The Biological Plausibility of a Relationship between Vaccines and Autism Spectrum Disorders David Berger, MD Medical Director Wholistic Pediatrics Tampa, FL (813) 960-3415 www.wholisticpeds.com A PRESENTATION TO THE FLORIDA GOVERNOR’S TASK FORCE ON AUTISM SPECTRUM DISORDERS

Discover Magazine, 3/14/07

IMMUNIZATIONS I am not suggesting that we abandon the use of vaccines I am concerned about the growing number of chronically ill children There are more children with learning disabilities and hyperinflammatory/ autoimmune disorders then there has ever been in the history of medicine.

I am not suggesting that we abandon the use of vaccines

I am concerned about the growing number of chronically ill children

There are more children with learning disabilities and hyperinflammatory/ autoimmune disorders then there has ever been in the history of medicine.

Concerns about Vaccines Are we unnaturally stressing underdeveloped immune systems beyond their capabilities in our effort to keep the children from becoming ill? There are inadequate safety studies for the vaccines that are currently on the market Are we giving too many vaccines too early in life or over a short time span? We do not have a clear understanding of the effects of some of the vaccine components such as Thimerosal, aluminum, formaldehyde, and human fetal tissue. Nor do we understand how they interact with atypical immune systems and toxins that people are more commonly being exposed to.

Are we unnaturally stressing underdeveloped immune systems beyond their capabilities in our effort to keep the children from becoming ill?

There are inadequate safety studies for the vaccines that are currently on the market

Are we giving too many vaccines too early in life or over a short time span?

We do not have a clear understanding of the effects of some of the vaccine components such as Thimerosal, aluminum, formaldehyde, and human fetal tissue. Nor do we understand how they interact with atypical immune systems and toxins that people are more commonly being exposed to.

Mercury levels in Certain Fish Species (PPM) (EPA 2006; http://www.cfsan.fda.gov/~frf/sea-mehg.html) MACKEREL KING 0.730 SHARK 0.988 SWORDFISH 0.976 TILEFISH (Gulf of Mexico) 1.450 BASS (SALTWATER, BLACK, STRIPED) 3 0.219 BASS CHILEAN 0.386 BLUEFISH 0.337 CARP 0.14 GROUPER (ALL SPECIES) 0.465 HALIBUT 0.252 LOBSTER (NORTHERN/AMERICAN) 0.310 MACKEREL SPANISH (Gulf of Mexico) 0.454 MARLIN * 0.485 ORANGE ROUGHY 0.554 SNAPPER 0.189 TUNA (CANNED, ALBACORE) 0.353 TUNA(FRESH/FROZEN, ALL) 0.383 TUNA (FRESH/FROZEN,ALBACORE) 0.357 TUNA (FRESH/FROZEN, BIGEYE) 0.639 TUNA (FRESH/FROZEN, SKIPJACK) 0.205 TUNA (FRESH/FROZEN, YELLOWFIN) 0.325 TUNA (FRESH/FROZEN, Species Unknown) 0.414

On average, for each 1000 lb of environmentally released mercury, there was a 43% increase in the rate of special education services and a 61% increase in the rate of autism. Palmer et al. Health & Place 12 (2006) 203–209 Total toxicity Autism rates Proximity to point sources of environmental mercury release as a predictor of autism prevalence. Palmer et al. Health & Place 2008

Dental Amalgams as a source of Mercury Harris 2008, University of Sydney, “ Migration of mercury from dental amalgam through human teeth ”: Most importantly the detection of Hg in areas of the tooth that once contained an active bloodstream and in calculus indicates that both exposure pathways should be considered as significant. Austin 2008, Swinburne University of Technology, Melbourne, “An epidemiological analysis of the ‘autism as mercury poisoning’ hypothesis”: to examine the autism as mercury poisoning hypothesis, this paper reviews the existing scientific literature within the context of established epidemiological criteria and finds that the evidence for a causal relationship is compelling. Exposure to mercury (via vaccines and maternal dental amalgam) in utero and during infant years is confirmed; ….. given the severity, devastating lifelong impact and extremely high prevalence of autism, it would be negligent to continue to expose pregnant and nursing mothers and infant children to any amount of avoidable mercury.

Harris 2008, University of Sydney, “ Migration of mercury from dental amalgam through human teeth ”: Most importantly the detection of Hg in areas of the tooth that once contained an active bloodstream and in calculus indicates that both exposure pathways should be considered as significant.

Austin 2008, Swinburne University of Technology, Melbourne, “An epidemiological analysis of the ‘autism as mercury poisoning’ hypothesis”: to examine the autism as mercury poisoning hypothesis, this paper reviews the existing scientific literature within the context of established epidemiological criteria and finds that the evidence for a causal relationship is compelling. Exposure to mercury (via vaccines and maternal dental amalgam) in utero and during infant years is confirmed; ….. given the severity, devastating lifelong impact and extremely high prevalence of autism, it would be negligent to continue to expose pregnant and nursing mothers and infant children to any amount of avoidable mercury.

Children with Autism are Prone to Decreased Ability to Detoxify Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. James SJ , Cutler P , Melnyk S , Jernigan S , Janak L , Gaylor DW , Neubrander JA . Department of Pediatrics, University of Arkansas for Medical Sciences Am J Clin Nutr. 2004 Dec;80(6):1611-7 BACKGROUND: Autism is a complex neurodevelopmental disorder that usually presents in early childhood and that is thought to be influenced by genetic and environmental factors. Although abnormal metabolism of methionine and homocysteine has been associated with other neurologic diseases, these pathways have not been evaluated in persons with autism. OBJECTIVE: The purpose of this study was to evaluate plasma concentrations of metabolites in the methionine transmethylation and transsulfuration pathways in children diagnosed with autism. DESIGN: Plasma concentrations of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), adenosine, homocysteine, cystathionine, cysteine, and oxidized and reduced glutathione were measured in 20 children with autism and in 33 control children. On the basis of the abnormal metabolic profile, a targeted nutritional intervention trial with folinic acid, betaine, and methylcobalamin was initiated in a subset of the autistic children. RESULTS: Relative to the control children, the children with autism had significantly lower baseline plasma concentrations of methionine, SAM, homocysteine, cystathionine, cysteine, and total glutathione and significantly higher concentrations of SAH, adenosine, and oxidized glutathione. This metabolic profile is consistent with impaired capacity for methylation (significantly lower ratio of SAM to SAH) and increased oxidative stress (significantly lower redox ratio of reduced glutathione to oxidized glutathione) in children with autism. The intervention trial was effective in normalizing the metabolic imbalance in the autistic children. CONCLUSIONS: An increased vulnerability to oxidative stress and a decreased capacity for methylation may contribute to the development and clinical manifestation of autism.

Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism.

James SJ , Cutler P , Melnyk S , Jernigan S , Janak L , Gaylor DW , Neubrander JA .

Department of Pediatrics, University of Arkansas for Medical Sciences

Am J Clin Nutr. 2004 Dec;80(6):1611-7

BACKGROUND: Autism is a complex neurodevelopmental disorder that usually presents in early childhood and that is thought to be influenced by genetic and environmental factors. Although abnormal metabolism of methionine and homocysteine has been associated with other neurologic diseases, these pathways have not been evaluated in persons with autism. OBJECTIVE: The purpose of this study was to evaluate plasma concentrations of metabolites in the methionine transmethylation and transsulfuration pathways in children diagnosed with autism. DESIGN: Plasma concentrations of methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), adenosine, homocysteine, cystathionine, cysteine, and oxidized and reduced glutathione were measured in 20 children with autism and in 33 control children. On the basis of the abnormal metabolic profile, a targeted nutritional intervention trial with folinic acid, betaine, and methylcobalamin was initiated in a subset of the autistic children. RESULTS: Relative to the control children, the children with autism had significantly lower baseline plasma concentrations of methionine, SAM, homocysteine, cystathionine, cysteine, and total glutathione and significantly higher concentrations of SAH, adenosine, and oxidized glutathione. This metabolic profile is consistent with impaired capacity for methylation (significantly lower ratio of SAM to SAH) and increased oxidative stress (significantly lower redox ratio of reduced glutathione to oxidized glutathione) in children with autism. The intervention trial was effective in normalizing the metabolic imbalance in the autistic children. CONCLUSIONS: An increased vulnerability to oxidative stress and a decreased capacity for methylation may contribute to the development and clinical manifestation of autism.

SAM SAH MTase SAHH Homocysteine B6 THF MS CBS B12 Protein synthesis BHMT Choline Betaine Effect of Oxidative Stress on Methionine Transsulfuration THF: tetrahydrofolate 5-CH 3 THF Methylation of DNA, RNA, proteins, membrane phospholipids, creatine, neurotransmittors Cystathionine Cysteine GSH GSSG Methionine Adenosine ( AK and/or ADA) MAT B6

Neurotoxicity of Thimerosal in Human Brain Cells is Associated with Glutathione Depletion: Protective Effect of Cysteine or Glutathione Supplementation Neurotoxicology. 2005 Jan;26(1):1-8 S. Jill James, William Slikker, Elizabeth New, Stefanie Jernigan, Stepan Melnyk Department of Pediatrics University of Arkansas for Medical Sciences Little Rock, AR

0 2.5 5 10 20 VIABILITY OF GLIOBLASTOMA AND NEUROBLASTOMA CELLS WITH INCREASING DOSE OF THIMEROSAL Viability (MTT OD) Glioblastoma Cells Neuroblastoma Cells ( 48 hr Exposure ) ( 3 hr Exposure ) 0 2.5 5 10 20 0 2.5 5 10 20  M Thimerosal  M Thimerosal

Control Thimerosal +GSH + Cystine +NAC + Methionine O.D. (Viability) Viability of Glioblastoma cells exposed to 15  M Thimerasol in the presence of GSH-ester, Cystine, N-acetylcysteine (NAC), or Methionine

Control Thimerosal +GSH + Cystine +NAC + Methionine O.D. (Viability) Viability of Neuroblastoma cells exposed to 15  M Thimerosal Pretreated with 100  M GSH-ester, Cystine, N-acetylcysteine (NAC), or Methionine

So, Why is this happening? Certain toxins such as mercury can inhibit the enzymes of this pathway. Dr James has looked at the DNA sequences that code for the proteins that make up these enzymes and has found that autistic children have up to 3 times as many single DNA mutations (polymorphisms, SNPs) as do children without autism We have identified these SNPs in children with other neurodevelopmental disorders

Certain toxins such as mercury can inhibit the enzymes of this pathway.

Dr James has looked at the DNA sequences that code for the proteins that make up these enzymes and has found that autistic children have up to 3 times as many single DNA mutations (polymorphisms, SNPs) as do children without autism

We have identified these SNPs in children with other neurodevelopmental disorders

Jill James, 2004 Assessment of Single Nucleotide Polymorphins in Children with Autism vs. Controls

What’s Going On? Social deficits, shyness, social withdrawal Repetitive, perseverative, stereotypic behaviors; obsessive-compulsive tendencies Irritability, aggression, temper tantrums Lacks eye contact; impaired visual fixation Loss of speech, delayed language, failure to develop speech Speech comprehension deficits Sound sensitivity; mild to profound hearing loss Abnormal touch sensations; touch aversion Flapping, myoclonal jerks, choreiform movements, circling, rocking, toe walking, unusual postures Poor concentration, attention, response inhibition Self injurious behavior, e.g. head banging ADHD traits Sleep difficulties Diarrhea; abdominal pain/discomfort, constipation ALL SIGNS AND SYMPTOMS OF… MERCURY TOXICITY (Bernard, 2000)

Social deficits, shyness, social withdrawal

Repetitive, perseverative, stereotypic behaviors; obsessive-compulsive tendencies

Irritability, aggression, temper tantrums

Lacks eye contact; impaired visual fixation

Loss of speech, delayed language, failure to develop speech

Speech comprehension deficits

Sound sensitivity; mild to profound hearing loss

Abnormal touch sensations; touch aversion

Flapping, myoclonal jerks, choreiform movements, circling, rocking, toe walking, unusual postures

Poor concentration, attention, response inhibition

Self injurious behavior, e.g. head banging

ADHD traits

Sleep difficulties

Diarrhea; abdominal pain/discomfort, constipation

ALL SIGNS AND SYMPTOMS OF…

MERCURY TOXICITY

(Bernard, 2000)

The developing fetus and young children are thought to be disproportionately affected by mercury exposure, because many aspects of development, particularly brain maturation, can be disturbed by the presence of mercury. Minimizing mercury exposure is, therefore, essential to optimal child health ….. Mercury in all of its forms is toxic to the fetus and children, and efforts should be made to reduce exposure to the extent possible to pregnant women and children as well as the general population . _______________________________________________________________ Vaccine inserts would typically say “0.01% thimerosal as a preservative”, which to anyone would sound like an extremely small amount. When called to testify in front of the Institute of Medicine, an independent group formed by our government to monitor safety issues, Dr. Neil Halsey of Johns Hopkins University, and head of the vaccine recommendation committee that reports to the CDC, went on record as saying “No one ever did the math…. No one knows what dose of mercury, if any, from vaccines is safe. We can say there is no evidence of harm but the truth is no one has looked” MERCURY Statement: Pediatrics 2001 Jul, American Academy of Pediatrics: Committee on Environmental Health.

Mercury/Thimerosal Thimerosal is Ethylmercury, a neurotoxin Mercury was found in the blood of newborns even before Hepatitis B shot, and higher levels after the shot. Journal of Pediatrics, May 2000 In some pre-term infants, mercury levels were 10 times that of term infants Pre-term babies are vaccinated according to chronological age, not gestational age.

Thimerosal is Ethylmercury, a neurotoxin

Mercury was found in the blood of newborns even before Hepatitis B shot, and higher levels after the shot.

Journal of Pediatrics, May 2000

In some pre-term infants, mercury levels were 10 times that of term infants

Pre-term babies are vaccinated according to chronological age, not gestational age.

Mercury/Thimerosal Typical Thimerosal Exposure for 2 month old infant: Hep B 12.5 mcg DTaP 25 mcg Hib 25 mcg Total 62.5 mcg of which 50% is ethylmercury = 31.25mcg Total “safe” dose for 10 pound (2 month old) baby by EPA standards: 0.5 mcg. The average 2 month old received ~60x the EPA limit

Typical Thimerosal Exposure for 2 month old infant:

Hep B 12.5 mcg

DTaP 25 mcg

Hib 25 mcg

Total 62.5 mcg

of which 50% is ethylmercury = 31.25mcg

Total “safe” dose for 10 pound (2 month old) baby by EPA standards: 0.5 mcg. The average 2 month old received ~60x the EPA limit

Mercury/Thimerosal By 6 months of age, a fully vaccinated infant would have received: 3 DTP 75 mcg thimerosal 3 Hib 75 mcg thimerosal 3 Hep B 37.5 mcg thimerosal Total 187.5 mcg thimerosal 93.75 mcg mercury 1999 FDA Center for Biologics Evaluation and Research

By 6 months of age, a fully vaccinated infant would have received:

3 DTP 75 mcg thimerosal

3 Hib 75 mcg thimerosal

3 Hep B 37.5 mcg thimerosal

Total 187.5 mcg thimerosal

93.75 mcg mercury

1999 FDA Center for Biologics Evaluation and Research

Heavy Metal Exposures After exposure to mercury, the length of time to be eliminated varies for different organs: Blood and Hair: 4-6 months Non CNS organs: several years Brain: 20 years (Boyd Haley, PhD, University of Kentucky, Dept of Chemistry) Lead typically deposits into brain and bone. After exposure to lead, within several months the blood and urine levels will be normal even if the lead is still in the bone and brain (Clarkson, 2002)

After exposure to mercury, the length of time to be eliminated varies for different organs:

Blood and Hair: 4-6 months

Non CNS organs: several years

Brain: 20 years

(Boyd Haley, PhD, University of Kentucky, Dept of Chemistry)

Lead typically deposits into brain and bone. After exposure to lead, within several months the blood and urine levels will be normal even if the lead is still in the bone and brain (Clarkson, 2002)

Some Children with Autism Do Not Clear Mercury Mercury in first-cut baby hair of children with autism versus typically-developing children J. B. Adams; J. Romdalvik; K. E. Levine; Lin-Wen Hu (Arizona State University) Toxicological & Environmental Chemistry , May 2, 2008 Children with autism were examined to determine amounts of mercury (Hg) in their baby hair and the factors that might affect Hg body burden. US children with autism ( n = 78) and matched controls ( n = 31) born between 1988 and 1999 were studied. Hg in first-cut baby hair was determined using cold vapor atomic fluorescence spectrometry (CVAFS). Twenty samples were split and also measured with Neutron Activation Analysis (NAA). Logistic regression analysis showed that compared to children with higher levels of mercury (above 0.55 mcg g-1), children with lower levels of mercury in their hair (below 0.55 mcg g-1) were 2.5-fold more likely to manifest with autism . Children with autism had similar mercury exposure as controls from maternal seafood and maternal dental amalgams. Children with autism also had 2.5-fold higher incidence of oral antibiotic use during their first 18 months of life. Their mothers were possibly more likely to use oral antibiotics during pregnancy or nursing. The amount of Hg in the baby hair of children with autism showed a significant correlation with the number of maternal dental amalgams. The lower level of Hg in the baby hair of children with autism indicates an altered metabolism of Hg, and may be due to a decreased ability to excrete Hg. This is consistent with usage of higher amounts of oral antibiotics, which are known to inhibit Hg excretion in rats due to alteration of gut flora, and may exert a similar effect in humans. Higher usage of oral antibiotics in infancy may also partially explain the high incidence of chronic gastrointestinal problems seen in individuals with autism.

Heavy Metals Accumulate in Children with Autism Austin (2008), Swinburne University of Technology, Melbourne, “An investigation of porphyrinuria in Australian children with autism.”: Two recent studies, from France (Nataf et al., 2006) and the United States (Geier & Geier, 2007), identified atypical urinary porphyrin profiles in children with an autism spectrum disorder (ASD). These profiles serve as an indirect measure of environmental toxicity generally, and mercury (Hg) toxicity specifically, with the latter being a variable proposed as a causal mechanism of ASD (Bernard et al., 2001; Mutter et al., 2005). To examine whether this phenomenon occurred in a sample of Australian children with ASD, an analysis of urinary porphyrin profiles was conducted. A consistent trend in abnormal porphyrin levels was evidenced when data was compared with those previously reported in the literature. The results are suggestive of environmental toxic exposure impairing heme synthesis. Three independent studies from three continents have now demonstrated that porphyrinuria is concomitant with ASD, and that Hg may be a likely xenobiotic to produce porphyrin profiles of this nature.

Austin (2008), Swinburne University of Technology, Melbourne, “An investigation of porphyrinuria in Australian children with autism.”: Two recent studies, from France (Nataf et al., 2006) and the United States (Geier & Geier, 2007), identified atypical urinary porphyrin profiles in children with an autism spectrum disorder (ASD). These profiles serve as an indirect measure of environmental toxicity generally, and mercury (Hg) toxicity specifically, with the latter being a variable proposed as a causal mechanism of ASD (Bernard et al., 2001; Mutter et al., 2005). To examine whether this phenomenon occurred in a sample of Australian children with ASD, an analysis of urinary porphyrin profiles was conducted. A consistent trend in abnormal porphyrin levels was evidenced when data was compared with those previously reported in the literature. The results are suggestive of environmental toxic exposure impairing heme synthesis. Three independent studies from three continents have now demonstrated that porphyrinuria is concomitant with ASD, and that Hg may be a likely xenobiotic to produce porphyrin profiles of this nature.

Mitochondria, Vaccines and Autism The case of Hannah Polling: “ According to the concession report, this case involved a toddler who was developing normally until 18 months of age. Several days after the 18 month shots her development began to decline to the point that she eventually displayed many features of autism. Doctors also discovered that the child had a mitochondrial disorder (a disorder of metabolism that makes a child pre-disposed to developmental and medical problems). The court decided that there was enough evidence to show that the vaccines may have aggravated the mitochondrial disorder and triggered problems consistent with autistic-like behavior.” Dr Robert Sears, Author of “The Vaccine Book: Making the right decision for your child”

The case of Hannah Polling:

“ According to the concession report, this case involved a toddler who was developing normally until 18 months of age. Several days after the 18 month shots her development began to decline to the point that she eventually displayed many features of autism. Doctors also discovered that the child had a mitochondrial disorder (a disorder of metabolism that makes a child pre-disposed to developmental and medical problems). The court decided that there was enough evidence to show that the vaccines may have aggravated the mitochondrial disorder and triggered problems consistent with autistic-like behavior.”

Dr Robert Sears, Author of “The Vaccine Book: Making the right decision for your child”

Mitochondrial Dysfunction and Autism Evidence of Mitochondrial Dysfunction in Autism and Implications for Treatment Daniel A. Rossignol, J. Jeffrey Bradstreet, International Child Development Resource Center Abstract: Classical mitochondrial diseases occur in a subset of individuals with autism and are usually caused by genetic anomalies or mitochondrial respiratory pathway deficits. However, in many cases of autism, there is evidence of mitochondrial dysfunction (MtD) without the classic features associated with mitochondrial disease . MtD appears to be more common in autism and presents with less severe signs and symptoms. It is not associated with discernable mitochondrial pathology in muscle biopsy specimens despite objective evidence of lowered mitochondrial functioning. Exposure to environmental toxins is the likely etiology for MtD in autism. This dysfunction then contributes to a number of diagnostic symptoms and comorbidities observed in autism including: cognitive impairment, language deficits, abnormal energy metabolism, chronic gastrointestinal problems, abnormalities in fatty acid oxidation, and increased oxidative stress . MtD and oxidative stress may also explain the high male to female ratio found in autism due to increased male vulnerability to these dysfunctions. Biomarkers for mitochondrial dysfunction have been identified, but seem widely under-utilized despite available therapeutic interventions . Nutritional supplementation to decrease oxidative stress along with factors to improve reduced glutathione, as well as hyperbaric oxygen therapy (HBOT) represent supported and rationale approaches . The underlying pathophysiology and autistic symptoms of affected individuals would be expected to either improve or cease worsening once effective treatment for MtD is implemented.

Mercury Inhibits Mitochondrial Energy Metabolism At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish Cambier S , Bénard G , Mesmer-Dudons N , Gonzalez P , Rossignol R , Brèthes D , Bourdineaud JP . Int J Biochem Cell Biol. 2008 Aug 13 The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13mug of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish

Cambier S , Bénard G , Mesmer-Dudons N , Gonzalez P , Rossignol R , Brèthes D , Bourdineaud JP .

Int J Biochem Cell Biol. 2008 Aug 13

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13mug of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Vaccines can Induce Th2 weighted Immunity Delay in diphtheria, pertussis, tetanus vaccination is associated with a reduced risk of childhood asthma McDonald et al(2008), University of Manitoba J Allergy Clin Immunol. 2008 Mar;121(3):626-31 “ Many early childhood vaccinations have been viewed as promoters of asthma development by stimulating a Th2-type immune response, shifting the cytokine balance” (Johnston, et al, 2002) “ At birth the newborn immune system has a limited ability to produce Th1 cytokines but levels increase over the period of the next 6 months” (Marodi, 2002) CONCLUSION: We found a negative association between delay in administration of the first dose of whole-cell DPT immunization in childhood and the development of asthma; the association was greater with delays in all of the first 3 doses. The mechanism for this phenomenon requires further research.

Delay in diphtheria, pertussis, tetanus vaccination is associated with a reduced risk of childhood asthma

McDonald et al(2008), University of Manitoba

J Allergy Clin Immunol. 2008 Mar;121(3):626-31

“ Many early childhood vaccinations have been viewed as promoters of asthma development by stimulating a Th2-type immune response, shifting the cytokine balance” (Johnston, et al, 2002)

“ At birth the newborn immune system has a limited ability to produce Th1 cytokines but levels increase over the period of the next 6 months” (Marodi, 2002)

CONCLUSION: We found a negative association between delay in administration of the first dose of whole-cell DPT immunization in childhood and the development of asthma; the association was greater with delays in all of the first 3 doses. The mechanism for this phenomenon requires further research.

Vaccine Induced Autoimmunity (Cohen&Shoenfeld, Tel Aviv University, Journal of Autoimmunity , 1996)

Mercury Induced Autoimmunity IL-12 Down-Regulates Autoantibody Production in Mercury-Induced Autoimmunity Lee M. Bagenstose, Padmini Salgame 2 and Marc Monestier 2 Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140 In genetically susceptible H-2 s mice , subtoxic doses of mercuric chloride (HgCl 2 )induce a complex autoimmune syndrome characterized by the production of anti-nucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1 and IgE Abs, and renal Ig deposits. Mercury-induced autoimmunity in H-2 s mice provides a useful model for chemically related autoimmunity in humans . The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in this syndrome . The IL-12 cytokine induces T cell proliferation and IFN- production and is necessary for differentiation of naive T cells into the Th1 subset. To gain an understanding of T cell control in this syndrome and, in particular, Th1/Th2 regulation, we assessed the effect of IL-12 administration in mercury-induced autoimmunity. Groups of A.SW mice (H-2 s ) received HgCl 2 plus IL-12, HgCl 2 alone, or IL-12 alone. IL-12 treatment resulted in a dramatic reduction of the anti-nucleolar Ab titers. IL-12 also inhibited the HgCl 2 -induced serum IgG1 increase, but, in contrast, did not significantly affect IgE induction in this model. This observation may be related to our unexpected finding that IL-12 further potentiated the HgCl 2 -triggered IL-4 induction in this model. The levels of renal Ig deposits were similar in mice receiving HgCl 2 alone or HgCl 2 plus IL-12. Our results indicate that IL-12 can down-regulate the autoimmune component of this experimental syndrome and that the various manifestations of mercury-induced autoimmunity are independently regulated.

IL-12 Down-Regulates Autoantibody Production in Mercury-Induced Autoimmunity

Lee M. Bagenstose, Padmini Salgame 2 and Marc Monestier 2

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140

In genetically susceptible H-2 s mice , subtoxic doses of mercuric chloride (HgCl 2 )induce a complex autoimmune syndrome characterized by the production of anti-nucleolar IgG Abs, lymphoproliferation, increased serum levels of IgG1 and IgE Abs, and renal Ig deposits. Mercury-induced autoimmunity in H-2 s mice provides a useful model for chemically related autoimmunity in humans . The increase in serum IgG1 and IgE, which are under IL-4 control, suggests a role for the Th2 subset in this syndrome . The IL-12 cytokine induces T cell proliferation and IFN- production and is necessary for differentiation of naive T cells into the Th1 subset. To gain an understanding of T cell control in this syndrome and, in particular, Th1/Th2 regulation, we assessed the effect of IL-12 administration in mercury-induced autoimmunity. Groups of A.SW mice (H-2 s ) received HgCl 2 plus IL-12, HgCl 2 alone, or IL-12 alone. IL-12 treatment resulted in a dramatic reduction of the anti-nucleolar Ab titers. IL-12 also inhibited the HgCl 2 -induced serum IgG1 increase, but, in contrast, did not significantly affect IgE induction in this model. This observation may be related to our unexpected finding that IL-12 further potentiated the HgCl 2 -triggered IL-4 induction in this model. The levels of renal Ig deposits were similar in mice receiving HgCl 2 alone or HgCl 2 plus IL-12. Our results indicate that IL-12 can down-regulate the autoimmune component of this experimental syndrome and that the various manifestations of mercury-induced autoimmunity are independently regulated.

Children with Autism are prone to autoimmunity Prevalence of serum antibodies to caudate nucleus in autistic children Vijendra K. Singh , and Wyatt H. Rivas Department of Biology, Biotechnology Center Building, Utah State University Neuroscience Letters, October 2003 Autism may involve autoimmunity to brain. We studied regional distribution of antibodies to rat caudate nucleus, cerebral cortex, cerebellum, brain stem and hippocampus. The study included 30 normal and 68 autistic children. Antibodies were assayed by immunoblotting. Autistic children, but not normal children, had antibodies to caudate nucleus (49% positive sera), cerebral cortex (18% positive sera) and cerebellum (9% positive sera). Brain stem and hippocampus were negative. Antibodies to caudate nucleus were directed towards three proteins having 160, 115 and 49 kD molecular weights. Since a significant number of autistic children had antibodies to caudate nucleus, we propose that an autoimmune reaction to this brain region may cause neurological impairments in autistic children. Thus, the caudate nucleus might be involved in the neurobiology of autism.

Prevalence of serum antibodies to caudate nucleus in autistic children

Vijendra K. Singh , and Wyatt H. Rivas

Department of Biology, Biotechnology Center Building, Utah State University

Neuroscience Letters, October 2003

Autism may involve autoimmunity to brain. We studied regional distribution of antibodies to rat caudate nucleus, cerebral cortex, cerebellum, brain stem and hippocampus. The study included 30 normal and 68 autistic children. Antibodies were assayed by immunoblotting. Autistic children, but not normal children, had antibodies to caudate nucleus (49% positive sera), cerebral cortex (18% positive sera) and cerebellum (9% positive sera). Brain stem and hippocampus were negative. Antibodies to caudate nucleus were directed towards three proteins having 160, 115 and 49 kD molecular weights. Since a significant number of autistic children had antibodies to caudate nucleus, we propose that an autoimmune reaction to this brain region may cause neurological impairments in autistic children. Thus, the caudate nucleus might be involved in the neurobiology of autism.

Children with Autism are prone to autoimmunity Abnormal Measles-Mumps-Rubella Antibodies and CNS Autoimmunity in Children with Autism Vijendra K. Singh, Sheren X. Lin, Elizabeth Newell, Courtney Nelson Department of Biology and Biotechnology Center, Utah State University, Logan, Utah, USA Journal of Biomedical Science 9:4:2002, 359-364. Autoimmunity to the central nervous system (CNS), especially to myelin basic protein (MBP), may play a causal role in autism, a neurodevelopmental disorder. Because many autistic children harbor elevated levels of measles antibodies, we conducted a serological study of measles-mumps-rubella (MMR) and MBP autoantibodies. Using serum samples of 125 autistic children and 92 control children , antibodies were assayed by ELISA or immunoblotting methods . ELISA analysis showed a significant increase in the level of MMR antibodies in autistic children . Immunoblotting analysis revealed the presence of an unusual MMR antibody in 75 of 125 (60%) autistic sera but not in control sera . This antibody specifically detected a protein of 73-75 kD of MMR. This protein band, as analyzed with monoclonal antibodies , was immunopositive for measles hemagglutinin (HA) protein but not for measles nucleoprotein and rubella or mumps viral proteins. Thus the MMR antibody in autistic sera detected measles HA protein, which is unique to the measles subunit of the vaccine. Furthermore, over 90% of MMR antibody-positive autistic sera were also positive for MBP autoantibodies, suggesting a strong association between MMR and CNS autoimmunity in autism. Stemming from this evidence, we suggest that an inappropriate antibody response to MMR, specifically the measles component thereof, might be related to pathogenesis of autism

Abnormal Measles-Mumps-Rubella Antibodies and CNS Autoimmunity in Children with Autism

Vijendra K. Singh, Sheren X. Lin, Elizabeth Newell, Courtney Nelson Department of Biology and Biotechnology Center, Utah State University, Logan, Utah, USA Journal of Biomedical Science 9:4:2002, 359-364. Autoimmunity to the central nervous system (CNS), especially to myelin basic protein (MBP), may play a causal role in autism, a neurodevelopmental disorder. Because many autistic children harbor elevated levels of measles antibodies, we conducted a serological study of measles-mumps-rubella (MMR) and MBP autoantibodies. Using serum samples of 125 autistic children and 92 control children , antibodies were assayed by ELISA or immunoblotting methods . ELISA analysis showed a significant increase in the level of MMR antibodies in autistic children . Immunoblotting analysis revealed the presence of an unusual MMR antibody in 75 of 125 (60%) autistic sera but not in control sera . This antibody specifically detected a protein of 73-75 kD of MMR. This protein band, as analyzed with monoclonal antibodies , was immunopositive for measles hemagglutinin (HA) protein but not for measles nucleoprotein and rubella or mumps viral proteins. Thus the MMR antibody in autistic sera detected measles HA protein, which is unique to the measles subunit of the vaccine. Furthermore, over 90% of MMR antibody-positive autistic sera were also positive for MBP autoantibodies, suggesting a strong association between MMR and CNS autoimmunity in autism. Stemming from this evidence, we suggest that an inappropriate antibody response to MMR, specifically the measles component thereof, might be related to pathogenesis of autism

Children with Autism are prone to autoimmunity Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders. Connolly AM , Chez MG , Pestronk A , Arnold ST , Mehta S , Deuel RK . Departments of Neurology and Pediatrics, Washington University, St. Louis Children's Hospital, St Louis, Missouri,. J Pediatr. 1999 May;134(5):607-13. OBJECTIVE : Etiologically unexplained disorders of language and social development have often been reported to improve in patients treated with immune-modulating regimens. Here we determined the frequency of autoantibodies to brain among such children. DESIGN: We collected sera from a cohort of children with (1) pure Landau-Kleffner syndrome (n = 2), (2) Landau-Kleffner syndrome variant (LKSV, n = 11), and (3) autistic spectrum disorder (ASD, n = 11). None had received immune-modulating treatment before the serum sample was obtained. Control sera (n = 71) were from 29 healthy children, 22 with non-neurologic illnesses (NNIs), and 20 children with other neurologic disorders (ONDs). We identified brain autoantibodies by immunostaining of human temporal cortex and antinuclear autoantibodies using commercially available kits. RESULTS: IgG anti-brain autoantibodies were present in 45% of sera from children with LKSV, 27% with ASD, and 10% with ONDs compared with 2% from healthy children and control children with NNIs. IgM autoantibodies were present in 36% of sera from children with ASD, 9% with LKSV, and 15% with ONDs compared with 0% of control sera. Labeling studies identified one antigenic target to be endothelial cells. Antinuclear antibodies with titers >/=1:80 were more common in children with ASD and control children with ONDs. CONCLUSION: Children with LKSV and ASD have a greater frequency of serum antibodies to brain endothelial cells and to nuclei than children with NNIs or healthy children. The presence of these antibodies raises the possibility that autoimmunity plays a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders.

Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders.

Connolly AM , Chez MG , Pestronk A , Arnold ST , Mehta S , Deuel RK .

Departments of Neurology and Pediatrics, Washington University, St. Louis Children's Hospital, St Louis, Missouri,.

J Pediatr. 1999 May;134(5):607-13.

OBJECTIVE : Etiologically unexplained disorders of language and social development have often been reported to improve in patients treated with immune-modulating regimens. Here we determined the frequency of autoantibodies to brain among such children. DESIGN: We collected sera from a cohort of children with (1) pure Landau-Kleffner syndrome (n = 2), (2) Landau-Kleffner syndrome variant (LKSV, n = 11), and (3) autistic spectrum disorder (ASD, n = 11). None had received immune-modulating treatment before the serum sample was obtained. Control sera (n = 71) were from 29 healthy children, 22 with non-neurologic illnesses (NNIs), and 20 children with other neurologic disorders (ONDs). We identified brain autoantibodies by immunostaining of human temporal cortex and antinuclear autoantibodies using commercially available kits. RESULTS: IgG anti-brain autoantibodies were present in 45% of sera from children with LKSV, 27% with ASD, and 10% with ONDs compared with 2% from healthy children and control children with NNIs. IgM autoantibodies were present in 36% of sera from children with ASD, 9% with LKSV, and 15% with ONDs compared with 0% of control sera. Labeling studies identified one antigenic target to be endothelial cells. Antinuclear antibodies with titers >/=1:80 were more common in children with ASD and control children with ONDs. CONCLUSION: Children with LKSV and ASD have a greater frequency of serum antibodies to brain endothelial cells and to nuclei than children with NNIs or healthy children. The presence of these antibodies raises the possibility that autoimmunity plays a role in the pathogenesis of language and social developmental abnormalities in a subset of children with these disorders.

Children with Autism are prone to autoimmunity Antibrain antibodies in children with autism and their unaffected siblings. Singer HS , Morris CM , Williams PN , Yoon DY , Hong JJ , Zimmerman AW . Department of Neurology, Johns Hopkins University School of Medicine, J Neuroimmunol. 2006 Sep;178(1-2):149-55. Serum autoantibodies to human brain , identified by ELISA and Western immunoblotting, were evaluated in 29 children with autism spectrum disorder (22 with autistic disorder), 9 non-autistic siblings and 13 controls . More autistic subjects than controls had bands at 100 kDa in caudate, putamen and prefrontal cortex (p<0.01) as well as larger peak heights of bands at 73 kDa in the cerebellum and cingulate gyrus. Both autistic disorder subjects and their matched non-autistic siblings had denser bands (peak height and/or area under the curve) at 73 kDa in the cerebellum and cingulate gyrus than did controls (p<0.01). Results suggest that children with autistic disorder and their siblings exhibit differences compared to controls in autoimmune reactivity to specific epitopes located in distinct brain regions.

Antibrain antibodies in children with autism and their unaffected siblings.

Singer HS , Morris CM , Williams PN , Yoon DY , Hong JJ , Zimmerman AW .

Department of Neurology, Johns Hopkins University School of Medicine,

J Neuroimmunol. 2006 Sep;178(1-2):149-55.

Serum autoantibodies to human brain , identified by ELISA and Western immunoblotting, were evaluated in 29 children with autism spectrum disorder (22 with autistic disorder), 9 non-autistic siblings and 13 controls . More autistic subjects than controls had bands at 100 kDa in caudate, putamen and prefrontal cortex (p<0.01) as well as larger peak heights of bands at 73 kDa in the cerebellum and cingulate gyrus. Both autistic disorder subjects and their matched non-autistic siblings had denser bands (peak height and/or area under the curve) at 73 kDa in the cerebellum and cingulate gyrus than did controls (p<0.01). Results suggest that children with autistic disorder and their siblings exhibit differences compared to controls in autoimmune reactivity to specific epitopes located in distinct brain regions.

Thimerosal Induces Autistic Symptoms in Mice prone to Autoimmune diseases Neurotoxic effects of postnatal thimerosal are mouse strain dependent. Hornig M , Chian D , Lipkin WI . Jerome L and Dawn Greene Infectious Disease Laboratory, Department of Epidemiology, Mailman School of Public Health, Columbia University Mol Psychiatry. 2004 Sep;9(9):833-45 The developing brain is uniquely susceptible to the neurotoxic hazard posed by mercurials. Host differences in maturation, metabolism, nutrition, sex, and autoimmunity influence outcomes. How population-based variability affects the safety of the ethylmercury-containing vaccine preservative, thimerosal, is unknown. Reported increases in the prevalence of autism, a highly heritable neuropsychiatric condition, are intensifying public focus on environmental exposures such as thimerosal. Immune profiles and family history in autism are frequently consistent with autoimmunity. We hypothesized that autoimmune propensity influences outcomes in mice following thimerosal challenges that mimic routine childhood immunizations. Autoimmune disease-sensitive SJL/J mice showed growth delay; reduced locomotion; exaggerated response to novelty; and densely packed, hyperchromic hippocampal neurons with altered glutamate receptors and transporters. Strains resistant to autoimmunity, C57BL/6J and BALB/cJ, were not susceptible. These findings implicate genetic influences and provide a model for investigating thimerosal-related neurotoxicity.

Neurotoxic effects of postnatal thimerosal are mouse strain dependent.

Hornig M , Chian D , Lipkin WI .

Jerome L and Dawn Greene Infectious Disease Laboratory, Department of Epidemiology, Mailman School of Public Health, Columbia University

Mol Psychiatry. 2004 Sep;9(9):833-45

The developing brain is uniquely susceptible to the neurotoxic hazard posed by mercurials. Host differences in maturation, metabolism, nutrition, sex, and autoimmunity influence outcomes. How population-based variability affects the safety of the ethylmercury-containing vaccine preservative, thimerosal, is unknown. Reported increases in the prevalence of autism, a highly heritable neuropsychiatric condition, are intensifying public focus on environmental exposures such as thimerosal. Immune profiles and family history in autism are frequently consistent with autoimmunity. We hypothesized that autoimmune propensity influences outcomes in mice following thimerosal challenges that mimic routine childhood immunizations. Autoimmune disease-sensitive SJL/J mice showed growth delay; reduced locomotion; exaggerated response to novelty; and densely packed, hyperchromic hippocampal neurons with altered glutamate receptors and transporters. Strains resistant to autoimmunity, C57BL/6J and BALB/cJ, were not susceptible. These findings implicate genetic influences and provide a model for investigating thimerosal-related neurotoxicity.

Mercury Removal: Chelation Agents EDTA, Dimercaprol (BAL), DMSA, DMPS, and DMPA all have heavy metal binding activity Marked specificity for heavy metals, but also can cause decreases in trace elements and micronutrients (and these should be tested for periodically) Mercury is essentially irreversibly bound to DMSA, so mercury is not deposited in other tissues, even the kidney. DMSA/DMPS works through increasing urinary excretion. DMSA/DMPS does not cross the blood-brain barrier, so no risk of delivering bound mercury to the brain Very low toxicity. Side effects may include anorexia, nausea, vomiting, diarrhea, rash and a transient increase in liver enzymes. There are no known adverse drug interactions with DMSA/DMPS. EDTA: disodium vs Calcium disodium. Disodium EDTA given rapidly by IV can suddenly drop serum calcium levels. Only should use CaNa2 EDTA

Agents

EDTA, Dimercaprol (BAL), DMSA, DMPS, and DMPA all have heavy metal binding activity

Marked specificity for heavy metals, but also can cause decreases in trace elements and micronutrients (and these should be tested for periodically)

Mercury is essentially irreversibly bound to DMSA, so mercury is not deposited in other tissues, even the kidney.

DMSA/DMPS works through increasing urinary excretion.

DMSA/DMPS does not cross the blood-brain barrier, so no risk of delivering bound mercury to the brain

Very low toxicity. Side effects may include anorexia, nausea, vomiting, diarrhea, rash and a transient increase in liver enzymes.

There are no known adverse drug interactions with DMSA/DMPS.

EDTA: disodium vs Calcium disodium. Disodium EDTA given rapidly by IV can suddenly drop serum calcium levels. Only should use CaNa2 EDTA

Mercury Removal: Chelation oral (DMPS and DMSA) IV (DMPS and CaNa2 EDTA) Rectal (DMPS, DMSA, CaNa2 EDTA [detoxamin]). best to have stool passage before insertion CaNa2 EDTA seems to cause less yeast exacerbation TD – emu oil seems the best vehicle seen very little benefit/movement with DMPS seen some positive excretions with DMSA CaNa2 very difficult to keep in suspension without precipitation

oral (DMPS and DMSA)

IV (DMPS and CaNa2 EDTA)

Rectal (DMPS, DMSA, CaNa2 EDTA [detoxamin]).

best to have stool passage before insertion

CaNa2 EDTA seems to cause less yeast exacerbation

TD – emu oil seems the best vehicle

seen very little benefit/movement with DMPS

seen some positive excretions with DMSA

CaNa2 very difficult to keep in suspension without precipitation

Single dose chelation challenge baseline urine taken before dose given 8 hour urine collection regardless of type/route empty bladder before giving dose DMSA (oral or rectal 25mg/kg) DMPS (3mg/kg for IV, 10mg/kg for rectal, 5-10mg/kg oral) CaNA2 EDTA (25-50mg/kg regardless of rout, maximum of 1500mg) May need to do more than 1 challenge with different agents/routes (Defeat Autism Now! 2005 Consensus Paper)

baseline urine taken before dose given

8 hour urine collection regardless of type/route

empty bladder before giving dose

DMSA (oral or rectal 25mg/kg)

DMPS (3mg/kg for IV, 10mg/kg for rectal, 5-10mg/kg oral)

CaNA2 EDTA (25-50mg/kg regardless of rout, maximum of 1500mg)

May need to do more than 1 challenge with different agents/routes

(Defeat Autism Now! 2005 Consensus Paper)

Antonio (7 y/o) on first chelation challenge with DMSA

Antonio, after 4 cycles of DMSA

Antonio, after 8 cycles of DMSA

Antonio After Chelation with DMSA Has bad gas during the DMSA days, and is moody, then this goes away when the DMSA is finished. Doing better and better in speech therapy If he does not want to do things he cries. Teachers are reporting improvements seen on a month-to-month basis More hand gesturing In a more advanced class. The mimicry behavior has stopped. After finishing chelation, language is the major barrier, but behaviors and stemming are under control

Baseline

DMPS/Glutathione-IV

DMPS/Glutathione-Rectal

We Need More Research! The Bottom Line: ----The Bottom Line----The Bottom Line----The Bottom Line----The Bottom Line---