross-react with epitopes on
Purkinje cells.
28
Immune Response to Dietary Proteins, Gliadin and
Cerebellar Peptides in Children with Autism
Nutritional Neuroscience, 7(3):151-161, June 2004
“We conclude that, similar to patients with gluten ataxia, a subgroup of
patients with autism produce antibodies against Purkinje cells and
gliadin peptides. They cross-react with epitope of 8 amino acid on
cerebellar Purkinje cells, which consist of EDVPLLED with 50% similarity
to gliadin peptide (EQVPLVQQ).”
29
29
PERCENT POSITIVE FOR GLIADIN AND
CEREBELLAR ANTIBODIES
45
40
35
30
25
20
15
10
5
0
IgG
IgM
IgA
Percent positive sera from patients with Autism for IgG, IgM, and IgA
antibodies against gliadin
and cerebellar peptides
.
30
Percent Binding of Different Antibodies to Gliadin
Peptide
120
100
80
60
40
20
0
AntiGliadin
Peptide
AntiCrude
Gliadin
AntiBrain
MBP
AntiCerebellar
Anti-Milk
Proteins
AntiEgg
AntiCorn
AntiSoy
Reaction of antibody to: gliadin peptide, crude gliadin, brain, cerebellar,
milk, egg, corn, and soy with gliadin peptide coated plates.
31
Percent Binding of Different Antibodies to Cerebellar
Peptide
120
100
80
60
40
20
0
AntiCerebellar
AntiBrain
MBP
AntiCrude
Gliadin
AntiGliadin
Peptide
Anti-Milk
Proteins
AntiEgg
AntiCorn
AntiSoy
Reaction of antibody to: cerebellar, brain, crude gliadin, milk protiens,
egg, corn, and soy with cerebellar peptide coated plates.
32
Infections, Toxic Chemicals and Dietary Peptides
Binding to Lymphocyte Receptors and Tissue Enzymes
are Major Instigators of Autoimmunity in Autism
Aristo Vojdani, Jon B. Pangborn, Elroy Vojdani, Edwin L. Cooper;
International Journal of Immunopathology and Pharmacology, Vol. 16, 189-199 (2003)
“This study is the first to demonstrate that dietary peptides,
bacterial toxins and xenobiotics bind to lymphocyte
receptors and/or tissue enzymes, resulting in autoimmune
reaction in children with Autism.
33
33
Simultaneous Detection of Antibodies Against CD26,
CD69, Gliadin (Gli), Casein Peptides (CA), SK, and
Ethyl Mercury (Hg) in Children with Autism.
IgG
Specimen # CD26 CD69
3

-
4
-
5
+

+


-
1
2
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20


+

-

-
Gli



+



-
IgM
CA



+

+


+
SK


+


+


+
Hg


-
CD26 CD69
+
+
+
+

+
+
+
+

+
+

-
IgA
Gli
CA
SK
Hg


+
+
+
+
+


+
+
+
-
+
+
+

+
+
+

+
+
+
+
+
+
+

-
CD26 CD69



+




+
-








-
Gli





+



+
-
CA




+



+
-
SK


+

+

-
Hg

34
FUNCTION OF TISSUE ENZYMES AND
LYMPHOCYTE RECEPTORS
 CD 69 – is a lymphocyte activation marker involved in
apoptosis of autoreactive T-cells and hence autoimmunity
 Peptidases- Dipeptidylpeptidase (DPPIV) or CD 26
Aminopeptidase - N or CD 13
Aminopeptidase I (DPPI)
 Peptidases function as:
1. Cleaving Peptides
2. Receptors
3. Adhesion Molecules
4. Signal transduction molecules
5. Immune regulators
6. T-cell mediators of immune response
7. Inducers of cytokine production
8. Proenzymes in cytotoxic lymphocytes
35
Characterization of Human Serum Dipeptidyl
Peptidase IV (CD26) and Analysis of its
Autoantibodies in Patients with Rheumatoid
Arthritis and other Autoimmune Diseases
Cuchanovich et al., Clinical and Experimental Rheumatology
19:673, 2001.

Streptokinase Promotes Development of Dipeptidyl
Peptidase IV (CD26) Autoantibodies after
Fibrinolytic Therapy in Myocardial Infarction
Patients
Cuchanovich et al., Clinical and Diagnostic Laboratory Immunology
9:1253, 2002.
36
Searching for a mechanism underlying autoimmunity in autism, we
postulated that gliadin peptides, heat shock protein (HSP-60) and
streptokinase (SK) bind to different peptidases. Binding results in
autoantibody production against gliadin peptides, HSP-60, SK and tissue
peptidases. We assessed this hypothesis in patients with autism and in
those with mixed connective tissue diseases. Concomitant with the
appearance of anti-gliadin and anti-HSP antibodies, children with autism and
patients with autoimmune disease developed anti-DPP I, anti-DPP IV and
anti-CD13 autoantibodies… Furthermore, addition of anti-SK, anti-HSP-60
and anti-gliadin to DPP IV+ peptides caused 18-20% enhancement of
antigen-antibody reaction. These results further support binding of SK,
gliadin and HSP to DPP IV. We propose that superantigens (e.g. SK, HSP60), dietary proteins (eg. gliadin peptides) in individuals with predisposing
HLA molecules bind to aminopeptidases and induce autoantibodies against
peptides and tissue antigens.
37
Percent Elevation of Antibodies Against DPP IV, DPPI,
CD13, Gliadin Peptidase and HSP-60 in
Controls and Patients with Autism and Autoimmune
Disease
% IgG Elevation in:
Adults
Autism
Control
Antigens
Children
Control
DPP IV
10
54
14
64
DPP I
8
56
14
60
CD13
8
40
6
28
Gliadin
Peptide
12
42
18
62
HSP-60
16
36
22
52
Autoimmune
38
Accumulation of peptides and
opening of the tight junctions
39
Conclusions:
 Autism is a disorder of the immune system and the nervous
system, induced by the combination of genes plus
environmental factors such as infections, toxic chemicals
and certain dietary proteins and peptides.
 Since the gut maintains an extensive and highly active
immune system, environmental factors can induce
dysregulation of the immune system and potentially
damage the tissue.
 In the healthy gut, Th1 and Th2 responses are carefully
regulated by regulatory T cells (CD4+CD25+) expressing IL10 and TGF-b.
 Depletion of IL-10- and TGF-b-producing regulatory T cells,
or homing of CD4+CD25RBHIGH T cells in the GI tissue of
children with autism, may be responsible for GI pathology
reported by different investigators in autism.
40
Conclusions:
 Regulatory T cells and TGF-b production measured in the
blood of children with autism were found to be elevated in
one subgroup and low in the second subgroup.
 Immune function abnormalities, in particular, low natural
killer cell activity, low glutathione and abnormal cytokine
production, is part of the illness in autism, with a potential
for becoming a biological test for autism at birth.
 Abnormal levels of neurotransmitters such as serotonin,
dopamine, epinephrine, and norepinephrine are detected in
children with autism.
 A gluten- and casein-free diet, with omega-3 oil probiotics,
and high doses of vitamin-A can be extremely helpful
towards recovery for a subgroup of children with autism.
41