Understanding The Puzzle of Complex Diseases

1. The Role of Environmental Factors in AutismAristo Vojdani, Ph.D., M.T. Immunosciences Lab., Inc.822 S Robertson Blvd, Ste 312Los Angeles, California 90035Tel: 310-657-1077E-mail: drari@msn.comAutism OneMay 20-24, 2009 Chicago, Illinois 1

2. 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.

3. 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 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, 2007 3

4. 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

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6. 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

7. Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children A.J. Wakefield et al., The Lancet, 1998, 351: 637-641 Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children A.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. 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. 7

8. Autistic Enterocolitis Crohn’s Disease 8

9. 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

10. 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

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12. 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 offspringShi L., Fatemi S. H., Sidewell R., Patterson, P. H., J. Neurosciences, 2003, 23:297.

13. Infectious Agents Maternal Exposure to Infection Abnormal Brain Development Child with Autism Child with Schizophrenia Limin Shi, et al., The Journal of Neuroscience, 23(1):297-302 (2003) 13

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15. Patient #1 20

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17. 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

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20. 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 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. Hg 20

21. 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 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 AUTOIMMUNE DISEASE AUTOIMMUNE DISEASE NO AUTOIMMUNE DISEASE 21

22. 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. 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

23. Exposure to Methyl Mercury Results in Serum Autoantibodies to Neurotypic and Gliotypic ProteinsEl-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

24. 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 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. 24

25. 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 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-g 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

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28. The Humoral Response in the Pathogenesis of Gluten AtaxiaHadjivassiliou 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

29. Immune Response to Dietary Proteins, Gliadin andCerebellar Peptides in Children with AutismNutritional 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

30. Percent positive sera from patients with Autism for IgG, IgM, and IgA antibodies against gliadin and cerebellar peptides . 30

31. Reaction of antibody to: gliadin peptide, crude gliadin, brain, cerebellar, milk, egg, corn, and soy with gliadin peptide coated plates. 31

32. Reaction of antibody to: cerebellar, brain, crude gliadin, milk protiens, egg, corn, and soy with cerebellar peptide coated plates. 32

33. 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. 33

34. Simultaneous Detection of Antibodies Against CD26, CD69, Gliadin (Gli), Casein Peptides (CA), SK, and Ethyl Mercury (Hg) in Children with Autism.

35. 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: • Cleaving Peptides • Receptors • Adhesion Molecules • Signal transduction molecules • Immune regulators • T-cell mediators of immune response • Inducers of cytokine production • Proenzymes in cytotoxic lymphocytes

36. Characterization of Human Serum Dipeptidyl Peptidase IV (CD26) and Analysis of its Autoantibodies in Patients with Rheumatoid Arthritis and other Autoimmune DiseasesCuchanovich et al., Clinical and Experimental Rheumatology19:673, 2001.Streptokinase Promotes Development of Dipeptidyl Peptidase IV (CD26) Autoantibodies after Fibrinolytic Therapy in Myocardial Infarction PatientsCuchanovich et al., Clinical and Diagnostic Laboratory Immunology 9:1253, 2002.

37. 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. 37

38. Percent Elevation of Antibodies Against DPP IV, DPPI, CD13, Gliadin Peptidase and HSP-60 in Controls and Patients with Autism and Autoimmune Disease

39. Accumulation of peptides and opening of the tight junctions 39

40. 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-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

41. 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