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The Role of Environmental Factors in Autism
Aristo Vojdani, Ph.D., M.T. Immunosciences Lab., Inc.
822 S Robertson Blvd, Ste 312Los Angeles, California 90035
Tel: 310-657-1077E-mail: [email protected]
Autism OneMay 20-24, 2009 Chicago, Illinois
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Understanding The Puzzle of Complex 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.
Understanding mechanisms of action responsible for the development of complex diseases including gastrointestinal, cardiovascular and autoimmune diseases.
Autism’s origin? Neuroligins and neurexins, proteins crucial for aligning and activat-ing 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
Autism’s Cause May Reside in Abnormalities at the SynapseNew genetic evidence is leading researchers to home in on the cleft
Separating neurons as the site where the disorder may originate.
BackgroundAutism spectrum disorder is a heritable developmental disorder in which chromosomal abnormalities are thought to play a role.
ConclusionsWe 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.
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N Engl J Med 2008;358
Gastrointestinal microflora studiesin 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.
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Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental
disorder in childrenA.J. Wakefield et al., The Lancet, 1998, 351: 637-641
FINDINGS: Onset of behavioral symptoms was associated, by the parents, with measles, mumps, and rubella vaccination in eight of the 12 children, with measles infection in one child, and otitis media in another. All 12 children had intestinal abnormalities, ranging from lymphoid nodular hyperplasia to apthoid ulceration. Histology showed patchy chronic inflammation in the colon in 11 children and reactivce ileal lymphoid hyperplasia in seven, but no granulomas. Behavioral disorders included autism (nine), disintegrative psychosis (one), and possible postviral or vaccinal encephalitis (two). Results showed low haemoglobin and a low serum IgA in four children.
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Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental
disorder in childrenA.J. Wakefield et al., The Lancet, 1998, 351: 637-641
FINDINGS: Onset of behavioral symptoms was associated, by the parents, with measles, mumps, and rubella vaccination in eight of the 12 children, with measles infection in one child, and otitis media in another. All 12 children had intestinal abnormalities, ranging from lymphoid nodular hyperplasia to apthoid ulceration. Histology showed patchy chronic inflammation in the colon in 11 children and reactive ileal lymphoid hyperplasia in seven, but no granulomas. Behavioral disorders included autism (nine), disintegrative psychosis (one), and possible postviral or vaccinal encephalitis (two). Results showed low haemoglobin and a low serum IgA in four children.
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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.
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AID deficiency leads to hyperplasia of isolated lymphoid follicles in the small intestine
A duodenal segment of the small intestine of AID-/- mouse showing many protruding follicles and an enlarged Peyer’s patch.
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Peyer’s patch
Isolated lymphoid follicles
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Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly in adulthood: implications for genesis of autism and
schizophreniaFatemi 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.
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Maternal Exposure to Infection
Abnormal Brain
DevelopmentChild with
Autism
Child with Schizophrenia
Infectious Agents
Limin Shi, et al., The Journal of Neuroscience, 23(1):297-302 (2003)
Patient #1
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Normal 2.0
Normal 40.0
1- B. b.2- C2+C63- LFA4 - OspA+OspC5 - OspE6 - VMP7- B. afz.8 - B. s. s.
9 - B. gar.10 - Babe11 - Bart12 - Ehr13 - Un Spir14 - MBP15 - Nf16 - BBB
IgG IgM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1620
Patient #2
0
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Normal 2.0
Normal 40.0
1- B. b.2- C2+C63- LFA4 - OspA+OspC5 - OspE6 - VMP7- B. afz.8 - B. s. s.
9 - B. gar.10 - Babe11 - Bart12 - Ehr13 - Un Spir14 - MBP15 - Nf16 - BBB
IgG IgM
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1621
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.
Polyreactive myelin oligodendrocyte glycoprotein antibodies: Implications for systemic autoimmunity in progressive
experimental autoimmune encephalomyelitisPeterson LK et al., J Neuroimmunol, 2007; 183:69-80
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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 side-chain; 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 character-istic 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 side-chains, such as those of cysteine and methionine; a methionine sulfoxide is shown.
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Immunogenicity of Metals by Interactionwith Amino Acids of Proteins
Hg
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)
SIGNIFICANT INCREASE
IL-4
IgG1 Antibodies
ANA
Immune Complexes
Abnormal Histology
SIGNIFICANT INCREASE
No IL-4
IgG1 Antibodies
ANA
Immune Complexes
Abnormal Histology
SIGNIFICANT INCREASE
IL-4
No IFN-
Low ANA
Low Immune Complexes
Normal Histology
WILD-TYPE MOUSE IL-4 GENE KNOCKOUT IFN- GENE KNOCKOUT
INJECT WITH 40 g MERCURY CHLORIDE TWICE A WEEK FOR 2 WEEKS
AUTOIMMUNEDISEASE
AUTOIMMUNEDISEASE
NOAUTOIMMUNE
DISEASE21
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.
Analysis of the Autoantibody Response to Fibrillarin in Human Disease and Murine
Models of AutoimmunityTakeuchi et al., The Journal of Immunology, 1995, 154:961-971.
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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.
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Exposure to Methyl Mercury Results in Serum Autoantibodies to Neurotypic and
Gliotypic ProteinsEl-Fawal et al., Neurotoxicity 17 (1) 267-276, 1996.
Detection of low-level environmental chemical allergy by a long-term
sensitization methodFukuyama 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 chemical-related 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 antigen-specific serum IgE levels.
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Detection of low-level environmental chemical allergy by a long-term
sensitization methodFukuyama et al. Toxicology Letters, 2008; vol. 180
TMA and TDI induced marked increases in levels of antigen-specific 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-) 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.
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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.
The Humoral Response in the Pathogenesis of Gluten Ataxia
Hadjivassiliou et al., Neurology 2002;58:1221-1226.
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Immune Response to Dietary Proteins, Gliadin andCerebellar 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).”
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Percent positive sera from patients with Autism for IgG, IgM, and IgA antibodies against gliadin and cerebellar peptides .
0
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PE
RC
EN
T P
OS
ITIV
E F
OR
GL
IAD
IN A
ND
C
ER
EB
EL
LA
R A
NT
IBO
DIE
S
IgG IgM IgA
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Reaction of antibody to: gliadin peptide, crude gliadin, brain, cerebellar, milk, egg, corn, and soy with gliadin peptide coated plates.
0
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40
60
80
100
120P
erc
en
t B
ind
ing
of
Dif
fere
nt
An
tib
od
ies
to
Gli
ad
in
Pe
pti
de
Anti-Gliadin Peptide
Anti-Crude Gliadin
Anti-BrainMBP
Anti-Cerebellar
Anti-Milk Proteins
Anti-Egg
Anti-Corn
Anti-Soy
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Reaction of antibody to: cerebellar, brain, crude gliadin, milk protiens, egg, corn, and soy with cerebellar peptide coated plates.
0
20
40
60
80
100
120P
erc
en
t B
ind
ing
of
Dif
fere
nt
An
tib
od
ies
to
Ce
reb
ella
r P
ep
tid
e
Anti-Gliadin Peptide
Anti-Crude Gliadin
Anti-BrainMBP
Anti-Cerebellar
Anti-Milk Proteins
Anti-Egg
Anti-Corn
Anti-Soy
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Infections, Toxic Chemicals and Dietary Peptides Binding to Lymphocyte Receptors and Tissue Enzymes
are Major Instigators of Autoimmunity in AutismAristo 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.
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Simultaneous Detection of Antibodies Against CD26, CD69, Gliadin (Gli), Casein Peptides (CA), SK, and
Ethyl Mercury (Hg) in Children with Autism.
CD26 CD69 Gli CA SK Hg CD26 CD69 Gli CA SK Hg CD26 CD69 Gli CA SK Hg
1 - - - + - - + - -2 - - - - - + - 3 - - - - + - + + - + + + - + -4 - - - - + -5 - - - - - - - - - - + + - - - - - -6 - - - - - - + + - - + - - -7 - - - - - - - - - - - - - - - - - -8 + - + - - - - - - - - - - - + + - -9 - - - + - - - + - - - - - - - - - -10 - - - - - - - - - - - - - - - - - -11 - + - - - - + - - - - - - - - - - -12 - - - - - - + - - - + + - - - - + -13 - -14 - - - - - - - - + + - - - -15 + - - + + - - - + + + - - - - - - -16 - - + - - - + - - -17 - - - - - - - - + + - - - - - - - -18 - - - - - - - - - - - - - - - - - -19 - - - - + - + - + + - -20 - - - + + - + - + - + - - - - - - -
IgM IgA
Specimen #
IgG
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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 26Aminopeptidase - N or CD 13Aminopeptidase I (DPPI)
Peptidases function as:1. Cleaving Peptides2. Receptors3. Adhesion Molecules4. Signal transduction molecules5. Immune regulators 6. T-cell mediators of immune response 7. Inducers of cytokine production8. Proenzymes in cytotoxic lymphocytes
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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.
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, HSP-60), dietary proteins (eg. gliadin peptides) in individuals with predisposing HLA molecules bind to aminopeptidases and induce autoantibodies against peptides and tissue antigens.
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Percent Elevation of Antibodies Against DPP IV, DPPI, CD13, Gliadin Peptidase and HSP-60 in
Controls and Patients with Autism and Autoimmune Disease
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
Antigens% IgG Elevation in:
AutismChildren Control
Adults Control
Auto- immune
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 IL-10 and TGF-.
Depletion of IL-10- and TGF--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.
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Conclusions:
Regulatory T cells and TGF-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.
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