Maternal Gene-Environment Interactions S. Jill James, PhD During Pregnancy: Risk of Autism and Potential for Prevention

Reduced Folate Carrier (RFC1) variant G allele RFC1 Genotype Frequencies : Mother, Father, Child is increased in mothers: Metabolic Impact Case

Methionine

THF SAM Methyl acceptor MS DMG 5,10-CH2-THF MTase Folate Cycle MTRR MTHFR B12 DNA Methylation 5-CH3-THF SAH SAHH Homocysteine Adenosine Cell Membrane RFC1 G allele B6 CBS Cystathionine B6 RFC1 Cysteine

GSH GSSG Redox Balance

RFC1 Transmission Disequilibrium Test (TDT) MAXIMUM LIKELIHOOD RATIO TEST The LRT is capable of differenang whether the genec risk is operang (relative transmission of G allele from each parent) through the case child and/or through the mother.

Genetic 95% Confidence Overall Maternal transmission Paternal transmission Relative Risk p-value Interval R1 (offspring inheriting 1 G allele) 1.23 0.12 0.94, 1.73 Transmitted Not Transmitted Not Transmitted Not Transmitted R2 (offspring inheriting 2 G alleles) 1.18 0.38 0.81, 1.70 Transmitted Transmitted S1 (mother carrying 1 G allele) 1.60 0.007 1.13, 2.26 275 258 118 96 92 97 S2 (mother carrying 2 G alleles 1.52 0.02 1.07, 2.15

p=0.4883 p=0.1510 p=0.7712 Offspring Likelihood Ratio 2.71 NS

Maternal Likelihood Ratio 7.76 0.02 Requires genotyping mother, father and child

Genetic Relative Risk Folate-Methylation-Redox Pathways are a Continuum: Imbalance in one will affect activity of the others Genetic Relative Risk p Value Child RFC1 AG 1.2 NS Methionine Child RFC1 GG 1.1 NS THF SAM Methyl acceptor DMG 5,10-CH2-THF MS MTase Maternal RFC1 AG 1.60 0.007 Folate Cycle MTRR DNA Methylation MTHFR B12 5-CH3-THF SAH Maternal RFC1 GG 1.52 0.01 SAHH Homocysteine Cell Membrane Adenosine B6 CBS Offspring Effect 2.71 NS Cystathionine Maternal Effect 7.76 0.02 B6 RFC1 Cysteine

For RFC1, the genotype of the mother, not the child, GSH GSSG is the RFC1 determinant of risk Redox Balance Maternal Gene-Environment Interactions S. Jill James, PhD During Pregnancy: Risk of Autism and Potential for Prevention

16% DECREASE IN DNA METHYLATION IN ASSOCIATION OF DNA METHYLATION AND SAM/SAH MOTHERS HOMOZYGOUS FOR RFC1 GG IN MOTHERS OF CHILDREN WITH AUTISM

DNA Methylaon (% 5mC) P< 0.02 SAM/SAH rao

P=0.005 P=0.01 n = 40 n = 40

Maternal Methionine Cycle Metabolites Maternal Transsulfuration Metabolites

Control Mothers Autism Mothers p value Control Mothers Autism Mothers p value n=80 n=57 n=80 n=57 Methionine (µmol/L) 23.9 ± 4.4 21.0 ± 3.8 0.001 Homocysteine (µmol/L) 7.2 ± 1.3 9.2 ± 1.8 0.001

SAM (nmol/L) 70.0 ± 12 69.9 ± 21 NS Cysteine (µmol/L) 252 ± 20 247 ± 27 NS

SAH (nmol/L) 23.9 ± 6.6 32.3 ± 10 0.001 fGSH (µmol/L) 2.0 ± 0.4 1.8 ± 0.4 0.05

SAM/SAH ratio 3.2 ± 1.0 2.4 ± 1.3 0.01 GSSG (µmol/L) 0.20 ± 0.08 0.26 ± 0.13 0.001

Adenosine (µmol/L) 0.27 ± 0.1 0.36 ± 0.2 0.01 GSH/GSSG ratio 11.1 ± 4.4 8.5 ± 4.5 0.01

Homocysteine (µmol/L) 7.2 ± 1.3 9.2 ± 1.8 0.001 Nitrotyrosine (µmol/L) 68 ± 22 104 ± 43 0.01

Folate (nmol/L) 17.4 ± 6.2 15.4 ± 4.0 0.01 24% of mothers had homocysteine levels > 10 µmol/L

HOMOCYSTEINE: BRANCH POINT METABOLITE HOMOCYSTEINE: BRANCH POINT METABOLITE

Methionine Methionine METHYLATION METHYLATION THF SAM THF SAM MTase Cellular MTase Cellular 5,10-CH2THF MS Methylation 5,10-CH2THF MS Methylation B12 SAH Reactions B12 SAH Reactions

5-CH3THF 5-CH3THF Purines and Adenosine Purines and Adenosine Thymidylate Thymidylate HOMOCYSTEINE HOMOCYSTEINE

B6 B6 DNA SYNTHESIS Cystathionine REDOX DNA SYNTHESIS Cystathionine REDOX Cysteine HOMEOSTASIS Cysteine HOMEOSTASIS PROLIFERATION PROLIFERATION GSH GSSG GSH GSSG Maternal Gene-Environment Interactions S. Jill James, PhD During Pregnancy: Risk of Autism and Potential for Prevention

HOMOCYSTEINE: BRANCH POINT METABOLITE HOMOCYSTEINE: BRANCH POINT METABOLITE

Methionine Methionine METHYLATION METHYLATION THF SAM THF SAM MTase Cellular MTase Cellular 5,10-CH2THF MS Methylation 5,10-CH2THF MS Methylation B12 SAH Reactions B12 SAH Reactions

5-CH3THF 5-CH3THF Purines and Adenosine Purines and Adenosine Thymidylate Thymidylate HOMOCYSTEINE HOMOCYSTEINE

B6 B6 DNA SYNTHESIS Cystathionine REDOX DNA SYNTHESIS Cystathionine REDOX Cysteine HOMEOSTASIS Cysteine HOMEOSTASIS PROLIFERATION PROLIFERATION GSH GSSG GSH GSSG

Both genes and environment affect Homocysteine

Maternal Genecs/Epigenecs Important Caveat

PRE-NATAL Because the plasma metabolic profile was taken from Autism mothers 3- 10 years after the birth of their child, these results cannot be extrapolated to their PRE-NATAL ASDs metabolic profile during pregnancy.

We recently measured the plasma metabolic profile in Child Genecs Environment high risk mothers (who already had one child with autism) /Epigenecs POST-NATAL in collaboration with the MARBLES study at UC Davis. Homocysteine is a commercially available biomarker of abnormal one-carbon metabolism

Percent of Mothers with Functionally Significant Alterations in Methylation and Redox Metabolites During Pregnancy Interestingly, the percent of mothers with elevated homocysteine (~20%) in the third trimester is close the Functionally Significant 1st Trimester 2nd Trimester 3rd Trimester Change n=23 n=47 n=47 recent percent risk of a second autism outcome. SAM < 60 nmol/L 22% 27% 27% SAH > 32 nmol/L 4% 15% 25% SAM/SAH < 1.8 13% 13% 23% Homocysteine >10 µmol/L 17% 23% 23% We do not yet know if this metabolic imbalance GSH < 1.5 µmol/L 13% 13% 23% GSSG > 0.3 µmol/L 4% 21% 25% will be associated with an autism outcome. GSH/GSSG < 6.5 9% 23% 21% It will take 2-3 years before all siblings are evaluated Homocysteine levels normally drop during pregnancy to 6-7 μmol/L and we know final answers. Maternal homocysteine crosses the placenta and predicts fetal and neonatal homocysteine Maternal Gene-Environment Interactions S. Jill James, PhD During Pregnancy: Risk of Autism and Potential for Prevention

Risk factors associated with elevated Homocysteine: Conditions associated with elevated Homocysteine: Common Polymorphisms in one-carbon metabolism (MTHFR, MTRR, MTR, TCN2, RFC1) Cardiovascular Disease Age Alzheimer’s Disease B vitamin insufficiency Bipolar Disorder Alcohol Schizophrenia Smoking Osteoporosis Diabetes Birth defects Preeclampsia

Homocysteine levels are easily normalized by B vitamins: Potential for Autism Prevention: Folate, B12, B6, Riboflavin, Choline) We cannot control our genetic inheritance but we do have some control over environmental exposures that may increase risk of an autism Have your (total) homocysteine level checked: pregnancy (as they come to be known) Now Before conception As soon as you know you are pregnant Every trimester Fasting level should be between 7 and 8 μmol/L if you are under 35

RISK FACTORS DO NOT ACT IN ISOLATION Pung it all into Perspecve……we see what we know Epigenetics SNPs Elevated Homocysteine Cellular Metabolic Pathways Mutations Dietary deficiencies Mitochondrial dysfunction You Are Here Estrogen Free radicals Testosterone Toxins Thyroxin Viral/Bacterial infections Genes Emotional stress Environment Hormones Oxidative Stress Epigenetic FINAL COMMON PATHWAY Dysfunction Timing, duration, developmental stage, severity

Neuroinflammation Microbiome pathology Abnormal circuitry Gut dysfunction Immune Dysfunction Maternal Gene-Environment Interactions S. Jill James, PhD During Pregnancy: Risk of Autism and Potential for Prevention

Acknowledgements Autism Metabolic Genomics Laboratory Stepan Melnyk, PhD Stefanie Jernigan Teresa Evans Tim Trusty Shannon Palmer Sarah Blossom, PhD Dr. Moms and Dads Lesya Pavliv

Study Coordinators Researchers And Physicians Lisa Seidel, Laura Walters, Margaret Pauly

Funding: NICHD, DoD, ACHRI Foundation Folate Receptor Autoimmunity in Dan Rossignol, MD Autism Spectrum Disorders: Research Update

Disclaimer Folate Receptor Autoimmunity in While Dr. Rossignol has attempted to make the Autism Spectrum Disorders: information in this presentation as accurate as Research Update possible, the information is provided without any expressed or implied warranty. The purpose of this Dan Rossignol, MD FAAFP lecture is to provide information about different International Child Development Resource Center conditions or treatments that may affect individuals 321-259-7111 www.icdrc.org [email protected] with autism and other conditions. Please be advised that Dr. Rossignol is not giving medical www.danrossignolmd.com advice and that circumstances may dictate different ARI Conference treatments. All of the reviewed treatments in this October 16, 2011 lecture are considered off-label and not FDA- approved. Before beginning any treatment, please consult with your or your child’s physician.

Types of Folate 5MTHF Transport into Brain

. Folate = folic acid, inactive and oxidized form . 5MTHF binds to a Folate Receptor . 5-methyltetrahydrofolate (5MTHF): form of (FR1) in the choroid plexus and then folate that crosses the blood brain barrier undergoes endocytosis, storage and and is concentrated 2 to 4 times higher in then delivery into the CSF. It is then cerebral spinal fluid (CSF) than in the blood transported into neurons by the . Methylenetetrahydrofolate reductase reduced folate carrier (RFC) (MTHFR) converts folic acid into 5MTHF . 5-formyltetrahydrofolate (folinic acid) can . In this process, folate transport by FR1 also cross the blood brain barrier is ATP-dependent . There are over 100 folate analogues

Folate Receptors (FR)

. FR1: found at the luminal surface of intestinal cells, choroid plexus epithelium, pulmonary alveolar cells, thyroid cells (may play a role in hypothyroidism), and renal tubular cells (does not affect kidney function as antibodies do not cross there) . FR2: found in mesenchymal cells such as red blood cells Folate Receptor Autoimmunity in Dan Rossignol, MD Autism Spectrum Disorders: Research Update

Reduced Folate Carrier (RFC) FR Autoantibodies

. The Reduced Folate Carrier (RFC) carries . Autoantibodies to FR1 were first reported in folate into cells, including into neuronal 2005 and can be measured in the blood cells . Block the folate binding site on the plasma surface of choroid plexus epithelial cells, . Is located at the blood brain barrier as impairing folate transport into the CNS well, but has low folate affinity and does . FR autoantibodies might block transplacental not normally play much of a role in transport of folate by neural crest cells in the transporting folate into the brain developing embryo . Some children with ASD have genetic . Are associated with Cerebral Folate Deficiency variants in the RFC (CFD) syndrome, low-functioning autism, and Rett syndrome

Types of FR Autoantibodies

. Blocking antibodies: blocks the binding of 5MTHF to FR1. As in the case of all autoimmune diseases, the presence of autoantibody should be considered as abnormal. However, a low titer in some may not be pathological. Typically, lower CSF folate levels have been associated with higher titer but sometimes, low CSF folate values have been observed in patients with low titer . Binding antibody: may also be pathological by a yet unidentified mechanism

Raemaekers and Blau, 2004 Dev Med Child Neurol 46(12): 843-51

Prevalence of Folate Receptor Autoantibodies . Blocking antibody – United States: 10-15% (low titer) – Spain: 7% – Ireland: 13% . Binding antibody – United States: 15-17% – Ireland: 32%

Ramaekers et al., 2008 Dev Med Child Neurol 50(5):346-52 Folate Receptor Autoimmunity in Dan Rossignol, MD Autism Spectrum Disorders: Research Update

Cerebral Folate Deficiency (CFD)

. Definition: any neurological or psychiatric Twenty-eight patients with different mitochondrial condition associated with lowered CSF disorders and fulfilling the previously defined 5MTHF in the presence of normal folate levels diagnostic criteria were recruited from the outside the CSF (a neurometabolic syndrome) Hospital Sant Joan de De´u, Barcelona, Spain (21 patients), the University Clinic Aachen, Germany . Most common cause is autoimmune (5 patients), and Hospital 12 de Octubre, Madrid, mechanism (antibodies to the folate receptor) Spain (2 patients). Despite normal serum folate . First described in 2002; in autism in 2004 levels, 14 patients (mean age: 9.5 years; range: 3 . Most studies report a male-to-female ratio of months–34 years; Patients 1–14, table) had low 2.5-3:1 CSF 5-MTHF concentrations (mean: 22.1 nmol/L; range: 0.6– 48.8 nmol/L).

Garcia-Cazorla et al., 2008 Neurology 70(16):1360-1362

Milk and Folate Receptor Autoantibodies . Soluble FR antigen from human, bovine and goat’s milk are structurally homologous; Human and bovine milk FR antigen are 91% similar . Serum folate receptor autoantibodies cross-react with milk-derived soluble folate receptor antigens . Elimination of cow’s milk significantly lowers FR autoantibody production

Ramaekers et al., 2008 Dev Med Child Neurol 50(5):346-52

Synergistic Effects?

. Things that can go wrong: – Inadequate folate intake (poor nutrition) – Poor GI absorption of folate due to celiac disease Folate Receptor Autoimmunity or inflammatory bowel disease – Drugs that deplete folate in Autism Spectrum Disorders – Autoantibodies to the FR – Cow’s milk intake – Genetic defects in RFC and other rare genetic defects – MTHFR defects – Dihydrofolate reductase defects – Too much folic acid: inactive form Folate Receptor Autoimmunity in Dan Rossignol, MD Autism Spectrum Disorders: Research Update

Twenty-five patients with early-onset low-functioning autism with or without neurological deficits, were evaluated for serum folate, cerebrospinal fluid (CSF) 5MTHF, and Described 20 children with CFD, 7 had autism (ADOS). serum FR autoantibodies of the blocking type to determine 18 of 20 children had normal development during the the significance of folate receptor (FR) autoantibodies with first 4 months of life, followed by deceleration of head respect to folate transport across the blood-CSF barrier. In growth from 4 to 6 months, marked unrest, irritability spite of normal serum folate, CSF 5MTHF was low in 23 of and sleep disturbances. 9 of 20 children had reduced 25 patients. The reduced CSF folate in 19 of these 23 CSF 5-hydroxy-indolacetic acid (5HIAA) and normal patients could be explained by serum FR autoantibodies homovanillic acid (HVA) levels. Almost all children blocking the folate binding site of the membrane-attached had normalization of 5HIAA levels after folinic acid FR on the choroid epithelial cells. Oral folinic acid treatment. supplements led to normal CSF 5MTHF and partial or complete clinical recovery after 12 months. Ramaekers and Blau, 2004 Dev Med Child Neurol 46(12):843-851 Ramaekers et al., 2007 Neuropediatrics 38(6):276-81

“Two patients (patients 2, 4) who were diagnosed early and received treatment were cured with full recovery from autism and neurological deficits. In the whole group these two patients were among the youngest and were detected at 2 years 8 months and at 3 years and 2 months. Three older patients (patients 11, 23, 25), diagnosed and treated from the age of 4.9, 8 and 11.9 years, did not recover from autism but showed improvement of their neurological Current Data deficits. The remaining thirteen patients in the age range of three and seven years showed a good response after treatment with improvement of most neurological deficits, but only partial recovery from their autism. The partial recovery in the latter group of 13 patients consisted of amelioration of social impairment in 4 of 13 patients, reversal of impaired communication in 9 of 13 patients and disappearance of perseverative behavior and restricted interests in 6 of 13 patients.” Ramaekers et al., 2007 Neuropediatrics 38(6):276-81 Folate Receptor Autoimmunity in Dan Rossignol, MD Autism Spectrum Disorders: Research Update Update: Autism Brain Studies S. Jill James, PhD

Functional Consequence of Oxidative Stress in the Autism Brain

Autism Metabolic Genomics Laboratory S. Jill James PhD, Director Arkansas Children’s Hospital Research Institute Little Rock, Arkansas

GLUTATHIONE SYNTHESIS IN THE BRAIN IS DEPENDENT ASTROCYTE GLUTATHIONE SYNTHESIS IS DEPENDENT ON HEPATIC CYSTEINE SYNTHESIS AND EXPORT ON LIVER GLUTATHIONE SYNTHESIS Methionine GGT LIVER Glutathione Cysteine Cystine PLASMA THF 2 SAM Blood Brain Barrier B6 Methylated products: DNA, RNA, protein, BRAIN B12 Methionine Synthase 5,10-CH2THF phospholipids, neuro- 1 SAH transmiors GGT ASTROCYTE Glutathione Cys-Gly NEURON

5-CH3THF Adenosine Cystine Homocysteine Cysteine Cysteine B6 Cystathionine β Synthase Glutathione 3 Cystathionine Glutathione Cystathionine Lyase expression is low in brain; B6 Cystathionine Lyase therefore brain cells are dependent on plasma cysteine (derived primarily from the liver) for Cysteine LIVER intracellular glutathione synthesis. NEURON GLUTATHIONE SYNTHESIS IS DEPENDENT GLUTATHIONE ON ASTROCYTE GLUTATHIONE SYNTHESIS

Similar to neurons, T cells depend on macrophage synthesis and export of glutathione Brain Regions Altered in Autism

Cysne Methionine • Prefrontal cortex • Temporal lobe T Cell • Amygdala Macrophage • Cerebellum Glutathione Cysteine • Differences in total brain volume • Differences in early growth trajectories Update: Autism Brain Studies S. Jill James, PhD

Cerebellum Brodmann’s Area 22 (BA22) • In the superior temporal • Reduced cerebellar vermis gyrus (STG) volumes – Auditory processing • Differences in total – Wernicke’s area (left) cerebellum volume • Increased volume of the right • Reducon in the number of STG in autism Purkinje cells • More rightward asymmetry of • Altered expression of the STG in individuals with proteins involved with autism GABA neurotransmission • Increase in transcript levels • Autoanbodies to of many immune-related cerebellar proteins genes associated with the late recovery phase of an auto-immune disorder

Oxidave Stress in Ausm Brain Recent Studies

• Increased 3-nitrotyrosine (3-NT) in Wernicke’s area and CB Evidence of oxidative stress and functional in a study of 2 autism and 2 control brains (Sajdel- consequence in autism brain Sulkowska et al, 2011)

• A trend of increased 8-hydroxydeoxyguanosine (8-OH-dG), in the CB in a postmortem analysis of 5 cases with autism Autism Metabolic Genomics Laboratory compared to 6 controls (Sajdel-Sulkowska et al, 2009) Manuscript in preparation • Increased lipid hydroperoxides and decreased expression of electron transport chain complexes in the cerebellum and temporal cortex in a study of 8 cases with autism compared to 8 controls (Chauhan et al, 2011)

Tissues Examined Methods • 15 autism and 15 control • Aconitase Activity Assay tissues from ~ 100 mg of frozen tissue was minced and homogenized with a dounce cerebellar cortex homogenizer; supernatant sonicated on ice • 12 autism and – 0.5 mg/ml samples analyzed using kit (Cayman) 12 control • HPLC quantification of transsulfuration metabolites, 3- tissues from BA22 were Nitrotyrosine and 3-Chlorotyrosine studied ~ 200 mg of frozen tissue was minced and homogenized in PBS; free and total metabolites measured by HPLC; normalize to protein content • Autism and control cases • HPLC/MS quantification of 8-OH-dG were chosen in – DNA was extracted from brain tissues with phenol chloroform pairs, matched methodology, treated with RNase A, digested into component for gender, race, nucleotides using nuclease P1, snake venom phosphodiesterase and PMI, age and alkaline phosphatase cause of death Update: Autism Brain Studies S. Jill James, PhD

ACONITASE INACTIVATION BY SUPEROXIDE Aconitase Acvity PROMOTES FREE RADICAL GENERATION

15 ++ p<0.01 Fe + H2O2

Citrate 10 Hydroxyl TCA ACONITASE Inactive Aconitase Cycle [4Fe-4S] [3Fe-4S] Radical

Isocitrate 5 (nmol/min/ml)

Superoxide Aconitase Acvity

GSH/GSSG 0 III II IV Control Autism Control Autism I V ATP CB CB BA22 BA22

MITOCHONDRIA

Mitochondrial damage LITERATURE SUPPORT

• Consistent with aconitase inactivation: increased citrate and decreased isocitrate has been found in autism (Kaluzna-Czaplinska et al, Clin Biochem. 2011) Aconitase inactivation GSH/GSSG • Aconitase activity also found to be decreased in brains of patients with schizophrenia, Huntington’s disease, Parkinson’s disease, and Friedreich’s Ataxia.

ROS

Metabolites and Oxidative Stress Markers Cerebellum Metabolite Correlaons Cerebellum BA22 16 Control 16 Control Autism Autism 18 120 14 14 16 # fGSH/GSSG 100 3-NT 12 12 14 *** 12 80 10 10 10 *** 60 8 8 * 8 * 6 40 6 6 4 ** 20 2 4 4 0 0 2 2 0 3 6 9 12 0 5 10 15 20 0 0 Aconitase Activity fGSH/GSSG fGSH GSSG(x10) fGSH/GSSG fGSH GSSG(x10) fGSH/GSSG

140 160 120 *** 120 3-NT 3-NT ** *p<0.0001 100 120 140 100 120 80 100 80 60 * 100 **p<0.001 60 # 80 40 80 40 60 ** 20 20 60 ** ***p<0.01 0 0 40 40 # p<0.05 0 3 6 9 12 0 20 40 60 80 20 20 Aconitase Activity 8-OH-dG 0 0 3-NT 3-CT 8-OH-dG 3-NT 3-CT 8-OH-dG Update: Autism Brain Studies S. Jill James, PhD

BA22 Metabolite Correlaons Behavior Correlaons

20 120 3-NT fGSH/GSSG 100 • ADI-R (Autistic Diagnostic Interview-Revised) 15 80 10 60 scores were available on 5 cases that had both 40 5 cerebellum and BA22 samples 20 0 0 0 5 10 15 0 5 10 15 20 Aconitase Activity fGSH/GSSG • GSH/GSSG was positively associated with verbal communication (p<0.04) and repetitive 120 3-NT 120 3-NT behaviors (p<0.01). 100 100 80 80 60 60 • Aconitase activity was negatively correlated with 40 40 20 20 social communication (p<0.007) 0 0 0 5 10 15 0 20 40 60 80 100 Aconitase Activity 8-OH-dG

Summary of Brain Results Summary of Brain Results

• Largest sample size in a study of oxidative • GSH/GSSG redox status is compromised in stress in autism brain cerebellum and Wernicke’s area and is associated with decreased aconitase activity • Decrease in Aconitase activity is an indicator of excessive mitochondrial superoxide release • Protein oxidative damage (3-NT) and DNA oxidative damage (8-OH-dG) are elevated in CB • Excessive superoxide release indicates and Wernicke’s area in autism mitochondrial dysfunction in the cerebellum and • GSH/GSSG positively correlated with ADI-R Wernicke’s area verbal communication and aconitase negatively correlated with ADI-R social communication Overview of the State of Cytokine/ Judy Van de Water, PhD Chemokine Analysis

Overview of the State of Cytokine/Chemokine Analysis Judy Van de Water, PhD. •Plasma levels of IL-6, IL-8,IL-1β and IL-12p40 are significantly higher in the ASD (n=97) group when compared to TD (n=87) and DD (n=39) controls.

Altered T cell responses in children with Plasma cytokine levels

autism: association with behavior Cytokine Typically Developmental Ausm (pg/ml) Developing Delay Spectrum • When peripheral blood T cells were stimulated, GM-CSF, (n=87) (n=39) Disorders TNFα, and IL-13 were significantly increased whereas (n=97) IL-12p40 was decreased in ASD relative to TD controls. IL-1β 62.8 46.1 110.6* • Increased pro-inflammatory or TH1 cytokines were IL-2 8 7.3 22.9 associated with greater impairments in core features of IL-4 36.7 39.9 35.5 ASD as well as aberrant behaviors. IL-5 9.8 8.5 9.9 • In contrast, production of GM-CSF and TH2 cytokines IL-6 11.8 3 20.3*# were associated with better cognitive and adaptive IL-8 3.9 4.9 14.1* function. IL-10 16.4 12.4 11.9 IL-12 p40 171.7 169.7 196.2*# • Ashwood P, Krakowiak P, Hertz-Picciotto I, Hansen R, IL-13 20.9 18 20 Pessah IN, Van de Water, J. GM-CSF 54.2 52.3 63 Brain Behav Immun. 2010 Sep 9. [Epub ahead of print] IFNγ 62.8 84.3 70.2 TNFα 63.9 51.6 81.8

Onset status – relationship with cytokines Cytokine Typically Early Onset Regression Conclusions (pg/ml) Developing (n=53) (n=40) (n=87) • Plasma cytokines can be informative IL-1β 62.8 61 144.3*‡ – Fairly stable over time with multiple samples IL-2 8 17.7 19.3 • Very variable from subject to subject both IL-4 36.7 28.8 39.4 AU and controls IL-5 9.8 9.2 11.5 IL-6 11.8 15.1 32.6* • See several profiles within autism IL-8 3.9 6.8* 14.5* – Elevated inflammatory IL-10 16.4 7.5 15.6 – Reduced profile IL-12 p40 171.7 192.3* 198.6* – Normal profile IL-13 20.9 14.1 29.4 – What appears to be a somewhat AU specific GM-CSF 54.2 51.3 101.2*‡ profile IFNγ 62.8 51.2 94.1 – Onset status driven TNFα 63.9 56.2 111.1* Raw data Plasma- all AU, ages 2-4 yrs IFN IL-12 GM- IL-12 TNF MIP-1 gamma IL-2 IL-4 IL-6 IL-7 IL-8 IL-10 (p70) IL-13 MCP- eotaxiCSF IL-1a IL-1B (p40) IL-17 IP-10 MP-1 a IL-1ra B sil-2ra (20) (28) (32) (36) (38) (40) (44) (48) (50) 1 (58) n (4) (14) (22) (24) (46) (54) (56) a (64) (80) (26) (66) (76) AUT Age 4 OOR yrs 5.27< 190 14.6 173.4 39.4 70.2 1.4 16.6 286.5 84.7 835. 119 15.4 401 0.95 86.4 62.1 3.2 142 59.7 144.7

Age 7.52 OOR < 498.49 19.24 626.7 80.42 129.56*0.99 22.61275.41 126.7 1329.72 153.42 25.96 488.02*1.97 101.56 3.09 289.7 64.73 375.12 3 yrs

AUT 212.8 49.05 80.64 946.5 114.33 77.22 32.89 10.69 45.67 247.2 63.2 497.9 434.7 15.13 268.2 194.6 122.6 519.1 3.96 127.2 724.5 125.81

OOR 113.0 AUT 1.89 8.25 8.43 2.88< 4.43 4.79 6.66 1.88 300.9 120.1 363.4 12.14 2.36 46.95 4.19 3 14.46 4.21 60.39 33.17 119.57

1888. 1777. TYP 78.18 18.9 855.6 327.6 926.2 111.4 307.3 37.4 128.5 313.2 146.3 7 716.2 132.15 1 28.6 167.1 320.2 9.2 898.4 235.1 146.8

TYP 18.2 8.76 8.9 2.31 23.62 11.85 2.22 5.74 1.15 355.8 157.1 480.4 35.68 7.2 42.78 16.01 139.7 67.89 3.2 85.66 52.67OOR < GLOSSARY

Acidosis an abnormal condition characterized by reduced alkalinity of the blood and of the body tissues. Amino acids are the building blocks of proteins. While the body can make certain amino acids, others are re- quired from the food we eat in order to manufacture proteins in the liver. Ingested proteins are broken down into amino acids, and then absorbed, in the small intestine. Anaerobic living or existing in the absence of free oxygen. Antipyretic an agent that reduces fever. Antibodies (immunoglobulins [Ig]) are specialized proteins produced by white blood cells that circulate in the blood, hunting for and binding to foreign proteins, microorganisms or toxins in order to disable them. They are a critical part of the immune response. (See also Autoantibody.) Antifungals are medications that target fungus (yeast) via absorption into the blood stream, allowing the com- pound to permeate all tissues. Examples are Diflucan (Fluconazole), Sporonox (Itraconazole), Nizoral (Keto- conazole) and Lamisil (Terbinafine). These differ from Nystatin, which does not enter the bloodstream and has yeast-killing effects only in the gut (with benefits that may, however, extend beyond the gut). Antioxidants are beneficial substances that inhibit oxidation, or reactions promoted by oxygen, peroxides, or free radicals. They also support detoxification. Beta-carotene and vitamin C are examples. Apraxia loss or impairment of the ability to execute complex coordinated movements without muscular or sen- sory impairment. Auditory of, relating to, or experienced through hearing. Autoantibodies are antibodies that mistakenly attack the body’s own tissues as if they were pathogens. Autonomic Nervous System a part of the nervous system that innervates muscle and glandular tissues and gov- erns involuntary actions; consists of the sympathetic nervous system and the parasympathetic nervous sys- tem. Biofilms are colonies of connected organisms that can be a mix of different species. Examples vary from dental plaque to certain specialized bacterial colonies used to clean up oil spills. Biofilms also line the human bowel. Candida albicans is a fungus (“yeast”) naturally found in small amounts in the body; it can cause infection if the natural flora is thrown out of balance (usually by the use of antibiotic medication). These infections can be localized (e.g. a vaginal “yeast infection” or a rash on the bottom), or systemic. (Candidiasis is the name of an infection of a body part with a fungus from the genus Candida.) Carbohydrates are any member of a very abundant and widespread class of natural organic compounds that includes sugars, starch, and cellulose. They are commonly classified as monosaccharides (simple sugars, e.g., glucose, fructose), disaccharides (e.g., sucrose, lactose), oligosaccharides, and polysaccharides. Carbo- hydrates are the quickly accessible reservoir of energy, and oxidation of glucose in tissues supplies energy for metabolism. Casein is one of several proteins found in all milk from humans and other mammals. Chelation is the combination of a metal with an organic molecule to form a ring-like structure known as a chelate. Chelation of a metal may inhibit or enhance its bioavailability, but in medical terms usually refers to one of various methods used to remove toxic heavy metals such as lead or mercury from the body. Creatine is an amino acid that is biosynthesized in the body by the use of parts from three amino acids (argin- ine, glycine, and methionine), with about half from food sources, mainly from fish and meat. It helps to sup- ply energy to muscle and nerve cells. Without this process, some cells cannot “listen” (receive messages) and cannot “speak” (send out messages or release neurotransmitters). Cysteine is an amino acid that binds heavy metals such as lead and mercury, thereby helping to detoxify them. Cytokines are signaling compounds that deliver messages from one cell to another. They play a central role in the immune system, but are also involved in several stages of development as a baby embryo is formed; some of them promote inflammation, some dampen it. Cytotoxic poisonous to cells. Dysbiosis is a state of living (due to putrefaction, fermentation, deficiency, or sensitization) with intestinal flora that have harmful effects. A number of inflammatory diseases within the bowel, but also perhaps involving skin and connective tissue, have been reported in association with dysbiosis. Endogenous caused by factors inside the body.

GLOSSARY

Enzymes are biological catalysts that increase the speed of a chemical reaction without themselves being changed in the overall process. Enzymes are vitally important to the regulation of the chemistry of cells and organisms. In digestion, they break food down so it can be absorbed by the body. Epigenetics is the science that studies processes that change gene activity without changing the DNA of the gene. Methylation of DNA is the most-studied factor so far, and known or suspected drivers behind epige- netic processes include many agents: heavy metals, pesticides, diesel exhaust, tobacco smoke, hydrocarbons, hormones, radioactivity, viruses, bacteria, and basic nutrients. An example of epigenetic influence is the commonly observed difference in appearance between identical twins as they grow older. Some epigenetic changes can be passed in the gene code from parent to child. Epithelial cells line the inner portion of any structure of the body such as the GI system, lungs or urinary tract. Essential Fatty Acids: Omega-3, Omega-6, and Omega-9 fatty acids are essential nutrients. A balance is neces- sary between the three, with the best source of Omega-3 usually coming from purified fish oils, and Omega-6 from vegetable oil. Etiology means the cause of a disease. Excitotoxins are substances that cause damage or death to living cells. First discovered by some scientists in the 1950s, who noted that when they fed monosodium glutamate (commonly used at the time in Chinese res- taurants to stimulate taste buds) to baby mice, it destroyed the nerve cells in their eye retinas. Some excito- toxins are produced by the body, some come from outside. Exogenous caused by factors (i.e., food or a traumatic factor) or an agent (a disease-producing organism) from outside the body. Folate and folic acid are forms of water-soluble B vitamins. Folic acid is the synthetic vitamin and folate is the form found in foods (the term comes from a Latin word that also gives us “foliage”-- folate is found in leafy vegetables). Critical for the proper development and maintenance of cells, especially during times of rapid cell-division such as fetal development and childhood, it was discovered several years ago to be critically important for avoiding neural tube birth defects, so folic acid supplementation is recommended for all women who are pregnant or may become pregnant. Glutathione (GSH) is the major antioxidant in cells; along with cysteine, it is critical for binding and detoxify- ing heavy metals and free radicals. GSH is also required for synthesis of methylB12, so low levels can con- tribute to reduced methionine and therefore to impaired methylation. (See Methylation.) Gluten is protein found in the grass family of wheat, oats, barley, rye and triticale. Derivatives of these grains might contain gluten, including malt, grain starches, hydrolyzed vegetable/plant proteins, textured vegetable proteins, grain vinegar, soy sauce, grain alcohol, flavorings and the binders and fillers found in vitamins and medications. Homocysteine is an amino acid that is an intermediate in the metabolism of another sulfur-containing amino acid, methionine. Hormone an organic compound (often a steroid or peptide) that is produced in one part of the body and travels to another part to exert its action. Hormones regulate physiological activities including growth, reproduction, and homeostasis. Most hormones originate in specialized tissues and are carried to their targets through the circulation. Hypoperfusion decreased blood flow through an organ. Ileo-colonoscopy is the examination of the colon and the distal part of the small bowel with a CCD camera or a fiber optic camera on a flexible tube passed through the anus; including biopsies, it is the first-line investi- gation in the diagnosis, management, and monitoring of inflammatory bowel disease (IBD). Immunoglobulins (Ig) (antibodies) are proteins produced by plasma cells. They are designed to control the body’s immune response by binding to substances in the body that are recognized as foreign antigens. Anti- bodies are diverse, with more than 1010 possible variations, yet each is designed to recognize only a specific antigen. The antibodies are released into the intercellular fluid, where they bind to the infecting antigen, iden- tifying it for destruction by the immune system. Immunoglobulins also play a central role in allergies by binding to antigens that are not necessarily a threat to health, thus provoking an inflammatory reaction. There are five main types of antibody; IgA, IgD, IgE, IgG and IgM are the most common.

GLOSSARY

Inflammation normally, inflammation is part of a healthy immune response, an orchestrated onslaught of cells and chemicals that heal injury and fight infection—think redness, pain, swelling. But chronic "hidden" in- flammation occurs throughout the body when something kick-starts the immune system and disengages the shut-off button. Regardless of the cause, the end result is the same: an endless trickle of immune cells inter- feres with the body's healthy tissues. “Leaky Gut” Syndrome is an increase in intestinal permeability that allows passage into the bloodstream of food elements that don’t belong there. Lymphocytes determine the type of immune response; they defend the body against disease either by attacking bacteria and toxins directly (B-cells), or attacking the body’s own cells that have been taken over by viruses or become cancerous (T-cells). Together, these and lesser types of lymphocytes make up about 30% of the white blood cell count. Natural killer (NK) cells are lymphocytes. Lymphonodular hyperplasia (LNH) is an overgrowth of the cells in the lymph nodes. If found in the gut lin- ing, it’s described in one study as “common, but not an innocent bystander. Significantly related to a diagno- sis of GI food allergy in this study, it is an expression of mucosal immune response...if present in the termi- nal ileum, it may be related to food allergy but also to a variety of other immunologically active disease states.” Metabolism the chemical changes in living cells by which energy is provided for vital processes and activities and new material is assimilated. Metabolite a substance essential to the metabolism of a particular organism or to a particular metabolic process. Methylation is the transfer of a methyl group from one to another molecule, thus changing their structure and function; for instance, it makes epinephrine (adrenaline) from norepinephrine, and melatonin from serotonin. It is crucial for the transcription of DNA, turning the expression of genes on and off, and is a metabolic pathway that produces molecules affecting detoxification and anti-oxidation. If methylation is impaired, then neurotransmitter function, protein and cell membrane structure and function, fatty acid metabolism, allergic responses, myelination, and cellular energy transfer are all adversely affected. Mitochondria are very small energy-producing units within our cells that take part in cell growth and death, signaling, and differentiation, among other functions. They make ATP (adenosine triphosphate) as fuel for the cell. (See adenosine.) N-Acetyl Cysteine (NAC) is a precursor to intracellular glutathione. It is a known nucleophile and free radical scavenger; a potent and important antioxidant. Neurodegenerative relating to or marked by degeneration of nervous tissue. Neurotransmitter a substance that transmits nerve impulses across a synapse (a synapse is the point at which a nervous impulse passes from one neuron to another). Opioid any of a group of endogenous neural polypeptides that bind especially to opiate receptors and mimic some of the pharmacological properties of opiates (e.g., morphine) -- called also opioid peptide. Oxidative stress is caused by the normal by-products of metabolism (energy use), or the result of environmental and lifestyle insults like smoking cigarettes and chemicals like insecticides. It can cause damage to cells, premature aging, and disease, and is counteracted by supplying antioxidants from good food sources or sup- plements, as well as removal of factors that are not good for the body. Oxalate is a very simple sort of molecule; it links up with calcium and crystallizes under some conditions, in- cluding encounters with damaged tissues. The crystals formed this way can be painful to tissues in which they cause or increase inflammation. Oxytocin is a hormone that also acts as a neurotransmitter in the brain; it is thought to be released during hug- ging and touching in both sexes. It is involved in social recognition and bonding, and may be involved in the formation of trust between people. Paradigm a philosophical and theoretical framework of a scientific school or discipline within which theories, laws, and generalizations and the experiments performed in support of them are formulated. PBDEs polybrominated diphenylethers are a class of toxic chemicals in wide use to prevent the spread of fire and are likely to be found in dozens of products in your home, from the padding below your carpet, to your bed, couch or television screen. They are most commonly found in polyurethane foam products and electron- ics.

GLOSSARY

PCBs Polychlorinated Biphenyl belongs to a broad family of man-made organic chemicals; they were domestically manufactured from 1929 until1979, and used in hundreds of industrial and commercial applications including electrical, heat transfer, and hydraulic equipment; as plasticizers in paints, plastics, and rubber products; in pigments, dyes, and carbonless copy paper; and many other industrial applications. Peptide organic compound composed of a series of amino acids linked by peptide bonds between the carbon of one and the nitrogen of the next. Peptide chains longer than a few dozen amino acids are proteins. Many hormones, antibiotics, and other compounds that participate in life processes are peptides. PET scan a sectional view of the body constructed by positron-emission tomography. Phenols are similar to alcohols but form stronger hydrogen bonds with water (see bonding), so they dissolve more readily in water and boil at higher temperatures. They may be colorless liquids or white solids; many have a sharp, spicy odor. Some are found in essential oils. Phenotype of an individual is the class to which that person belongs as determined by the description of their physical and behavioral characteristics. The phenotype may change constantly throughout the life of an indi- vidual. In terms of children with ASDs, medical treatment can be approached by defining their similarities to children who have been successfully treated before them. Polymorphism genetic variation that results in the occurrence of several different forms or types of individuals among the members of a single species. Porphyrins are complex molecules in the body that combine with iron to produce heme, which is responsible for giving blood its red color, and helps form hemoglobin. Heme delivers oxygen through the circulatory sys- tem and is therefore important in metabolism. Disordered urinary porphyrins can indicate a toxic exposure. Urinary porphyrin concentrations vary substantially with age among children and adolescents; it’s important to be careful about reference ranges when interpreting lab results. Prebiotics non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways believed to be beneficial to health. Probiotics are certain friendly bacteria that are beneficial in preventing and treating some intestinal problems. They improve the environment in the intestinal tract by producing acids and other compounds that inhibit the growth of undesirable bacteria. They’re available in supplement form in the refrigerated section of health food stores. Salicylates chemicals that occur naturally in many plants, including many fruits vegetables, and herbs. Salicy- lates in plants act as a natural immune hormone and preservative, protecting the plants against diseases, in- sects, fungi, and harmful bacteria. Salicylates are also created synthetically and can be found in many medi- cines, perfumes and preservatives. SPECT scan a single-photon emission computerized tomography (SPECT) scan lets your doctor analyze the function of some of your internal organs. A SPECT scan is a type of nuclear imaging test, which means it uses a radioactive substance and a special camera to create 3-D pictures. While imaging tests such as X-rays can show what the structures inside your body look like, a SPECT scan produces images that show how your organs work. For instance, a SPECT scan can show how blood flows to your heart or what areas of your brain are more active or less active. Sulfation a phase-II detoxification pathway that occurs in the liver and in which compounds that contain sulfur combine with components to remove them from the body. Transsulfuration is a major detoxification pathway, the precursors of which are supplied by the methylation cycle. Transsulfuration involves the formation of glutathione as the primary detoxifier; cysteine and sulfate are also produced through this pathway.

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Uncover the Myths: Discover the Truth Epilepsy Awareness

Time the don’t put seizure with anything a watch in mouth as seizure ends, offer help Look For Medical alert don’t identification hold down

cushion head, loosen tight turn remove glasses clothing on side

MYTH: You must put something in someone’s mouth to hold MYTH: People with epilepsy are mentally challenged and need their tongue down when they have a seizure. to go to special schools. FACT: Never put anything in the mouth of someone who is FACT: Some of the smartest and most creative people in history having a seizure. are thought to have had epilepsy including Thomas Edison, Julius Caesar, Vincent Van Gogh, Socrates and MYTH: You can stop a seizure by holding someone down. Leonardo DaVinci. Most people with epilepsy have normal FACT: Never hold someone down while they’re having a seizure. IQs, attend regular colleges and live normal, full lives. This is very dangerous and can injure both of you. MYTH: All seizures involve falling on the ground and shaking MYTH: If someone with epilepsy sees flashing lights, they will (convulsions). have a seizure. FACT: Most people who have epilepsy don’t have seizures with FACT: A very small percentage of people with epilepsy convulsions. In fact, there are many kinds of seizures. have seizures triggered by flashing. This is called Some last only a few seconds, similar to “zoning photosensitivity. Most people with epilepsy can be out,” while others cause a person to be confused and around strobe lights, play video games and see flashing disoriented for a minute or two, sometimes with no lights without a problem. recollection of what might have just happened.

EFA 344

1-800-332-1000 This publication was made possible by a grant from the Centers for www.epilepsyfoundation.org Disease Control and Prevention (grant number 1 U58 DP000606-02) and its contents are solely the responsibility of its authors and do not necessarily represent the official views of the CDC. © 2008 Epilepsy Foundation of America, Inc. K1520-1004 8&9!:;<&9!=#8>?' &$#!:;'@?$!%&A

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