Biokjemisk Ubalanse Og Biomedisinsk Behandling Av Autisme: Gjennomgang Av Litteraturen

Biokjemisk ubalanse og biomedisinsk behandling av autisme: Gjennomgang av litteraturen.

Mars 2010 ©

Utarbeidet av: Hanne Bjørg Walker1 Kathrine Ebbesen Rør1 Dr. med. Karl Ludvig Reichelt, Ph.D2 Professor Eremitus Tore Midtvedt, Ph.D3 Susan Owens, MAIS4 Lege Geir Flatabø5 Professor Svein Eikeseth, Ph.D6

Bidragsytere: Dr. Jeff Bradstreet, MD, MD(H) FAAFP7 Dr. Elisabeth Mumper, MD8 Førsteamanuensis Bjørn Bolann, Ph.D9

1 Vår Vei, Parent network for children with autism, Norway 2 Pediatric Research Institute, Rikshospitalet University State Hospital, Oslo, Norway 3 Karolinska Institute, Stockholm, Sverige 4 Autism Research Center, San Diego, CA, USA 5 Medisinsk Helsesenter, Ulvik, Norge 6 Akershus University College, Lillestrøm, Norge 7 International Child Developmental Research Centre, Florida, USA 8 The Rimland Centre, USA 9 Institutt for indremedisin, Seksjon for medisinsk biokjemi, Haukeland Universitetssykehus, Bergen, Norway

Oppsummering & konklusjoner

Mandatet Mandatet er gitt av Helsedirektoratet i brev av 14. november 2006: Beskrive aktuelle biokjemiske ubalanser og biomedisinsk behandling for barn med autisme.

Autisme Autisme – Autism Specter Disorder(ASD) er et komplekst klinisk problem for alle som arbeider med mulige årsaksforhold og for alle som søker etter effektive behandlingsmetoder. Det tradisjonelle synspunktet som mange fagpersoner har er at autisme er en genetisk hjerneforstyrrelse som ikke kan behandles.

Antall barn med ASD øker. Forekomst av en ren genetisk betinget sykdom kan bare vokse i takt med popula- sjonsøkning. Selv om det er sannsynlig at gener spiller en rolle ved ASD, kan ikke økningen forklares ut fra endringer i gener eller i diagnosekriterier, men gjør det overveiende sannsynlig at andre faktorer også spiller en rolle.

I denne kunnskapsoppsummeringen vises til mange mulige faktorer. Det kan ikke understrekes sterkt nok at på- gående forskning stadig avdekker funn som kan bli verdifulle i fremtidig diagnostikk, terapi og prognose.

Kunnskapsoppsummeringen bør ikke sees på som statisk og den endelige sannhet, men som en sammen- stilling av faktorer som man må ha i mente når man utformer den videre veien som skal gås i behandlingen av barn med autisme.

Det nye synet om at autisme ikke er en hjerneforstyrrelse, men en forstyrrelse som påvirker hjernen kan beskrives som følger. Autisme er en multiorgan sykdom som innvolverer immunologiske og metabolske dysfunksjoner som kan bli forverret av miljøfaktorer i genetisk sårbare individer – såkalte subpopulasjoner.

Kunnskapsoppsummeringen trekker frem noen viktige immunologiske og metabolske faktorer og beskriver noen miljøfaktorer som kan være av viktighet for diagnosen. Blant disse miljøfaktorene inkluderes også dietære faktorer som gluten,kasein osv. Det er økende antall publiserte rapporter som beskriver markert bedring/ endring av autistiske symptomer i undergrupper av barn hvor slike faktorer er eliminert. Det sier seg selv at hver suksessfull historie representerer en fantastisk lettelse for familieliv samtidig som det har en substansiell økonomisk verdi for samfunnet.

Det er en diagnostisk utfordring å finne de forskjellige triggerne og undergruppene.

Basert på det vel etablerte, men fremdeles utfordrende paradigme skift at “autism kan behandles”, så fortjener hvert eneste barn fortjener å bli utredet og behandlet på optimal måte.

Forord Autisme kan beskrives som en atferdsforstyrrelse med underliggende biologiske faktorer, influert av gener og av interaksjon mellom gener og miljø. Det er holdepunkter for at de underliggende biologiske faktorer er av en omfattende størrelse. For å kunne påvirke tilstanden er det nødvendig med en god forståelse av mange typer ulike reaksjonsveier.

Ny viten slår fast at man i stedet for å se autisme som en hjernebasert skade, må ses på som en funksjonsforstyr- relse som påvirker hjernen (Herbert 2005).1

Det er mer enn et halvt århundre siden det ble tydelig demonstrert at forekomsten av en bestemt tarmflora økte sensitiviteten i vev til flere forskjellige nevrotransmittere (Strandberg et al. 19662, Gustafsson et al. 1970).3

Dette endrede synet på autisme fra “sterkt genetisk betinget” til “genetisk påvirket”, gir rom for epigenetikk, sto- kastiske effekter, pleiotropisme, epistase, variabel ekspressivitet og interaksjoner mellom gener og miljø (Veen- stra-Vanderweele et al. 2004)4, (Herbert 2005).1 Innhold

Oppsummering & konklusjoner...... 2 Forord...... 3 1. Innledning ...... 6 2. Mandat...... 7 2.1. Bakgrunn...... 7 2.2. Formål...... 7 2.3. Prosjektgruppe...... 7 2.4. Søkestrategi...... 7 2.5. Inklusjonskriterier...... 7 2.6. Innsamling og analyse av data...... 7 2.7. Prevalens...... 7 2.8. Miljøfaktorer...... 8 3. Biokjemiske og immunologiske faktorer...... 8 3.1. Nevrologi og immunologi...... 8 3.2. Th1/Th2...... 9 3.3. Autoimmunitet...... 10 3.4. Cytokiner i hjerne og tarm; NK-celler...... 12 3.5. Mage tarm...... 13 3.6. Mikrobiell metabolisme i mage–tarm...... 18 3.6.1. Neurotoxin...... 19 3.6.2. Endotoksin...... 20 3.6.3. Bakterielle korte frie fettsyre...... 20 3.6.4. Svovelmetabolisme...... 20 3.7. Oksalat...... 20 3.8. Oksidativt stress...... 22 3.9. Virus infeksjoner...... 24 4. Miljøstudier...... 25 4.1. Miljøgifter...... 25 5. Biomarkører...... 30 5.1. Blodbilde...... 31 5.1.1. Røde og hvite blodplater med differensiealtelling...... 31 5.1.2. Metabolsk panel...... 31 5.2. Lever funksjon...... 31 5.3. Aminosyrer...... 31 5.4. Tarmfunksjon og metabolisme...... 31 5.4.1. Peptidmarkører i urin...... 31 5.4.2. Permeabilitet i tarmen...... 32 5.4.3. Mikrobielle produkter urin...... 32 5.4.4. Mikrober og mikrobielle produkter i avføring...... 32 5.4.5. Mikrobielle metabolitter...... 32 5.5. Tungmetaller ...... 32 5.5.1. Tungmetallmarkører i blod...... 33 5.5.2. Tungmetallmarkører i urin...... 33 5.6. Oksidativt stress...... 33 5.6.1. Markører for oksidativt stress i blod...... 33 5.6.2. Markører for oksidativt stress i urin...... 33

– 4 – 5.7. Immun systemet...... 33 5.7.1. Autoantistoffer til endovaskulatur...... 33 5.7.2. Neopterin og biopterin...... 33 5.8.1. ASO (AST) og anti-DNase B...... 33 5.8.2. Immunoglobulinklasser: IgG (1–4), IgM, IgA og IgE...... 33 5.8.3. Antistoffer i hjernen...... 34 5.8.4. Vaksinetiter...... 34 5.8.5. Kalprotectin...... 34 5.9. Metylering og transulfatering...... 34 5.9.1. Metylerings- og transulfateringskapasitet, markører i blod...... 34 5.9.2. Metylerings- og transulfateringskapasitet, markører i urin...... 34 5.10. Fettsyrer...... 34 6. Aktuelle reaksjonsveier...... 35 6.1. Metylering...... 35 6.2. Transulfatering...... 35 7. Biomedisinske behandlinger...... 37 7.1. Vitaminer og mineraler generelt...... 37 7.2. Glutenfri og kaseinfri diett uten hvetestivelse...... 40 7.3. Essensielle fettsyrer...... 41 7.4. Metyleringssyklusene...... 42 7.5. Oksidativt stress...... 42 7.6. Fordøyelse...... 43 7.7. Tungmetaller...... 43 7.8. Hyperbar oksygen behandling (HBOT)...... 43 Konklusjoner...... 45 Referanser...... 46

– 5 – 1. Innledning Autisme – Autism Specter Disorder(ASD) er et komplekst klinisk problem for alle som arbeider med mulige årsaksforhold og for alle som søker etter effektive behandlingsmetoder.

Ny viten har gitt et paradigmeskifte der man ikke lenger ser autisme som en hjernebasert genetisk forstyrrelse, men i stedet som en systemisk forstyrrelse som påvirker mange organer inkludert hjernen.1 Denne presisering gjør det enklere å forstå og behandle autisme. Som et resultat av nyere forskning på biomarkører er det mulig å begynne å kartlegge forskjellige underliggende årsaker til autisme.

Nyere forskning har vist at barn med autisme kan ha endringer på følgende målbare områder som delvis overlap- per hverandre:

1. Økt oksidativt stress. 2. Lavt serumsulfat, begrenset transulfatering samt nedsatt metyleringskapasitet. 3. Endringer i den mikrobielle mage-tarm metabolismen. 4. Økt toksisk belastning, spesielt av tungmetaller og primært kvikksølv. 5. Dysregulering av immunsystemet med en unik form for inflammatorisk tarmsykdom samt markører for autoimmunitet i hjernen. 6. Endring av blodtilførselen til hjernen.

Slike underliggende ubalanser kan påvirke utviklingen i negativ retning. Forskning har vist at enkelte barn med autisme kan ha flere slike avvik samtidig. Det er dokumentert at målrettet håndtering av de underliggende ubalansene kan føre til forbedring i kliniske symptomer.

Sett i lys av nyere data fra USA og Storbritannia som viser at opptil 1–2 % gutter under 12 år kan ha ASD5, 6, 7 er det et stort behov av økt kunnskap. Dette er særs viktig for at flest mulig barn kan få behandling for sine biokjemiske avvik. Kunnskapen må også spres til leger som behandler barn med autisme.

– 6 – 2. Mandat Gjennomgang av publiserte studier og artikler om biokjemisk ubalanse etter oppdrag fra Helsedirektoratet i brev av 14. november 2006. Beskrive aktuelle biokjemiske ubalanser og biomedisinsk behandling for barn med autisme.

2.1. Bakgrunn ASD er en kompleks nevrologisk utviklingsforstyrrelse. Selve diagnosen betegnes av nedsatte evner til sosial samhandling og kommunikasjon. Videre kjennetegnes den av ritualistisk og stereotyp atferd (DSM-IV; APA, 1994). Diagnosen er utelukkende bygget på atferdsendringer og ikke på underliggende biologiske årsaksfaktorer. Både nyere og eldre forskning viser dog at det er sannsynlig at slike biologiske endringer er en underliggende årsak til atferdssymptomene som ligger til grunn for diagnosen.

2.2. Formål Gjennomgang av medisinsk litteratur.

2.3. Prosjektgruppe Prosjektgruppen besto av Dr. Karl L. Reichelt, Rikshospitalet, Professor Tore Midtvedt, Karolinska Institutet, Sverige, Professor Svein Eikeseth, Høgskolen i Akershus, Lege Geir Flatabø, Susan Owens, leder for Autism Oxalate Project, Autism Research Institute (ARI), USA Kathrine Ebbesen Rør, og Hanne Bjørg Walker, Vår Vei. I starten av prosjektet var Dr. Bjørn Bolann, Haukeland Sykehus med i gruppen.

2.4. Søkestrategi Det ble søkt i databasene MEDLINE og The Cochrane Library per august 2009. Noen studier som har blitt publisert etter er tatt med.

Studier, referanser og annen medisinsk litteratur fra Autism Research Institute, USA ble gjennomgått for hånd. Dr. James Jeffrey Bradstreet, ICDRC, Florida og Dr. Elizabeth Mumper, Rimland Center, Virginia ble konsultert.

Referanser i de forskjellige studiene ble gjennomgått og vi kontaktet enkelte forfattere for å be om ytterligere publiserte eller upubliserte data.

2.5. Inklusjonskriterier Vi valgte randomiserte eller delvis randomiserte kontrollerte studier, case-studier og enkeltcase-studier. Vi har også inkludert dyrestudier der disse hadde klar relevans.

Vi har søkt etter relevant forskning som er utført innenfor feltene immunologi, nevrologi, mikrobiologi og gastroenterologi.

2.6. Innsamling og analyse av data Deltakerne i prosjektgruppen vurderte hver artikkel individuelt etter å ha brukt de samme søkeordene, og vurderte også hvorvidt artiklene skulle inkluderes. Søkeordene er oppgitt i vedlegget.

2.7. Prevalens En ny rapport fra SINTEF antyder at prevalensen av ASD i Norge er høyere enn tidligere antatt (Hem et al. 2008).8 Ifølge Fombonne et al.9 er det beste estimatet 60–70 per 10.000. Ettersom kjønnsfordelingen er 4:1 for gutter versus jenter, vurderes forekomsten av ASD hos gutter til å være 1 av 114 barn i Norge.

– 7 – Chakrabarti, S. Fombonne, E. (June 2005) Pervasive Developmental Disorders in Preschool children: Confirmation of High Prevalence. Am J Psychiatry, 167, 1133-1141.7

Fombonne, E. (2005) Epidemiological studies of Pervasive Developmental Disorders. Chapter in Handbook of Autism and Pervasive Developmental Disorders. Editors: Volkmar, F. Rhea, P. Ami, K. Donald, C. Third Edition. John Wiley & Sons, Inc.9

2.8. Miljøfaktorer Det er enighet blant forskere at forekomsten av ASD øker. Økningen i autismediagnoser kan ikke bare forklares ved endringer i diagnosekriteriene, større bevissthet rundt tilstanden eller tendenser til overdiagnostisering. Øk- ningen gjør det sannsynlig at miljøfaktorer spiller en rolle , ettersom en genetisk endring vanligvis tar lengre tid å komme til uttrykk.

3. Biokjemiske og immunologiske faktorer 3.1. Nevrologi og immunologi I familier til autistiske barn er det en hyppig forekomst av familiær autoimmunitet. (Valicenti-McDermott, McVi- car et al. 2006).10 Pardo og medarbeidere11 hevdet at det forekom aktivering av det nevroimmune system i sentralnervesystemet hos pasienter med autisme. Vargas, Nascimbene et al. (2005) hevdet at nevrobetennelse kan spille en vesentlig rolle i å opprettholde og/eller initiere noen av abnormitetene i sentralnervesystemet hos barn med ASD12. Hva slags rolle aktivering av neuroglia og nevrobetennelse spiller krever videre undersøkelse. Potensiell behandling av ovennevnte krever også videre undersøkelser.

Hos barn med autisme fant Ashwood og medarbeidere et unikt mønster av intracellulære cytokiner i perifere lymfocytter fra blod og tarmmukosa, og hevdet at dette vev er i samsvar med signifikant dysregulering av immunsystemet (Ashwood og Wakefield 2006).35

Jyonouchi og medarbeidere publiserte en evaluering av forbindelsen mellom cytokinproduksjon og proteiner fra vanlig kosthold samt mage tarm symptomer hos autistiske barn. De noterte en høy forekomst av økt TNF alpha- og interleukin 12-produksjon hos barn med autisme og gastrointestinale symptomer (Jyonouchi, Geng et al. 2005).13 I et videre arbeid rapporterte Jyonouchi og medarbeidere om dysregulert immunrespons hos unge barn med autisme samt forholdet mellom mage tarm symptomer og kostholdsintervensjoner. Forfatternes konklusjon var at “...våre funn antyder iboende defekter i immunsystemet (hos barn med autisme og mage tarm symptomer)” men ikke hos barn med ikke-allergen matintoleranse eller asymtomatisk autisme. Forfatterne foreslår en “mulig sammenheng mellom mage tarm og atferdsmessige symptomer mediert av abnormiteter i immunsystemet” (Jyonouchi, Geng et al. 2005).14

Det finnes holdepunkter for dysregulering av immunsystemet hos barn med autisme. Blant annet omfatter økt nivå av IgE (Hamilton og Franklin Adkinson 2004)15, dårlig funksjon av NK-celler, endringer i T-celle-funksjon (Gupta, Aggarwal et al. 1998)16 samt andre tegn på betennelse. (Horning, Weissenböck et al. 1999)17. Når betennelse reduseres av behandling, utløses ofte samtidig kognitiv og atferdsmessig bedring hos barna. Det er blitt hevdet at neopterinnivået kan brukes som betennelsesmarkør for å følge effekten av en behandling. (Messahel, Phesant et al. 1998).18

Vi siterer tre publiserte studier som gir holdepunkter for at nevroimmune reaksjoner kan spille en rolle ved ASD:

Vargas DL et al. Neuroglial activation and neuroinflammation in the brain of patiens with autism. Ann Neurol 2005;57:67-81.12 Autism is a neurodevelopmental disorder characterized by impaired communication and social interaction and may be accompanied by mental retardation and epilepsy. Its cause remains unknown, despite evidence that genetic, environmental, and immunological factors may play a role in its pathogenesis. To investigate whether

– 8 – immune-mediated mechanisms are involved in the pathogenesis of autism, we used immunocytochemistry, cytokine protein arrays, and enzyme-linked immunosorbent assays to study brain tissues and cerebrospinal fluid (CSF) from autistic patients and determined the magnitude of neuroglial and inflammatory reactions and their cytokine expression profiles. Brain tissues from cerebellum, midfrontal, and cingulate gyrus obtained at autopsy from 11 patients with autism were used for morphological studies. Fresh-frozen tissues available from seven patients and CSF from six living autistic patients were used for cytokine protein profiling. We demonstrate an active neuroinflammatory process in the cerebral cortex, white matter, and notably in cerebellum of autistic patients. Immunocytochemical studies showed marked activation of microglia and astroglia, and cytokine profiling indicated that macrophage chemoattractant protein (MCP)-1 and tumour growth factor-beta1, derived from neuroglia, were the most prevalent cytokines in brain tissues. CSF showed a unique proinflammatory profile of cytokines, including a marked increase in MCP-1. Our findings indicate that innate neuroimmune reactions play a pathogenic role in an undefined proportion of autistic patients, suggesting that future therapies might involve modifying neuroglial responses in the brain.

Pardo CA,Vargas DL et al. Immunity, neuroglia and neuroinflammation in autism. International Review of Psychiatry 2005;17:485-95.11 The role of the immune system in the development of autism is controversial. Several studies showing peripheral immune abnormalities support immune hypotheses, however until recently there have been no immune findings in the CNS. We recently demonstrated the presence of neuroglial and innate neuroimmune system activation in brain tissue and cerebrospinal fluid of patients with autism, findings that support the view that neuroimmune abnormalities occur in the brain of autistic patients and may contribute to the diversity of the autistic phenotypes. The role of neuroglial activation and neuroinflammation are still uncertain but could be critical in maintaining, if not also in initiating, some of the CNS abnormalities present in autism. A better understanding of the role of neuroinflammation in the pathogenesis of autism may have important clinical and therapeutic implications.

Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression. Jyonouchi H, Sun S, Le H. 1: J Neuroimmunol. 2001 Nov 1;120(1-2):170-9.19 We determined innate and adaptive immune responses in children with developmental regression and autism spectrum disorders (ASD, N=71), developmentally normal siblings (N=23), and controls (N=17). With lipopolysaccharide (LPS), a stimulant for innate immunity, peripheral blood mononuclear cells (PBMCs) from 59/71 (83.1%) ASD patients produced >2 SD above the control mean (CM) values of TNF-alpha, IL-1beta, and/or IL-6 produced by control PBMCs. ASD PBMCs produced higher levels of proinflammatory/counter-regulatory cytokines without stimuli than controls. With stimulants of phytohemagglutinin (PHA), tetanus, IL-12p70, and IL-18, PBMCs from 47.9% to 60% of ASD patients produced >2 SD above the CM values of TNF-alpha depending on stimulants. Our results indicate excessive innate immune responses in a number of ASD children that may be most evident in TNF-alpha production.

3.2. Th1/Th2 T-cellene som har fått navn etter tymus, bidrar til å styre aktiviteten til immunsystemet . Det finnes flere typer T-celler. Noen bidrar til å fremme immunresponsen, andre slår av immunresponsen. Andre igjen dreper kreftcel- ler. T-celler som bidrar til å aktivere immunresponsen kalles T-hjelpeceller (Th-celler). Det finnes to hovedtyper Th-celler: Th-1 og Th-2.

Hos barn med ASD er det funnet at Th-celle-balansen er forskjøvet. Vi siterer fire studier som understøtter dette:

Th1- and Th2-like cytokines in CD4+ and CD8+ T cells in autism. Gupta S, Aggarwal S, Rashanravan B, Lee T. J Neuroimmunol. 1998 May 1;85(1):106-9.16 Th1-like (IL-2, IFN-gamma) and Th2-like (IL-4, IL-6, and IL-10) cytokines were examined in CD4+ and CD8+ T cells in children with autism. Intracellular cytokines were measured using specific antibodies to various cytokines and anti-CD4 or anti-CD8 monoclonal antibodies by FACScan. Proportions of IFN-gamma+CD4+ T cells and IL-2+CD4+ T cells (Th1), and IFN-gamma+CD8+ and IL-2+CD8+ T cells (TC1) were significantly lower in

– 9 – autistic children as compared to healthy controls. In contrast, IL-4+CD4+ T cells (Th2) and IL-4+CD8+ T cells (TC2) were significantly increased in autism. The proportions of IL-6+ CD4+, IL-6+CD8+ and IL-10+CD4+, IL- 10+CD8+ T cells were comparable in autism and control group. These data suggest that an imbalance of Th1- and Th2-like cytokines in autism may play a role in the pathogenesis of autism.

Deficiency of suppressor-inducer (CD4+CD45RA+) T cells in autism. Warren RP, Yonk LJ, et al.Burger RA, Cole P, Odell JD, Warren WL, White E, Singh VK. Immunol Invest. 1990 Jun;19(3):245-51.20 CD4+ cells are a heterogenous population of lymphocytes including at least two distinct subpopulations: CD45RA+ cells, inducers of suppressor T cells and CDw29+ cells, inducers of helper function for antibody production. To investigate the possibility that immune abnormalities in autism may involve abnormal distribution of these helper subpopulations, monoclonal antibodies were used in flow cytometric analysis to characterize peripheral blood lymphocytes of 36 subjects with autism. The autistic subjects as compared to a group of 35 healthy age-matched subjects had a significantly reduced number of lymphocytes, a decreased number of CD2+ T cells and reduced numbers of CD4+ and CD4+CD45RA+ lymphocytes. The numbers of B (CD20+) cells, suppressor T (CD8+) cells, inducers of helper function (CD4+CDw29+) and natural killer (CD56+) cells were not altered in the autistic subjects. Our results suggest that an alteration in the suppressor-inducer T-cell subset is associated with autism.

Immune abnormalities in patients with autism. Warren RP, Margaretten NC, Pace NC, Foster A. J Autism Dev Disord. 1986 Jun;16(2):189-97.21 We have begun an investigation on the immune systems of patients with autism in attempt to determine if immune mechanisms are involved in the development of this severe developmental disorder. A study of 31 autistic patients has revealed several immune-system abnormalities, including reduced responsiveness in the lymphocyte blastogenesis assay to phytohemagglutinin, concanavalin A, and pokeweed mitogen; decreased numbers of T lymphocytes; and an altered ratio of helper to suppressor T cells. Immune-system abnormalities may be directly related to underlying biologic processes of autism, or these changes may be an indirect reflection of the actual pathologic mechanism.

Elevated immune response in the brain of autistic patients. Li, X., et al., , J. Neuroimmunol. (2009), doi:10.1016/j. jneuroim.2008.12.002.22 This study determined immune activities in the brain of ASD patients and matched normal subjects by examining cytokines in the brain tissue. Our results showed that proinflammatory cytokines (TNF-α, IL-6 and GM- CSF), Th1 cytokine (IFN-γ) and chemokine (IL-8) were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients. Conclusion: ASD patients displayed an increased innate and adaptive immune response through the Th1 pathway, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD.

3.3. Autoimmunitet Det er vel etablert at 60–70 % av immunsystemet hos menneske ligger i fordøyelseskanalen med tilhørende organer. Dette gjør tarmen til kroppens desidert største immunologiske organ. Ettersom immunsystemet er så knyttet til mage-/tarmkanalen kan intestinal patologi bidra til immundysregulering og omvendt.

I det nyere fagfeltet psykonevroimmunologi, legger van Gent et al. (1997)23 frem hypotesen om at autoimmune og/eller virale prosesser påvirker nervesystemet og endrer nervesystemets aktivitet. Vojdani og medarbeidere rapporterer at “peptider fra kost, toksiner fra bakterier og xenobiotika bindes til lymfocyttreseptorer og/eller enzymer i kroppsvev, noe som fører til autoimmune reaksjoner hos barn med autisme.”24

El-Fawal og medarbeidere rapporterte at antistoffer til myelin protein og neuron-axon filament protein (NAFP) er funnet hos barn med autisme sammenlignet med friske kontroller (El-Fawal et al. 1999).25 Singh (2002) rapporterte at positive titre for meslingvirus var forbundet med antistoffer, spesielt til myelin protein hos barn med autisme, men ikke hos kontrollene.26

– 10 – Det er identifisert økt forekomst av visse infeksjonssykdommer hos autistiske barn. Dette omfatter bl.a.herpes simplex og roseola (forårsaket av viruset HHV6). Hos svært små barn kan en immunsystem forstyrrelse forhindre barnet i å eliminere et patogen på en betimelig og normal måte uten at barnet nødvendigvis virker særlig sykt. Denne situasjonen skaper en høyere risiko hos barnet for at patogenet kan hindre hjernens utvikling eller funksjon og/eller utløse autoimmun respons. Begge deler er sterkt identifisert som viktige symptomer ved ASD.

Vi siterer to publiserte studier som støtter standpunktet om autoimmune reaksjoner hos barn med ASD:

Dysregulated innate immune response in young children with autism spectrum disorders. Their relationship to gastrointestinal symptoms and dietary intervention. Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. Neuropsychobiology 2005;51:77-85.14 OBJECTIVE: Our previous study indicated an association between cellular immune reactivity to common dietary proteins (DPs) and excessive proinflammatory cytokine production with endotoxin (lipopolysaccharide, LPS), a major stimulant of innate immunity in the gut mucosa, in a subset of autism spectrum disorder (ASD) children. However, it is unclear whether such abnormal LPS responses are intrinsic in these ASD children or the results of chronic gastrointestinal (GI) inflammation secondary to immune reactivity to DPs. This study further explored possible dysregulated production of proinflammatory and counter-regulatory cytokines with LPS in ASD children and its relationship to GI symptoms and the effects of dietary intervention measures. METHODS: This study includes ASD children (median age 4.8 years) on the unrestricted (n = 100) or elimination (n = 77) diet appropriate with their immune reactivity. Controls include children with non-allergic food hypersensitivity (NFH; median age 2.9 years) on the unrestricted (n = 14) or elimination (n = 16) diet, and typically developing children (median age 4.5 years, n = 13). The innate immune responses were assessed by measuring production of proinflammatory (TNF-alpha, IL-1beta, IL-6, and IL-12) and counter-regulatory (IL-1ra, IL-10, and sTNFRII) cytokines by peripheral blood mononuclear cells (PBMCs) with LPS. The results were also compared to T-cell responses with common DPs and control T-cell mitogens assessed by measuring T-cell cytokine production. RE- SULTS: ASD and NFH PBMCs produced higher levels of TNF-alpha with LPS than controls regardless of dietary interventions. However, only in PBMCs from ASD children with positive gastrointestinal (GI(+)) symptoms, did we find a positive association between TNF-alpha levels produced with LPS and those with cow’s milk protein (CMP) and its major components regardless of dietary interventions. In the unrestricted diet group, GI(+) ASD PBMCs produced higher IL-12 than controls and less IL-10 than GI(‑) ASD PBMCs with LPS. GI(+) ASD but not GI(‑) ASD or NFH PBMCs produced less counter-regulatory cytokines with LPS in the unrestricted diet group than in the elimination diet group. There was no significant difference among the study groups with regard to cytokine production in responses to T-cell mitogens and other recall antigens. Conclusion: Our results revealed that there are findings limited to GI(+) ASD PBMCs in both the unrestricted and elimination diet groups. Thus our findings indicate intrinsic defects of innate immune responses in GI(+) ASD children but not in NFH or GI(‑) ASD children, suggesting a possible link between GI and behavioral symptoms mediated by innate immune abnormalities.

Infections, toxic chemicals and dietary peptides binding to lymphocyte receptors and tissue enzymes are major instigators of autoimmunity in autism. Vojdani A et al. Int J Immunopathol Pharmacol 2003;16:189-99.24 Similar to many complex autoimmune diseases, genetic and environmental factors including diet, infection and xenobiotics play a critical role in the development of autism. In this study, we postulated that infectious agent antigens such as streptokinase, dietary peptides (gliadin and casein) and ethyl mercury (xenobiotic) bind to different lymphocyte receptors and tissue enzyme (DPP IV or CD26). We assessed this hypothesis first by measuring IgG, IgM and IgA antibodies against CD26, CD69, streptokinase (SK), gliadin and casein peptides and against ethyl mercury bound to human serum albumin in patients with autism. A significant percentage of children with autism developed anti-SK, anti-gliadin and casein peptides and anti-ethyl mercury antibodies, concomitant with the appearance of anti-CD26 and anti-CD69 autoantibodies. These antibodies are synthesized as a result of SK, gliadin, casein and ethyl mercury binding to CD26 and CD69, indicating that they are specific. Immune absorp- tion demonstrated that only specific antigens, like CD26, were capable of significantly reducing serum anti-CD26 levels. However, for direct demonstration of SK, gliadin, casein and ethyl mercury to CD26 or CD69, microtiter wells were coated with CD26 or CD69 alone or in combination with SK, gliadin, casein or ethyl mercury and then reacted with enzyme labeled rabbit anti-CD26 or anti-CD69. Adding these molecules to CD26 or CD69

– 11 – resulted in 28-86% inhibition of CD26 or CD69 binding to anti-CD26 or anti-CD69 antibodies. The highest % binding of these antigens or peptides to CD26 or CD69 was attributed to SK and the lowest to casein peptides. We, therefore, propose that bacterial antigens (SK), dietary peptides (gliadin, casein) and Thimerosal (ethyl mercury) in individuals with pre-disposing HLA molecules, bind to CD26 or CD69 and induce antibodies against these molecules. In conclusion, this study is apparently the first to demonstrate that dietary peptides, bacterial toxins and xenobiotics bind to lymphocyte receptors and/or tissue enzymes, resulting in autoimmune reaction in children with autism.

3.4. Cytokiner i hjerne og tarm; NK-celler Det er foreslått at ASD er nært forbundet med betennelse i hjernen, såkalt nevrobetennelse, og at immunsystemet utløser denne betennelsen. Dette kan oppstå gjennom unormal produksjon av signalsubstanser i immunsystemet kalt cytokiner og kemokiner. Cerebral eller nevrologisk betennelse kan hindre funksjonen til kroppens nevrotransmittere, spesielt dopamin – en nevrotransmitter som er nært forbundet med godt humør og grad av grasiøsitet. Når én del av kroppen er betent, sender området ut signalsubstanser, cytokiner, som går gjennom kroppen, blant annet til leddene, slim- hinnene i luftveiene og mage-/tarmkanalen samt hjernen. Hvert av disse områdene sender så ut sine egne signalsubstanser, som skaper enda mer betennelse, slik at betennelsen sprer seg utover i kroppen.

Vi siterer to publiserte studier som støtter standpunktet om at betennelses mekanismer i sentralnervesystemet kan være forbundet med ASD:

Evaluation of an association between gastrointestinal symptoms and cytokine productions against common dietary proteins in children with autism spectrum disorders. Jyonouchi H et al. J Ped 2005;146:605-610.13 OBJECTIVE: To evaluate an association between cytokine production with common dietary proteins as a marker of non-allergic food hypersensitivity (NFH) and gastrointestinal (GI) symptoms in young children with autism spectrum disorders (ASD). STUDY DESIGN: Peripheral blood mononuclear cells (PBMCs) were obtained from 109 ASD children with or without GI symptoms (GI [+] ASD, N = 75 and GI (‑) ASD, N = 34], from children with NFH (N = 15), and control subjects (N = 19). Diarrhea and constipation were the major GI symptoms. We measured production of type 1 T-helper cells (Th1), type 2 T-helper cells (Th2), and regulatory cytokines by PBMCs stimulated with whole cow’s milk protein (CMP), its major components (casein, beta-lactoglobulin, and alpha- lactoalbumin), gliadin, and soy. RESULTS: PBMCs obtained from GI (+) ASD children produced more tumor necrosis factor-alpha (TNF-alpha)/interleukin-12 (IL-12) than those obtained from control subjects with CMP, beta-lactoglobulin, and alpha-lactoalbumin, irrespective of objective GI symptoms. They also produced more TNF-alpha with gliadin, which was more frequently observed in the group with loose stools. PBMCs obtained from GI (‑) ASD children produced more TNF-alpha/IL-12 with CMP than those from control subjects, but not with beta-lactoglobulin, alpha-lactoalbumin, or gliadin. Cytokine production with casein and soy were unremar- kable. CONCLUSION: A high prevalence of elevated TNF-alpha/IL-12 production by GI (+) ASD PBMCs with CMP and its major components indicates a role of NFH in GI symptoms observed in children with ASD.

Elevation of tumor necrosis factor-alpha in cerebrospinal fluid of autistic children. Chez MG, Dowling T, et al. Pediatr Neurol. 2007 Jun;36(6):361-5.27 Recent reports implicating elevated cytokines in the central nervous system in a small number of patients studied with autism have reported clinical regression. These studies have not focused on tumor necrosis factor-alpha as a possible marker for inflammatory damage. A series of 10 children with autism had clinical evaluation of their serum and spinal fluid for inflammatory changes and possible metabolic disease as part of their neurological evaluation. Elevation of cerebrospinal fluid levels of tumor necrosis factor-alpha was significantly higher (mean = 104.10 pg/mL) than concurrent serum levels (mean = 2.78 pg/mL) in all of the patients studied. The ratio of the cerebrospinal fluid levels to serum levels averaged 53.7:1. This ratio is significantly higher than the elevations reported for other pathological states for which cerebrospinal fluid and serum tumor necrosis factor-alpha levels have been simultaneously measured. This observation may offer a unique insight into central nervous system inflammatory mechanisms that may contribute to the onset of autism and may serve as a potential clinical marker. More controlled study of this potentially important observation may prove valuable.

– 12 – NK-celler er en type lymfocytter som forekommer i blodet og i lymfevevet. Til forskjell fra Th-cellene trenger de ikke antigenaktivering fra celleoverflaten for å bli cytotoksiske. I stedet aktiveres de av cytokine signaler som sendes fra Th-cellene som IL-2 og IFN-y. Forskning har vist at immuncellenes funksjon kan være annerledes hos barn med autisme. Mange rapporter har dokumentert økt antall lymfocytter, inklusive naive T-celler, cytotoksiske T-celler og B-celler. Warren et al. rapporterte allerede i 1987 å ha funnet at NK-celler er redusert hos mange autistiske barn28. Gupta og medarbeidere fant lave antall NK-celler og TH1-celler (Gupta et al. 1998)16

Vi siterer tre publiserte studier som støtter standpunktet om endret NK-celleaktivitet hos barn med ASD:

Reduced natural killer cell activity in autism.Warren RP, Foster A, Margaretten NC. J Am Acad Child Adolesc Psychiatry. 1987 May;26(3):333-5.28 Natural killer (NK) cells arc believed to afford protection against malignancy and viral infections. In addition, these cells may be involved in regulating the immune response because altered NK activity is often associated with autoimmune disorders. An investigation of the natural cytotoxic potential of peripheral blood mononuclear cells from 31 patients with autism has been carried out using K562 tumor cells as target cells. Cells of 12 of the patients induced significantly reduced levels of cytotoxicity; this was not correlated with a quantitative alteration in patient NK cells as determined by use of the Leu-11 monoclonal antibody. This observation of altered NK cell activity, and previously reported findings of other immune abnormalities in autism, suggest that immune changes may be directly related to underlying biological processes of autism or that these changes may be an indirect reflection of the actual pathological mechanism.

Lymphocyte subsets and interleukin-2 receptors in autistic children., Denney DR, Frei BW, Gaffney GR., J Autism Dev Disord. 1996 Feb;26(1):87-97.29 Blood samples were obtained from 10 male autistic children ages 7-15 years and 10 age-matched, male, healthy controls. Lymphocyte subsets (helper-inducer, suppressor-cytotoxic, total T, and total B cells) were enumerated using monoclonal antibodies and flow cytometry. Bound and soluble interleukin-2 receptors were assayed in unstimulated blood samples and in cell cultures following 72-hour stimulation with phytohemagglutinin. The children with autism had a lower percentage of helper-inducer cells and a lower helper:suppressor ratio, with both measures inversely related to the severity of autistic symptoms (r = - .56 and - .68, respectively). A lower percentage of lymphocytes expressing bound interleukin-2 receptors following mitogenic stimulation was also noted, and this too was inversely related to the severity of autistic symptoms.

Dysregulated immune system in children with autism: beneficial effects of intravenous immune globulin on autistic characteristics. Gupta S, Aggarwal S, Heads C. , J Autism Dev Disord. 1996 Aug;26(4):439-52.16 Children (ages 3-12) with autism (n=25) were given intravenous immune globulin (IVIG) treatments at 4-week intervals for at least 6 months. Marked abnormality of immune parameters was observed in subjects, compared to age-matched controls. IVIG treatment resulted in improved eye contact, speech, behavior, echolalia, and other autistic features.

3.5. Mage tarm Hyppigheten av mage – tarm symptomer hos barn med autisme har i den senere tid fått mer oppmerksomhet (Valicenti-McDermott, McVicar et al. 2006).10 En tidlig rapport publisert i The Lancet i februar 1998 beskrev 12 barn med gastrointestinal patologi og autisme (Wakefield, Murch et al. 1998).30 En økning i pankreatisk og biliær utskillelse etter sekretininfusjon, noe som antyder en oppregulering av sekretinreseptorene, ble observert av Horvath, Papadimitriou et al. 199931. De hevdet videre at “uidentifiserte GI-forstyrrelser, spesielt refluks, øsofagitt og disakkaridmalabsorpsjon, kan bidra til atferdsforstyrrelser hos ikke-verbale autistiske pasienter.” Videre arbeid beskrev pan-enterisk enterokolitt hos barn med ASD og konkluderte med forekomst av en ny variant av inflammatorisk tarmbetennelse i denne barnegruppen (Wakefield, Anthony et al. 2000).32

– 13 – Dr. Furlano beskrev CD8- og T-celleinfiltrasjon i kolon med epitelskade hos barn med autisme (Furlano, Anthony et al. 2001).33 Immunhistokjemisk undersøkelse viste en distinkt lymfocytær kolitt. Forfatterne konkluderte med at dette indikerte en dysfunksjon i tarmepitelet. Ashwood og hans medarbeidere publiserte holdepunkter for omfattende mukosal immunpatologi hos barn med regressiv autisme. De påviste fremtredende mukosal eosinofilinfiltrasjon hos affekterte barn. Denne var signifikant lavere hos barn på gluten- og kaseinfri diett selv om de totale lymfocytter forble upåvirket av dietten. CD3+ og CD3+ CD8+ intraepiteliale lymfocytter samt CD3+ lamina propria-lymfocytter var signifikant økt på alle steder hos affekterte barn sammenliknet med individer uten betennelse i kontrollgruppen” (Ashwood, Anthony et al. 2003).34 Ashwood og medarbeidere publiserte også en endret profil av mukosale lymfocytter hos barn med autisme og GI-symptomer. Disse barna viste mukosal immunaktivering med store økninger i inflammatoriske cytokiner som tumornekrosefaktor alfa, IL-2 og IL-4 samt IFN-gamma. IL-10 var redusert hos barn med autisme, noe som viser nedsatt evne til motregulering ved betennelse (Ashwood, Anthony et al. 2004).35 Vojdani påviste immunrespons til proteiner i kostholdet, gliadin og (peptider i cerebellum) cerebellare antigener hos barn med autisme. En undergruppe pasienter med autisme produserte antistoffer mot både purkinjeceller og gliadinpeptider; et videre indisium på en forbindelse mellom tarmen, hjernen og immunsystemet (Vojdani, O’Bryan et al. 2004).36

Vi siterer elleve publiserte studier som gir holdepunkter om en forbindelse mellom tarmen og hjernen:

Dysregulated innate immune responses in young children with autism spectrum disorders: their relationship to gastrointestinal symptoms and dietary intervention. Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. Neuropsychobiology. 2005;51(2):77-85.14 OBJECTIVE: Our previous study indicated an association between cellular immune reactivity to common dietary proteins (DPs) and excessive proinflammatory cytokine production with endotoxin (lipopolysaccharide, LPS), a major stimulant of innate immunity in the gut mucosa, in a subset of autism spectrum disorder (ASD) children. However, it is unclear whether such abnormal LPS responses are intrinsic in these ASD children or the results of chronic gastrointestinal (GI) inflammation secondary to immune reactivity to DPs. This study further explored possible dysregulated production of proinflammatory and counter-regulatory cytokines with LPS in ASD children and its relationship to GI symptoms and the effects of dietary intervention measures. METHODS: This study includes ASD children (median age 4.8 years) on the unrestricted (n = 100) or elimination (n = 77) diet appropriate with their immune reactivity. Controls include children with non-allergic food hypersensitivity (NFH; median age 2.9 years) on the unrestricted (n = 14) or elimination (n = 16) diet, and typically developing children (median age 4.5 years, n = 13). The innate immune responses were assessed by measuring production of proinflammatory (TNF-alpha, IL-1beta, IL-6, and IL-12) and counter-regulatory (IL-1ra, IL-10, and sTNFRII) cytokines by peripheral blood mononuclear cells (PBMCs) with LPS. The results were also compared to T-cell responses with common DPs and control T-cell mitogens assessed by measuring T-cell cytokine production. RE- SULTS: ASD and NFH PBMCs produced higher levels of TNF-alpha with LPS than controls regardless of dietary interventions. However, only in PBMCs from ASD children with positive gastrointestinal (GI(+)) symptoms, did we find a positive association between TNF-alpha levels produced with LPS and those with cow’s milk protein (CMP) and its major components regardless of dietary interventions. In the unrestricted diet group, GI(+) ASD PBMCs produced higher IL-12 than controls and less IL-10 than GI(‑) ASD PBMCs with LPS. GI(+) ASD but not GI(‑) ASD or NFH PBMCs produced less counter-regulatory cytokines with LPS in the unrestricted diet group than in the elimination diet group. There was no significant difference among the study groups with regard to cytokine production in responses to T-cell mitogens and other recall antigens. Conclusion: Our results revealed that there are findings limited to GI(+) ASD PBMCs in both the unrestricted and elimination diet groups. Thus our findings indicate intrinsic defects of innate immune responses in GI(+) ASD children but not in NFH or GI(‑) ASD children, suggesting a possible link between GI and behavioral symptoms mediated by innate immune abnormalities.

– 14 – Spontaneous mucosal lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms: mucosal immune activation and reduced counter regulatory interleukin-10. Ashwood P, Anthony A, Torrente F, Wakefield AJ., J Clin Immunol. 2004 Nov;24(6):664-73.35 A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3+ lymphocyte staining for spontaneous intracellular TNFalpha, IL-2, IL-4, IFNgamma, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3+ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3(+)TNFalpha+ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3(+)IL-2+ and CD3(+)IFNgamma+, and epithelial CD3(+)IL-4+ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3(+)IL-10+ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3(+)TNFalpha+ and CD3(+)IFNgamma+ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohn’s disease and non-Crohn’s colitis, LP and epithelial CD3(+)IL-10+ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3(+)TNFalpha+ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3+ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.

Frequency of gastrointestinal symptoms in children with autistic spectrum disorders and association with family history of autoimmune disease., Valicenti-McDermott M, McVicar K, Rapin I, Wershil BK, Cohen H, Shinnar S., J Dev Behav Pediatr. 2006 Apr;27(2 Suppl):S128-36.10 This is a cross-sectional study that compares lifetime prevalence of gastrointestinal (GI) symptoms in children with autistic spectrum disorders (ASDs) and children with typical development and with other developmental disabilities (DDs) and examines the association of GI symptoms with a family history of autoimmune disease. A structured interview was performed in 50 children with ASD and 2 control groups matched for age, sex, and ethnicity-50 with typical development and 50 with other DDs. Seventy-four percent were boys with a mean age of 7.6 years (SD, +/-3.6). A history of GI symptoms was elicited in 70% of children with ASD compared with 28% of children with typical development (p <.001) and 42% of children with DD (p =.03). Abnormal stool pattern was more common in children with ASD (18%) than controls (typical development: 4%, p =.039; DD: 2%, p =.021). Food selectivity was also higher in children with ASD (60%) compared with those with typical development (22%, p =.001) and DD (36%, p =.023). Family history of autoimmune disease was reported in 38% of the ASD group and 34% of controls and was not associated with a differential rate of GI symptoms. In the multivariate analysis, autism (adjusted odds ratio (OR), 3.8; 95% confidence interval (CI), 1.7-11.2) and food selectivity (adjusted OR, 4.1; 95% CI, 1.8-9.1) were associated with GI symptoms. Children with ASD have a higher rate of GI symptoms than children with either typical development or other DDs. In this study, there was no association between a family history of autoimmune disease and GI symptoms in children with ASD.

Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children.Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, Malik M, Berelowitz M, Dhillon AP, Thomson MA, Harvey P, Valentine A, Davies SE, Walker-Smith JA. Lancet. 1998 Feb 28;351(9103):637-41.30 BACKGROUND: We investigated a consecutive series of children with chronic enterocolitis and regressive developmental disorder. METHODS: 12 children (mean age 6 years [range 3-10], 11 boys) were referred to a paediatric gastroenterology unit with a history of normal development followed by loss of acquired skills, including language, together with diarrhoea and abdominal pain. Children underwent gastroenterological, neurological, and developmental assessment and review of developmental records. Ileocolonoscopy and biopsy sampling,

– 15 – magnetic-resonance imaging (MRI), electroencephalography (EEG), and lumbar puncture were done under se- dation. Barium follow-through radiography was done where possible. Biochemical, haematological, and immunological profiles were examined. FINDINGS: Onset of behavioural symptoms was associated, by the parents, with measles, mumps, and rubella vaccination in eight of the 12 children, with measles infection in one child, and otitis media in another. All 12 children had intestinal abnormalities, ranging from lymphoid nodular hyperplasia to aphthoid ulceration. Histology showed patchy chronic inflammation in the colon in 11 children and reactive ileal lymphoid hyperplasia in seven, but no granulomas. Behavioural disorders included autism (nine), disintegrative psychosis (one), and possible postviral or vaccinal encephalitis (two). There were no focal neurological abnormalities and MRI and EEG tests were normal. Abnormal laboratory results were significantly raised urinary methylmalonic acid compared with age-matched controls (p=0.003), low haemoglobin in four children, and a low serum IgA in four children. INTERPRETATION: We identified associated gastrointestinal disease and developmental regression in a group of previously normal children, which was generally associated in time with possible environmental triggers.

Colonic CD8 and gamma delta T-cell infiltration with epithelial damage in children with autism., Furlano RI, Anthony A, et al., J Pediatr. 2001 Mar;138(3):366-72.33 OBJECTIVES: We have reported colitis with ileal lymphoid nodular hyperplasia (LNH) in children with regressive autism. The aims of this study were to characterize this lesion and determine whether LNH is specific for autism. METHODS: Ileo-colonoscopy was performed in 21 consecutively evaluated children with autistic spectrum disorders and bowel symptoms. Blinded comparison was made with 8 children with histologically normal ileum and colon, 10 developmentally normal children with ileal LNH, 15 with Crohn’s disease, and 14 with ulcerative colitis. Immunohistochemistry was performed for cell lineage and functional markers, and histochemistry was performed for glycosaminoglycans and basement membrane thickness. RESULTS: Histology demonstrated lymphocytic colitis in the autistic children, less severe than classical inflammatory bowel disease. However, basement membrane thickness and mucosal gamma delta cell density were significantly increased above those of all other groups including patients with inflammatory bowel disease. CD8(+) density and intraepithelial lymphocyte numbers were higher than those in the Crohn’s disease, LNH, and normal control groups; and CD3 and plasma cell density and crypt proliferation were higher than those in normal and LNH control groups. Epithelial, but not lamina propria, glycosaminoglycans were disrupted. However, the epithelium was HLA-DR(‑), suggesting a predominantly T(H)2 response. INTERPRETATION: Immunohistochemistry confirms a distinct lymphocytic colitis in autistic spectrum disorders in which the epithelium appears particularly affected. This is consistent with increasing evidence for gut epithelial dysfunction in autism.

Intestinal lymphocyte populations in children with regressive autism: evidence for extensive mucosal immunopathology., Ashwood P, Anthony A, Pellicer AA, Torrente F, Walker-Smith JA, Wakefield AJ., J Clin Immunol. 2003 Nov;23(6):504-17.34 Inflammatory intestinal pathology has been reported in children with regressive autism (affected children). De- tailed analysis of intestinal biopsies in these children indicates a novel lymphocytic enterocolitis with autoimmune features; however, links with cognitive function remain unclear. To characterize further, the nature and extent of this disease we examined the mucosal infiltrate using flow cytometry. Duodenal, ileal, and colonic biopsies were obtained from 52 affected children, 25 histologically normal, and 54 histologically inflamed, developmentally normal controls. Epithelial and lamina propria lymphocyte populations were isolated and examined by multicolor flow cytometry. Adjacent biopsies were assessed by semiquantitative histopathology. At all sites, CD3(+) and CD3(+)CD8(+) IEL as well as CD3(+) LPL were significantly increased in affected children compared with developmentally normal noninflamed control groups (p<0.01) reaching levels similar to inflamed controls. In addition, two populations--CD3(+)CD4(+) IEL and LP CD19(+) B cells--were significantly increased in affected children compared with both noninflamed and inflamed control groups including IBD, at all sites examined (p<0.01). Histologically there was a prominent mucosal eosinophil infiltrate in affected children that was significantly lower in those on a gluten- and casein-free diet, although lymphocyte populations were not influenced by diet.The data provide further evidence of a pan-enteric mucosal immunopathology in children with regressive autism that is apparently distinct from other inflammatory bowel diseases.

– 16 – Autism and gastrointestinal symptoms., Horvath K, Perman JA., Curr Gastroenterol Rep. 2002 Jun;4(3):251-8.37 Recent clinical studies have revealed a high prevalence of gastrointestinal symptoms, inflammation, and dysfunction in children with autism. Mild to moderate degrees of inflammation were found in both the upper and lower intestinal tract. In addition, decreased sulfation capacity of the liver, pathologic intestinal permeability, increased secretory response to intravenous secretin injection, and decreased digestive enzyme activities were reported in many children with autism. Treatment of digestive problems appears to have positive effects on autistic behavior. These new observations represent only a piece of the unsolved autism “puzzle” and should stimulate more research into the brain-gut connection.

The significance of ileo-colonic lymphoid nodular hyperplasia in children with autistic spectrum disorder. Wakefield AJ, Ashwood P, Limb K, Anthony A. Eur J Gastroenterol Hepatol. 2005 Aug;17(8):827-36.38 BACKGROUND: Intestinal mucosal pathology, characterized by ileo-colonic lymphoid nodular hyperplasia (LNH) and mild acute and chronic inflammation of the colorectum, small bowel and stomach, has been reported in children with autistic spectrum disorder (ASD). AIM: To assess ileo-colonic LNH in ASD and control children and to test the hypothesis that there is an association between ileo-colonic LNH and ASD in children. PATIENTS AND METHODS: One hundred and forty-eight consecutive children with ASD (median age 6 years; range 2-16; 127 male) with gastrointestinal symptoms were investigated by ileo-colonoscopy. Macroscopic and histological features were scored and compared with 30 developmentally normal (non-inflammatory bowel disease, non-coeliac disease) controls (median age 7 years; range 1-11; 25 male) showing mild non-specific colitis in 16 cases (13 male) and normal colonic histology in 14 cases (12 male). Seventy-four ASD children and 23 controls also underwent upper gastrointestinal endoscopy. The influence on ileal LNH of dietary restriction, age at colonoscopy, and co-existent LNH elsewhere in the intestine, was examined. RESULTS: The prevalence of LNH was significantly greater in ASD children compared with controls in the ileum (129/144 (90%) vs. 8/27 (30%), P < 0.0001) and colon (88/148 (59%) vs. 7/30 (23%), P = 0.0003), whether or not controls had co-existent colonic inflammation. The severity of ileal LNH was significantly greater in ASD children compared with controls, with moderate to severe ileal LNH present in 98 of 144 (68%) ASD children versus 4 of 27 (15%) controls (P < 0.0001). Severe ileal LNH was associated with co-existent colonic LNH in ASD children (P = 0.01). The presence and severity of ileal LNH was not influenced by either diet or age at colonoscopy (P = 0.2). Isolated ileal LNH without evidence of pathology elsewhere in the intestine was a rare event, occurring in less than 3% of children overall. On histopathological examination, hyperplastic lymphoid follicles are significantly more prevalent in the ileum of ASD children (84/138; 61%) compared with controls (2/23; 9%, P = 0.0001). CONCLUSION: Ileo-colonic LNH is a characteristic pathological finding in children with ASD and gastrointestinal symptoms, and is associated with mucosal inflammation. Differences in age at colonoscopy and diet do not account for these changes. The data support the hypothesis that LNH is a significant pathological finding in ASD children.

Detection and Sequencing of Measles Virus from Peripheral Mononuclear Cells from Patients with Inflammatory Bowel Disease and Autism. Hisashi Kawashima, Takayuki Mori, Yasuyo Kashiwagi, Kouji Takekuma. Digestive Diseases and Sciences, 2000 Apr;45(4):723-9.39 It has been reported that measles virus may be present in the intestine of patients with Crohn’s disease. Ad- ditionally, a new syndrome has been reported in children with autism who exhibited developmental regression and gastrointestinal symptoms (autistic enterocolitis), in some cases soon after MMR vaccine. It is not known whether the virus, if confirmed to be present in these patients, derives from either wild strains or vaccine strains. In order to characterize the strains that may be present, we have carried out the detection of measles genomic RNA in peripheral mononuclear cells (PBMC) in eight patients with Crohn’s disease, three patients with ulcerative colitis, and nine children with autistic enterocolitis. As controls, we examined healthy children and patients with SSPE, SLE, HIV-1 (a total of eight cases). RNA was purified from PBMC by Ficoll-paque, followed by reverse transcription using AMV; cDNAs were subjected to nested PCR for detection of specific regions of the he- magglutinin (H) and fusion (F) gene regions. Positive samples were sequenced directly, in nucleotides 8393-8676 (H region) or 5325-5465 (from noncoding F to coding F region). One of eight patients with Crohn disease, one of three patients with ulcerative colitis, and three of nine children with autism, were positive. Controls were all negative. The sequences obtained from the patients with Crohn’s disease shared the characteristics with wild-strain

– 17 – virus. The sequences obtained from the patients with ulcerative colitis and children with autism were consistent with being vaccine strains. The results were concordant with the exposure history of the patients. Persistence of measles virus was confirmed in PBMC in some patients with chronic intestinal inflammation.

Small intestinal enteropathy with epithelial IgG and complement deposition in children with regressive autism., Torrente F, Ashwood P, Day R, Machado N, Furlano RI, Anthony A, Davies SE, Wakefield AJ, Thomson MA, Walker-Smith JA, Murch SH. Mol Psychiatry. 2002;7(4):375-82, 334. 40 We have reported lymphocytic colitis in children with regressive autism, with epithelial damage prominent. We now compare duodenal biopsies in 25 children with regressive autism to 11 with coeliac disease, five with cerebral palsy and mental retardation and 18 histologically normal controls. Immunohistochemistry was performed for lymphocyte and epithelial lineage and functional markers. We determined the density of intraepithelial and lamina propria lymphocyte populations, and studied mucosal immunoglobulin and complement C1q localisation. Standard histopathology showed increased enterocyte and Paneth cell numbers in the autistic children. Immunohistochemistry demonstrated increased lymphocyte infiltration in both epithelium and lamina propria with upregulated crypt cell proliferation, compared to normal and cerebral palsy controls. Intraepithelial lymphocytes and lamina propria plasma cells were lower than in coeliac disease, but lamina propria T cell populations were higher and crypt proliferation similar. Most strikingly, IgG deposition was seen on the basolateral epithelial surface in 23/25 autistic children, co-localising with complement C1q. This was not seen in the other conditions. These findings demonstrate a novel form of enteropathy in autistic children, in which increases in mucosal lymphocyte density and crypt cell prolferation occur with epithelial IgG deposition. The features are suggestive of an autoimmune lesion.

Focal-enhanced gastritis in regressive autism with features distinct from Crohn’s and Helicobacter pylori gastritis., Torrente F, Anthony A, Heuschkel RB, Thomson MA, Ashwood P, Murch SH. Am J Gastroenterol. 2004 Apr;99(4):598-605.41 BACKGROUND: Immunohistochemistry allowed recent recognition of a distinct focal gastritis in Crohn’s disease. Following reports of lymphocytic colitis and small bowel enteropathy in children with regressive autism, we aimed to see whether similar changes were seen in the stomach. We thus studied gastric antral biopsies in 25 affected children, in comparison to 10 with Crohn’s disease, 10 with Helicobacter pylori infection, and 10 histologically normal controls. All autistic, Crohn’s, and normal patients were H. pylori negative. METHODS: Snap- frozen antral biopsies were stained for CD3, CD4, CD8, gammadelta T cells, HLA-DR, IgG, heparan sulphate proteoglycan, IgM, IgA, and C1q. Cell proliferation was assessed with Ki67. RESULTS: Distinct patterns of gastritis were seen in the disease states: diffuse, predominantly CD4+ infiltration in H. pylori, and focal-enhanced gastritis in Crohn’s disease and autism, the latter distinguished by striking dominance of CD8+ cells, together with increased intraepithelial lymphocytes in surface, foveolar and glandular epithelium. Proliferation of foveolar epithelium was similarly increased in autism, Crohn’s disease and H. pylori compared to controls. A striking finding, seen only in 20/25 autistic children, was colocalized deposition of IgG and C1q on the subepithelial basement membrane and the surface epithelium. CONCLUSIONS: These findings demonstrate a focal CD8- dominated gastritis in autistic children, with novel features. The lesion is distinct from the recently recognized focal gastritis of Crohn’s disease, which is not CD8-dominated. As in the small intestine, there is epithelial deposition of IgG.

3.6. Mikrobiell metabolisme i mage–tarm Det er nå allment akseptert at at intestinal microbiota (IM) i mage/tarm utgjør kroppens største og mest aktive metabolske organ. Den påvirker mange av kroppens funksjoner på måter som vi tidligere ikke var klar over. Slike påvirkninger kan dels skje i tarmen, dels i andre av kroppens organer ved hjelp av signalstoffer som bakteriene har produsert og som er blitt absorbert.

Nyere og pågående forskning indikerer et komplekst samspill mellom de metabolske funksjonene til den intestinale flora hos barn med ASD.

– 18 – Ved å ta i bruk moderne molekylær metoder for å sammenligne forskjeller og likheter i sammensetning og funksjon av den intestinale flora hos barn med ASD og kontroller kan man kanskje finne nye metoder for å minimere symptomene hos barn med autisme.

Neurotransmisjon For mer enn et halvt århundre siden ble det klart påvist at IM i tarmen kunne øke vevssensitivitet til flere nev- rotransmittere2 og at kjente bakteriestammer i tarmen forårsaket delvis normalisering av denne sensitiviteten.3 Allerede da ble det klart etablert en akse mellom mikrober i tarmen og sensitiviteten til nevrotransmittere. Uten å gå i detalj kan det her være nyttig å nevne en nyere publikasjon på dette feltet42. En forskergruppe fra Japan har vist – gjennom komparative studier av mikrobefrie (GF = germfree) og konvensjonelle dyr – at GF-dyr viste redusert uttrykt hjernederivert nevrotropisk faktor i enkelte deler av hjernen, samt at de hadde overdreven reak- sjon på stress av hypothalamus-hypofyse-binyre-aksen (HPA). Verd å merke seg er at denne hyperaktiveringen av HPA-responsen hos GF-dyr kunne korrigeres ved etablering av mikroflora i GF-dyrene ved tidlig alder, dog ikke ved sen alder. Disse observasjonene taler til fordel for hypotesen at for å være funksjonelt optimal kan det finnes et “vindu for etablering” av mange bakteriearter i tarmen.

Endringer i bakteriefloraen og produksjon av metabolitter.

3.6.1. Neurotoxin Bolte43 la merke til at mange barn med ASD hadde en anamnese med omfattende antibiotikabruk, og la fram en hypotese om at i enkelte tilfeller kunne autisme være forårsaket av en overvekst av en klostridieart som det er kjent produserer et nevrotoksin. Bolte talte for behandling med antibiotika som hadde effekt på slike klostridier. Etter dette har mange publikasjoner fokusert på endringer i den klostride del av IM hos barn med ASD samt forekomst/mangel på forskjellige klostridiearter. Så langt er alle disse bakteriene strengt anaerobiske, og det meste av deres metabolisme kan kun finne sted ved omgivelser som er svært like de som finnes i enkelte deler av mitokondriene hos pattedyr.44, 45, 46, 47

Vi siterer tre publiserte studier som støtter dette standpunktet.

Autism and Clostridium tetani. Bolte ER. Med Hypotheses. 1998 Aug;51(2):133-44.43 Autism is a severe developmental disability believed to have multiple etiologies. This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals. A significant percentage of individuals with autism have a history of extensive antibiotic use. Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens. Clostridium tetani is an ubiquitous anaerobic bacil- lus that produces a potent neurotoxin. Intestinal colonization by C. tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells. The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS. This route bypasses TeNT’s normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident. Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism. Lab animals injected in the brain with TeNT have exhibited many of these behaviors. Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia. When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis. A review of atypical tetanus cases, and strategies to test the validity of this paper’s hypothesis, are included.

Gastrointestinal microflora studies in late-onset autism. Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe EM, Collins MD, Lawson PA, Summanen P, Baysallar M, Tomzynski TJ, Read E, Johnson E, Rolfe R, Nasir P, Shah H, Haake DA, Manning P, Kaul A. Clin Infect Dis. 2002 Sep 1;35(Suppl 1):S6-S16. 44 Some cases of late-onset (regressive) autism may involve abnormal flora because oral vancomycin, which is poorly absorbed, may lead to significant improvement in these children. Fecal flora of children with regressive autism

– 19 – was compared with that of control children, and clostridial counts were higher. The number of clostridial species found in the stools of children with autism was greater than in the stools of control children. Children with autism had 9 species of Clostridium not found in controls, whereas controls yielded only 3 species not found in children with autism. In all, there were 25 different clostridial species found. In gastric and duodenal specimens, the most striking finding was total absence of non-spore-forming anaerobes and microaerophilic bacteria from control children and significant numbers of such bacteria from children with autism. These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.

Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. Parracho HM, Bingham MO, Gibson GR, McCartney AL. J Med Microbiol. 2005 Oct;54(Pt 10):987-91.46 Children with autistic spectrum disorders (ASDs) tend to suffer from severe gastrointestinal problems. Such symptoms may be due to a disruption of the indigenous gut flora promoting the overgrowth of potentially pathogenic micro-organisms. The faecal flora of patients with ASDs was studied and compared with those of two control groups (healthy siblings and unrelated healthy children). Faecal bacterial populations were assessed through the use of a culture-independent technique, fluorescence in situ hybridization, using oligonucleotide probes targeting predominant components of the gut flora. The faecal flora of ASD patients contained a higher incidence of the Clostridium histolyticum group (Clostridium clusters I and II) of bacteria than that of healthy children. However, the non-autistic sibling group had an intermediate level of the C. histolyticum group, which was not significantly different from either of the other subject groups. Members of the C. histolyticum group are recognized toxin-producers and may contribute towards gut dysfunction, with their metabolic products also exerting systemic effects. Strategies to reduce clostridial population levels harboured by ASD patients or to improve their gut microflora profile through dietary modulation may help to alleviate gut disorders common in such patients.

3.6.2. Endotoksin En vesentlig del av IM produserer endotoksin, men dette blir vanligvis inaktivert i tarm og lever. Økt absorpsjon/ nedsatt inaktivering er blitt framholdt som mulige faktorer for utvikling av autisme Det foreligger nå åtte publikasjoner, men konklusjonene er usikre.

3.6.3. Bakterielle korte frie fettsyre En forskergrupp I Canada har fokusert på propionsyre, en kort fettsyre, som forbindelsesledd mellom IM og hjernefunksjon.48 Gruppen har testet påvirkningen av propionsyre (PPA) på hjernefunksjon hos rotte. og konklu- derer med at PPA “kan endre både hjernen og adferden hos laboratorierotter på en måte som er i samsvar med autisme hos menneske”. Det som gjør denne forskningen ekstra interessant er at PPA både er en viktig metabolitt i IM og også brukes som konserveringsmiddel i næringsmidler. Ved cøliaki bevares fettmønsteret selv etter at barnet er satt på en glutenfri diett (Tjellstrøm et.al 2007).49

3.6.4. Svovelmetabolisme. Mage–tarm har gruppens største innhold av svovel. IM kan både transulfatere, splitte svovelholdige konjugater og produsere store mengder svovelvannstoff (H2S). Det er nylig oppdaget av H2S er en viktig neuotransmitter i ulike organer, inklusivt hjerne.

3.7. Oksalat Oksalat – en organisk syre er et naturlig forekommende stoff i naturen. Den er grunnleggende og nødvendig for menneskets metabolisme.

Oksalats potensielle negative effekter er omfattende studert i nyrene, men det er gjort svært lite forskning som vurderer hvilken skade oksalat kan gjøre i resten av kroppen, når underliggende tilstander ikke er optimale. Omfattende litteratur viser at oksalat kan transporteres og blir transportert til hele kroppen (Doganavsargil et al 2009).50

– 20 – Ved en sjelden genetisk tilstand kalt primær hyperoksaluri er det kjent at leveren produserer for mye oksalat, slik at nivåene kan bli fatale. For å hindre dette, kan man utføre en levertransplantasjon. Etter transplantasjon tar det mange år før kroppen kvitter seg med overskudd av oksalat. Når dette skjer kan skadet vev få funksjoner tilbake (Cochet 1993).51

Så vidt vi kjenner til er det ingen som har utført forskning på å studere den parallelle tilstanden der høye ok- salatverdier kommer fra hyperabsorpsjon av oksalat fra en betent tarm, en tilstand kalt enterisk hyperoksaluri. En lav oksalat-diett blir bare i sjeldne tilfeller anbefalt ved enterisk hyperoksaluri etter at pasienten har utviklet nyreproblemer. Hvordan oksalat påvirker andre komorbide tilstander som det er kjent kan utvikles i resten av kroppen hos disse pasientene, er aldri blitt studert ved sykdommene forbundet med enterisk hyperoksaluri. Så vidt vi vet er det ikke utført noen studier på barn med ASD om forhøyet oksalat kan påvirke utviklingen av deres symptomer.

Enkelte bakterier, spesielt Oxalobacter formigenes, bruker oksalat som næring. Mangel på kolonisering av denne mikroben er forbundet med oksalatrelatert sykdom. Enkelte sopparter kan generere mer oksalat som respons på metaller som sink, kobber og kadmium. Candida albicans kan generere oksalat ved å danne mellomstadiet d-erytroaskorbinsyre fra sukkertyper som arabinose. Arabinose er ofte funnet forhøyet ved autisme. Årsaksfor- holdet bak de forhøyede nivåene er dog usikkert (Duncan et al 2002).52

Oksalater kan danne krystaller med enkelte aminosyrer som beta -alanin. I og med at de danner krystaller med kalsium, trekkes kalsiumet bort fra andre områder. Sitrat, sink, magnesium og A–vitamin beskytter mot dannelse av oksalatkrystaller. I og med at sink virker beskyttende betyr det også at oksalat kan føre til at mer sink skilles ut i urin. I og med at kobber ikke er involvert her kan det oppstå en ubalanse i forholdet mellom sink og kobber106 (Faber et al 2009).53

Oksalatproblemer kan forekomme på grunn av vitaminmangel. B6 er en viktig kofaktor ved konverteringen av svovel og er nødvendig i transulfateringsyklus for blant annet cystein, glutation, taurin og sulfat. Det er funnet unormale nivåer av alle disse stoffene hos personer med autisme. Ved å binde forløperne til oksalat kan tiolene i denne molekylgruppen beskytte kroppen mot at cellene produserer oksalat, men dette er ikke mulig ved mangel på disse tiolene (Nisjhijima et al 2006).54

Uansett er den mest direkte forbindelsen mellom B6 og oksalat forbundet med et enzym kalt glyoksylat aminotransferase (AGT), som sender forløperen til oksalat (glyoksylat) til dannelse av glysin. Ved B6- mangel vil ikke dette enzymet virke optimalt. Resultatet er at andre enzymer tar over og produserer oksalat fra glyoksylat (Nishi- jima et al. 2006).54 Et av disse enzymene, glykolat oksidase, hemmes av alfalinolensyre. (Holmes 1998).55

Vi siterer tre publiserte studier som støtter behovet for mer forskning om oksalat og oksalatets potensielle rolle ved autisme, spesielt forbindelsen mellom omsetningen av oksalat og svovel.

Effect of vitamin B6 deficiency on glyoxylate metabolism in rats with or without glyoxylate overload. Nishijima S, Sugaya K, Hokama S, Oshiro Y, Uchida A, Morozumi M, Ogawa Y. Biomed Res. 2006 Jun;27(3):93-8.54 We examined the effect of vitamin B6 deficiency on glyoxylate metabolism and hepatic alanine: glyoxylate aminotransferase (AGT) activity in rats with normal or high glyoxylate intake. Male rats were divided into four groups: a control group, a vitamin B6-free diet group, a glyoxylate water group, and a vitamin B6-free diet + glyoxylate water group. Each group was given special diet (control or vitamin B6-deficient diet) and drinking water (plain or 0.5% glyoxylate water) for 4 weeks, after which biochemical parameters and hepatic AGT mRNA level were measured. Compared with control rats, the urinary oxalate/creatinine ratio was higher in each of the other 3 groups. The urinary glycolate/creatinine ratio was also higher in the vitamin B6-free diet group and the vitamin B6-free diet + glyoxylate water group than the control group, while the urinary glycine/creatinine and citrate/ creatinine ratio was lower in both groups. The hepatic AGT mRNA level was reduced in the vitamin B6-free diet group, but was increased in the glyoxylate water group than the control group. These results suggest that vitamin B6 is necessary for glyoxylate metabolism as a coenzyme of AGT. Especially in the presence of a high glyoxylate intake, vitamin B6 deficiency leads to severe hyperoxaluria and hypocituria.

– 21 – Formation of the L-cysteine-glyoxylate adduct is the mechanism by which L-cysteine decreases oxalate production from glycollate in rat hepatocytes. Baker PW, Bais R, Rofe AM, Biochemical Journal 1994 Sep 15;302 ( Pt 3):753-7.56 Formation of thiazolidine-2,4-dicarboxylic acid, the L-cysteine-glyoxylate adduct, is the putative mechanism by which L-cysteine reduces hepatic oxalate production from glycollate [Bais, Rofe and Conyers (1991) J. Urol. 145, 1302-1305]. This was investigated in isolated rat hepatocytes by the simultaneous measurement of both adduct and oxalate formation. Different diastereoisomeric ratios of cis- and trans-adduct were prepared and characterized to provide both standard material for the enzymic analysis of adduct in hepatocyte supernatants and to investigate the stability and configuration of the adduct under physiological conditions. In the absence of L-cysteine, hepatocytes produced oxalate from 2 mM glycollate at a rate of 822 +/- 42 nmol/30 min per 10(7) cells. The addition of L-cysteine to the incubation medium at 1.0, 2.5 and 5.0 mM lowered oxalate production by 14 +/- 2, 25 +/- 3 (P < 0.05) and 38 +/- 3% (P < 0.01) respectively. These reductions were accompanied by almost stoichiometric increases in the levels of the adduct: 162 +/- 6, 264 +/- 27 and 363 +/- 30 nmol/30 min per 10(7) cells. Adduct formation is therefore confirmed as the primary mechanism by which L-cysteine decreases oxalate production from glycollate. As urinary oxalate excretion is a prime risk factor in the formation of calcium oxalate stones, any reduction in endogenous oxalate production is of clinical significance in the prevention of this formation.

Oxalate production from glyoxylate by lactate dehydrogenase in vitro: inhibition by reduced glutathione, cysteine, cysteamine. Sharma V, Schwille PO. Biochem Int. 1992 Jul;27(3):431-8.57 Lactate dehydrogenase is known to act as a dismutase converting glyoxylate to oxalate and glycolate. LDH (sources: human erythrocytes, human plasma; rabbit muscle; rat liver) activity was assayed at 340 nm using glyoxylate (5.0 mmol/l) and NADH. The LDH activity (approx. % of control) in all the cases decreased respectively in the presence of 5.0 and 10.0 mmol/l of cysteine (45 and 20), cysteamine (45 and 20), and GSH (55 and 30). This decrease in LDH activity resulted in decreased oxalate production from glyoxylate (0.5 mmol/l). A 50% inhibition in oxalate production was observed in presence of 0.3 mmol/l cysteine, 0.35 mmol/l cysteamine, and 2.0 mmol/l GSH. The results suggest that the net LDH activity towards oxalate production may be regulated by the free SH- groups in the cell. This possibility needs evaluation as a tool to lower endogenous oxalate production and the associated risk of stone formation.

3.8. Oksidativt stress Oksidativt stress ved autisme er studert på cellemembran nivå blant annet ved å måle produktene av lipid peroksidering, avgiftningsmekanismer samt antioksidanter. Markører for lipid peroksidering er generelt forhøyet ved autisme, noe som kan antyde at oksidativt stress er økt ved denne forstyrrelsen. Mengden viktige serumproteiner med antioksidante egenskaper, nærmere bestemt transferrin (et jernbindende protein) og ceruloplasmin (et kob- berbindende protein) er nedsatt hos barn med autisme. Det er en positiv korrelasjon mellom reduserte nivåer av disse proteinene og tap av eksisterende språkevne hos barn med autisme. Flere studier har foreslått endringer i aktivitetene til enzymer med antioksidant virkning som superoksiddismu- tase, glutationperoksidase og katalase ved autisme. Endrede glutationnivåer og homocystein/metioninmetabo- lisme, økt betennelse, eksitotoksisitet samt mitokondriell dysfunksjon og dysfunksjon i immunsystemet er også blitt foreslått ved autisme (Chauhan 2006).60

Vi siterer fire publiserte studer og et review som støtter standpunktet om at barn med ASD kan ha økt oksidativt stress:

Altered vascular phenotype in autism: correlation with oxidative stress. Yao Y, Walsh WJ, McGinnis WR, Praticò D.Department of Pharmacology, School of Medicine, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.58 BACKGROUND: Autism is a neurologic disorder characterized by impaired communication and social interaction. Results of previous studies showed biochemical evidence for abnormal platelet reactivity and altered blood flow in children with autism. OBJECTIVE: To evaluate the vascular phenotype in children with autism. DESIGN AND MAIN OUTCOME MEASURES: Urinary levels of isoprostane F(2alpha)-VI, a marker of lipid peroxidation;

– 22 – 2,3-dinor-thromboxane B(2), which reflects platelet activation; and 6-keto-prostaglandin F(1alpha), a marker of endothelium activation, were measured by means of gas chromatography-mass spectrometry in subjects with autism and healthy control subjects. SETTING AND SUBJECTS: Children with a clinical diagnosis of autism attending the Pfeiffer Treatment Center. RESULTS: Compared with controls, children with autism had significantly higher urinary levels of isoprostane F(2alpha)-VI, 2,3-dinor-thromboxane B(2), and 6-keto-prostaglandin F(1alpha). Lipid peroxidation levels directly correlated with both vascular biomarker ratios. CONCLUSION: Besides enhanced oxidative stress, platelet and vascular endothelium activation also could contribute to the development and clinical manifestations 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. Am J Clin Nutr. 2004 Dec;80(6):1611-7.59 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.

Oxidative Stress in Autism, Chauhan A, Chauhan, Pathophysiology. 2006Aug;13(3):171-81. Epub 2006 Jun 12.60 Oxidative stress in autism has been studied at the membrane level and also by measuring products of lipid peroxidation, detoxifying agents (such as glutathione), and antioxidants involved in the defense system against reactive oxygen species (ROS). Lipid peroxidation markers are elevated in autism, indicating that oxidative stress is increased in this disease. Levels of major antioxidant serum proteins, namely transferrin (iron-binding protein) and ceruloplasmin (copper-binding protein), are decreased in children with autism. There is a positive correlation between reduced levels of these proteins and loss of previously acquired language skills in children with autism. The alterations in ceruloplasmin and transferrin levels may lead to abnormal iron and copper metabolism in autism. The membrane phospholipids, the prime target of ROS, are also altered in autism. The levels of phosphatidylethanolamine (PE) are decreased, and phosphatidylserine (PS) levels are increased in the erythrocyte membrane of children with autism as compared to their unaffected siblings. Several studies have suggested alterations in the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase in autism. Additionally, altered glutathione levels and homocysteine/methionine metabolism, increased inflammation, excitotoxicity, as well as mitochondrial and immune dysfunction have been suggested in autism. Furthermore, environmental and genetic factors may increase vulnerability to oxidative stress in autism. Taken together, these studies suggest increased oxidative stress in autism that may contribute to the development of this disease. A mechanism linking oxidative stress with membrane lipid abnormalities, inflammation, aberrant immune response, impaired energy metabolism and excitotoxicity, leading to clinical symptoms and pathogenesis of autism is proposed.

Large Brains in Autism: The Challenge of Pervasive Abnormality. Herbert, Martha, Neuroscientist. 2005 Oct;11(5):417-40.61

– 23 – The most replicated finding in autism neuroanatomy – a tendency to unusually large brains – has seemed para- doxical in relation to the specificity of the abnormalities in three behavioral domains that define autism. We now know a range of things about this phenomenon, including that brains in autism have a growth spurt shortly after birth and then slow in growth a few short years afterward, that only younger but not older brains are larger in autism than in controls, that white matter contributes disproportionately to this volume increase and in a nonuniform pattern suggesting postnatal pathology, that functional connectivity among regions of autistic brains is diminished, and that neuroinflammation (including microgliosis and astrogliosis) appears to be present in autistic brain tissue from childhood through adulthood. Alongside these pervasive brain tissue and functional abnormalities, there have arisen theories of pervasive or widespread neural information processing or signal coordination abnormalities (such as weak central coherence, impaired complex processing, and underconnectivity), which are argued to underlie the specific observable behavioral features of autism. This convergence of findings and models suggests that a systems- and chronic disease–based reformulation of function and pathophysiology in autism needs to be considered, and it opens the possibility for new treatment targets.

Evidence of Toxicity, Oxidative Stress, and Neuronal Insult in Autism Kern JK, Jones AM.J Toxicol Environ Health B Crit Rev. 2006 Nov-Dec;9(6):485-99.62 According to the Autism Society of America, autism is now considered to be an epidemic. The increase in the rate of autism revealed by epidemiological studies and government reports implicates the importance of external or environmental factors that may be changing. This article discusses the evidence for the case that some children with autism may become autistic from neuronal cell death or brain damage sometime after birth as result of insult; and addresses the hypotheses that toxicity and oxidative stress may be a cause of neuronal insult in autism. The article first describes the Purkinje cell loss found in autism, Purkinje cell physiology and vulnerability, and the evidence for postnatal cell loss. Second, the article describes the increased brain volume in autism and how it may be related to the Purkinje cell loss. Third, the evidence for toxicity and oxidative stress is covered and the possible involvement of glutathione is discussed. Finally, the article discusses what may be happening over the course of development and the multiple factors that may interplay and make these children more vulnerable to toxicity, oxidative stress, and neuronal insult.

3.9. Virus infeksjoner Studier av betent tarmvev i en undergruppe av barn med ASD viste forekomst av virus i biopsier av tarmvevet. Meslingeviruset som ble funnet der var samme virusstamme som i vaksinen. Forskning har antydet forbindelser mellom ASD og forekomst av meslinger i tarmen. MMR-vaksinen inneholder levende virus som kan formere seg. Kawashima og hans medarbeidere rapporterte forekomst av vaksinestammen av meslingeviruset i mononukleæ- re celler i perifert blod hos en undergruppe med ASD (Kawashima et al. 2000)39, noe som kan bidra til betennelse i blod-hjernebarrieren og til lave nivåer av virusinfiltrasjon i sentralnervesystemet.

Vi siterer her tre publiserte studier som støtter standpunktet om at meslingevirus kan spille en rolle i utviklingen hos enkelte barn på autismespekteret.

Potential viral pathogenic mechanism for new variant inflammatory bowel disease. Uhlmann V, Martin CM, Sheils O, Pilkington L, Silva I, Killalea A, Murch SB, Walker-Smith J, Thomson M, Wakefield AJ, O’Leary JJ. Mol Pathol. 2002 Apr;55 (2):84-90.63 AIMS: A new form of inflammatory bowel disease (ileocolonic lymphonodular hyperplasia) has been described in a cohort of children with developmental disorder. This study investigates the presence of persistent measles virus in the intestinal tissue of these patients (new variant inflammatory bowel disease) and a series of controls by molecular analysis. METHODS: Formalin fixed, paraffin wax embedded and fresh frozen biopsies from the -ter minal ileum were examined from affected children and histological normal controls. The measles virus Fusion (F) and Haemagglutinin (H) genes were detected by TaqMan reverse transcription polymerase chain reaction (RT- PCR) and the Nucleocapsid (N) gene by RT in situ PCR. Localisation of the mRNA signal was performed using a specific follicular dendritic cell antibody. RESULTS: Seventy five of 91 patients with a histologically confirmed diagnosis of ileal lymphonodular hyperplasia and enterocolitis were positive for measles virus in their intestinal tissue compared with five of 70 control patients. Measles virus was identified within the follicular dendritic cells

– 24 – and some lymphocytes in foci of reactive follicular hyperplasia. The copy number of measles virus ranged from one to 300,00 copies/ng total RNA. CONCLUSIONS: The data confirm an association between the presence of measles virus and gut pathology in children with developmental disorder.

Detection of Measles Virus Genomic RNA in Cerebrospinal Fluid of Children with Regressive Autism: a Report of Three Cases. Bradstreet JJ., El Dahr J., Anthony A., Kartzinel J., Wakefield AJ. Journal of American Physicians and Surgeons 2004 9:39-45.64

In light of encephalopathy presenting as autistic regression (autistic encephalopathy, AE) closely following measles-mumpsrubella (MMR) vaccination, three children underwent cerebrospinal fluid (CSF) assessments including studies for measles virus (MV). All three children had concomitant onset of gastrointestinal (GI) symptoms and had already had MV genomic RNA detected in biopsies of ileal lymphoid nodular hyperplasia (LNH). Pre- sence of MV Fusion (F) gene was examined by TaqMan realtime quantitative polymerase chain reaction (RT- PCR) in cases and control CSF samples. The latter were obtained from three nonautistic MMR-vaccinated children with indwelling shunts for hydrocephalus. None of the cases or controls had a history of measles exposure other than MMR vaccination. Serum and CSF samples were also evaluated for antibodies to MV and myelin basic protein (MBP). MV F gene was present in CSF from all three cases, but not in controls. Genome copy number ranged from 3.7x10 to 2.42x10 per ng of RNA total. Serum anti-MBP autoantibodies were detected in all children with AE. Anti-MBP and MV antibodies were detected in the CSF of two cases, while the third child had neither anti-MBP norMVantibodies detected in hisCSF. Findings are consistent with both an MV etiology for the AE and active viral replication in these children. They further indicate the possibility of a virally driven cerebral immunopathology in some cases of regressive autism.

TaqMan RT-PCR Detection of Measles Virus Genomic RNA in Cerebrospinal Fluid in Children with Regressive Autism. IMFAR May 2004.65 In light of encephalopathy, presenting in children as autistic regression closely following MMR vaccination, affected (ASD) children (n = 28) underwent lumbar puncture and examination of cerebrospinal fluid (CSF) for measles virus (MV) genomic RNA. Presence of MV Fusion (F) gene was examined by TaqMan RT-PCR. Control CSF samples (n = 37) were obtained from children in remission from leukemia (n = 20), children undergoing shunt insertion for hydrocephalus (n = 3) and young adults with either multiple sclerosis (n = 7) or encephalitis (n = 7). All ASD cases and pediatric controls had received MMR vaccine. MV hemaglutinnin (H) gene allelic discrimination (AD) assay was performed on cases where adequate MV amplicon was obtained. MV F-gene was present in CSF from 19 of 28 (68%) cases and in one of 37 (3%) controls (RR = 25.90; CI 3.96-181.58, p<0.00001). Where data were available on CSF (5 cases), AD assay confirmed that the MV H-gene product was consistent with vaccine strain. The findings confirm a highly significant statistical association between the presence of MV RNA in CSF and autistic regression following MMR vaccination.

4. Miljøstudier 4.1. Miljøgifter Gruvearbeidere i Pennsylvania og West Virginias kullgruver tok ofte med seg kanarifugler ned i kullgruven for å bli varslet om forekomst av metangass. Så lenge kanarifuglene levde var gruvearbeiderne trygge. Når fuglene sluttet å synge og døde viste arbeiderne at de måtte skynde seg opp. Barn generelt og autistiske barn spesielt kan være dagens “kanarifugler” i vår kultur. De forteller oss allerede nå at kanskje noe av maten vi spiser, måten vi håndterer industrielt avfall og noen av materialene vi har brukt og bruker i jordbruk, tannpleie og vaksiner ikke alltid er trygge for oss. Det tar tid før mennesket skjønner hva som skjer, og vi fortsetter å bruke mange av disse stoffene som kan være skadelige.

Vitenskapen har lenge ment at autisme er en genetisk forstyrrelse. Genforskningen har dog ikke klart å identifise- re noe spesifikt kromosom eller sted på et gen som er en primær årsak til autisme. Selv om en spesifikk forståelse av hvordan genetikken bidrar til autismetilstander ennå ikke er fullstendig, er det åpenbart at det er sannsynlige genetiske predisposisjoner eller genetisk sårbarhet hos de fleste eller alle barn med ASD (Warren 1996)66. Det er mulig at en genetisk predisposisjon sammen med miljømessige påvirkninger utløser skaden.

– 25 – Mennesket utsettes for mange tungmetaller man vet er giftige i miljøet, blant annet bly, kvikksølv, antimon, arsenikk, kadmium, aluminium, krom og nikkel. Mange av disse metallene kan forstyrre forskjellige biokjemiske reaksjoner, som blant annet metyleringssyklusen (Waly 2004).70 De er også prooksidanter. Som et ledd i dette, kan de utløse autoimmune reaksjoner og kan også endre immunsystemet på andre måter. De kan forårsake direkte skade på følsomme celler i hjernen og i tarmen og forstyrre funksjonen til nevrotransmittere. Studier viser at mange barn med autisme har spesielt store problemer med å omsette og utskille tungmetaller.

Visse konserveringsmidler i vaksiner, spesielt tiomersal (kvikksølv) og aluminium, kan være giftige og skadelige for nervesystemet og immunsystemet hos barn under utvikling.

Toleransen og bivirkninger/reaksjoner på disse stoffene kan variere svært mye fra barn til barn avhengig av genetisk, ernæringsmessig og infeksjonsmessig status. Vi siterer elleve publiserte studier som støtter dette standpunktet:

Thimerosal Neurotoxicity is Associated with Glutathione Depletion: Protection with Glutathione Precursors. James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan S. Neurotoxicology, Jan 2005 Neurotoxicology. 2005 Jan;26(1):1-8.67 Thimerosal is an antiseptic containing 49.5% ethyl mercury that has been used for years as a preservative in many infant vaccines and in flu vaccines. Environmental methyl mercury has been shown to be highly neurotoxic, especially to the developing brain. Because mercury has a high affinity for thiol (sulfhydryl (-SH)) groups, the thiol- containing antioxidant, glutathione (GSH), provides the major intracellular defense against mercury-induced neurotoxicity. Cultured neuroblastoma cells were found to have lower levels of GSH and increased sensitivity to thimerosol toxicity compared to glioblastoma cells that have higher basal levels of intracellular GSH. Thimerosal- induced cytotoxicity was associated with depletion of intracellular GSH in both cell lines. Pretreatment with 100 microM glutathione ethyl ester or N-acetylcysteine (NAC), but not methionine, resulted in a significant increase in intracellular GSH in both cell types. Further, pretreatment of the cells with glutathione ethyl ester or NAC pre- vented cytotoxicity with exposure to 15 microM Thimerosal. Although Thimerosal has been recently removed from most children’s vaccines, it is still present in flu vaccines given to pregnant women, the elderly, and to children in developing countries. The potential protective effect of GSH or NAC against mercury toxicity warrants further research as possible adjunct therapy to individuals still receiving Thimerosal-containing vaccinations.

Uncoupling of ATP-mediated calcium signaling and dysregulated interleukin-6 secretion in dendritic cells by nanomolar thimerosal. Goth SR, Chu RA, Gregg JP, Cherednichenko G, Pessah IN. Environmental Health Perspectives, 2006 July;114(7):1083-91.68 Dendritic cells (DCs) , a rare cell type widely distributed in the soma, are potent antigen-presenting cells that initiate primary immune responses. DCs rely on intracellular redox state and calcium (Ca2+) signals for proper development and function, but the relationship between these two signaling systems is unclear. Thimerosal (THI) is a mercurial used to preserve vaccines and consumer products, and is used experimentally to induce Ca2+ release from microsomal stores. We tested adenosine triphosphate (ATP) -mediated Ca2+ responses of DCs transiently exposed to nanomolar THI. Transcriptional and immunocytochemical analyses show that murine myeloid im- mature DCs (IDCs) and mature DCs (MDCs) express inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) Ca2+ channels, known targets of THI. IDCs express the RyR1 isoform in a punctate distribution that is densest near plasma membranes and within dendritic processes, whereas IP3Rs are more generally distributed. RyR1 positively and negatively regulates purinergic signaling because ryanodine (Ry) blockade a) recruited 80% more ATP responders, b) shortened ATP-mediated Ca2+ transients > 2-fold, and c) produced a delayed and persistent rise (>/= 2-fold) in baseline Ca2+. THI (100 nM, 5 min) recruited more ATP responders, shortened the ATP-mediated Ca2+ transient (>/= 1.4-fold) , and produced a delayed rise (>/= 3-fold) in the Ca2+ baseline, mimicking Ry. THI and Ry, in combination, produced additive effects leading to uncoupling of IP3R and RyR1 signals. THI altered ATP-mediated interleukin-6 secretion, initially enhancing the rate of cytokine secretion but suppressing cytokine secretion overall in DCs.DCs are exquisitely sensitive to THI, with one mechanism involving the uncoupling of positive and negative regulation of Ca2+ signals contributed by RyR1.

– 26 – Aluminum adjuvant linked to Gulf War illness induces motor neuron death in mice. Petrik MS, Wong MC, Tabata RC, Garry RF, Shaw CA. Neuromolecular Med. 2007;9(1):83-100.69 Gulf War illness (GWI) affects a significant percentage of veterans of the 1991 conflict, but its origin remains unknown. Associated with some cases of GWI are increased incidences of amyotrophic lateral sclerosis and other neurological disorders. Whereas many environmental factors have been linked to GWI, the role of the anthrax vaccine has come under increasing scrutiny. Among the vaccine’s potentially toxic components are the adjuvants aluminum hydroxide and squalene. To examine whether these compounds might contribute to neuronal deficits associated with GWI, an animal model for examining the potential neurological impact of aluminum hydroxide, squalene, or aluminum hydroxide combined with squalene was developed. Young, male colony CD-1 mice were injected with the adjuvants at doses equivalent to those given to US military service personnel. All mice were subjected to a battery of motor and cognitive-behavioral tests over a 6-mo period postinjections. Following sacri- fice, central nervous system tissues were examined using immunohistochemistry for evidence of inflammation and cell death. Behavioral testing showed motor deficits in the aluminum treatment group that expressed as a progressive decrease in strength measured by the wire-mesh hang test (final deficit at 24 wk; about 50%). Signifi- cant cognitive deficits in water-maze learning were observed in the combined aluminum and squalene group (4.3 errors per trial) compared with the controls (0.2 errors per trial) after 20 wk. Apoptotic neurons were identified in aluminum-injected animals that showed significantly increased activated caspase-3 labeling in lumbar spinal cord (255%) and primary motor cortex (192%) compared with the controls. Aluminum-treated groups also showed significant motor neuron loss (35%) and increased numbers of astrocytes (350%) in the lumbar spinal cord. The findings suggest a possible role for the aluminum adjuvant in some neurological features associated with GWI and possibly an additional role for the combination of adjuvants.

Activation of Methionine Synthase by Insulin-like Growth Factor-1 and Dopamine: a Target for Neurodevelopmental Toxins and Thimerosal. Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC., Mol Psychiatry. 2004 Apr;9(4):358-70.70 Methylation events play a critical role in the ability of growth factors to promote normal development. Neuro- developmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)- and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH- SY5Y human neuroblastoma cells, via a PI3-kinase- and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylation-sensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+) and Al(3+) were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

Neurotoxic Effects of Postnatal Thimerosal are Mouse Strain Dependent., Hornig M, Chian D, Lipkin WI., Mol Psychiatry. 2004 Sep;9(9):833-45.71 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 varia- bility 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, hyperchro- mic hippocampal neurons with altered glutamate receptors and transporters. Strains resistant to autoimmunity,

– 27 – C57BL/6J and BALB/cJ, were not susceptible. These findings implicate genetic influences and provide a model for investigating thimerosal-related neurotoxicity.

Dette arbeidet fra Columbia University har sett på om den enkeltes gener er viktige når man skal bedømme om kvikksølv eksponering , som de man har brukt i vaksiner kan forstyrre utvikling av hjernen og funksjonen til nevrotransmittere. Dette er den første kjente studie som er gjort spesielt på etyl kvikksølv – administrert på en måte som speiler vaksinering for nyfødte. Dr. Hornig presenterte denne studie i den Amerikanske kongressen i september 2004. Utdrag:

“The premise of our research is that if mercury in vaccines creates risk for neurodevelopmental disorders such as autism, genetic differences are likely to contribute to that risk. Earlier studies, however, did not use the form of mercury present in vaccines, known as thimerosal, and did not consider whether intramuscular, repetitive administration during early postnatal development, when the brain and immune systems are still maturing, might intensify toxicity. Our predictions were confirmed. Using thimerosal dosages and timing that approximated the childhood immunization schedule, our model of postnatal thimerosal neurotoxicity demonstrated that the genes in mice that predict mercury-related immunotoxicity also predicted neurodevelopmental damage. Features re- miniscent of those observed in autism occurred in the mice of the genetically sensitive strain.”

Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.Baskin DS, Ngo H, Didenko VV. Toxicological Science, Aug;74(2):361-8. Epub 2003 May 2872 Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

Organic mercury compounds and autoimmunity., Havarinasab S, Hultman P. Autoimmun Rev. 2005 Jun;4(5):270-5. Epub 2005 Jan 5. Review.73 Based on in vitro studies and short-term in vivo studies, all mercurials were for a long time considered as pro- totypic immunosuppressive substances. Recent studies have confirmed that organic mercurials such as methyl mercury (MeHg) and ethyl mercury (EtHg) are much more potent immunosuppressors than inorganic mercury (Hg). However, Hg interacts with the immune system in the presence of a susceptible genotype to cause immu- nostimulation, antinucleolar antibodies targeting fibrillarin, and systemic immune-complex (IC) deposits, a syndrome called Hg-induced autoimmunity (HgIA). Recent studies in mice with a susceptible genotype has revealed that the immunosuppressive effect of MeHg and EtHg will within 1-3 weeks be superseded by immunostimu- lation causing an HgIA-like syndrome. At equimolar doses of Hg, MeHg has the weakest immunostimulating, autoimmunogen, and IC-inducing effect, while the effect of thimerosal is similar to that of inorganic mercury. The immunosuppression is caused by the organic mercurials per se. Since they undergo rapid transformation to inorganic Hg, studies are being undertaken to delineate the importance of the organic substances per se and the newly formed inorganic Hg for induction of autoimmunity.

– 28 – Retrograde degeneration of neurite membrane structural integrity of nerve growth cones following in vitro exposure to mercury. Leong CC, Syed NI, Lorscheider FL. Neuroreport. 2001 Mar 26;12(4):733-774 Inhalation of mercury vapor (Hg0) inhibits binding of GTP to rat brain tubulin, thereby inhibiting tubulin polymerization into microtubules. A similar molecular lesion has also been observed in 80% of brains from patients with Alzheimer disease (AD) compared to age-matched controls. However the precise site and mode of action of Hg ions remain illusive. Therefore, the present study examined whether Hg ions could affect membrane dyna- mics of neurite growth cone morphology and behavior. Since tubulin is a highly conserved cytoskeletal protein in both vertebrates and invertebrates, we hypothesized that growth cones from animal species could be highly susceptible to Hg ions. To test this possibility, the identified, large Pedal A (PeA) neurons from the central ring ganglia of the snail Lymnoea stagnalis were cultured for 48 h in 2 ml brain conditioned medium (CM). Following neurite outgrowth, metal chloride solution (2 microl) of Hg, Al, Pb, Cd, or Mn (10(-7) M) was pressure applied directly onto individual growth cones. Time-lapse images with inverted microscopy were acquired prior to, during, and after the metal ion exposure. We demonstrate that Hg ions markedly disrupted membrane structure and linear growth rates of imaged neurites in 77% of all nerve growth cones. When growth cones were stained with antibodies specific for both tubulin and actin, it was the tubulin/microtubule structure that disintegrated following Hg exposure. Moreover, some denuded neurites were also observed to form neurofibrillary aggregates. In contrast, growth cone exposure to other metal ions did not effect growth cone morphology, nor was their motility rate compromised. To determine the growth suppressive effects of Hg ions on neuronal sprouting, cells were cultured either in the presence or absence of Hg ions. We found that in the presence of Hg ions, neuronal somata failed to sprout, whereas other metalic ions did not effect growth patterns of cultured PeA cells. We conclude that this visual evidence and previous biochemical data strongly implicate Hg as a potential etiological factor in neurodegeneration.

Reduced levels of mercury in first baby haircuts of autistic children. Holmes AS, Blaxill MF, Haley BE. International Journal of Toxicology 2003 Jul-Aug;22(4):277-8575 Reported rates of autism have increased sharply in the United States and the United Kingdom. One possible factor underlying these increases is increased exposure to mercury through thimerosal-containing vaccines, but vaccine exposures need to be evaluated in the context of cumulative exposures during gestation and early in- fancy. Differential rates of postnatal mercury elimination may explain why similar gestational and infant exposures produce variable neurological effects. First baby haircut samples were obtained from 94 children diagnosed with autism using Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM IV) criteria and 45 age- and gender-matched controls. Information on diet, dental amalgam fillings, vaccine history, Rho D immunoglobulin administration, and autism symptom severity was collected through a maternal survey questionnaire and clinical observation. Hair mercury levels in the autistic group were 0.47 ppm versus 3.63 ppm in controls, a significant difference. The mothers in the autistic group had significantly higher levels of mercury exposure through Rho D immunoglobulin injections and amalgam fillings than control mothers. Within the autistic group, hair mercury levels varied significantly across mildly, moderately, and severely autistic children, with mean group levels of 0.79, 0.46, and 0.21 ppm, respectively. Hair mercury levels among controls were significantly correlated with the number of the mothers’ amalgam fillings and their fish consumption as well as exposure to mercury through childhood vaccines, correlations that were absent in the autistic group. Hair excretion patterns among autistic infants were significantly reduced relative to control. These data cast doubt on the efficacy of traditional hair analysis as a measure of total mercury exposure in a subset of the population. In light of the biological plau- sibility of mercury’s role in neurodevelopmental disorders, the present study provides further insight into one possible mechanism by which early mercury exposures could increase the risk of autism.

Biomarkers supporting mercury toxicity as the major exacerbator of neurological illness, recent evidence via the urinary porphyrin tests. Haley,B.E., and Small,T., Medical Veritas 3 (2006) 1-1476 In the recent past, several biological findings have supported the hypothesis that early exposure of infants to Thimerosal was the major exacerbation factor in the increase in autism-related disorders since the advent of the mandated vaccine program. These initially included the observations of a genetic susceptibility impairing the excretion of mercury and the increased retention of mercury by autistic children. This was followed by data indi-cating that autistics have low levels of the natural compound glutathione that is necessary for the bilary excretion of mercury, possibly explaining the genetic susceptibility. Other observations clearly point out that

– 29 – various biochemical processes are inhibited at exceptionally low nanomolar levels of Thimerosal, including the killing of neurons in culture, the inhibition of the enzyme that makes methyl-B12, the inhibition of phagocytosis (the first step in the innate and acquired immune system), the inhibition of nerve growth factor function at levels not cytotoxic, and the negative effect on brain dendritic cells. It is also now quite clear from primate studies that Thimerosal, or more correctly, the ethylmercury from Thimerosal delivers mercury to the brain, and causes brain inorganic mercury levels higher than equal levels of methylmercury. Most recently, one study showed that 53% of autistic children had aberrant prophyrin profiles similar to mercury toxic individuals. Treatment of these children with a mercury chelator brought these porphyrins back towards normal levels indicating mercury toxicity was the cause, not genetic impairment. Porphyrin profiles are one of the most sensitive methods of measuring toxic mercury exposures. Recently, in a major advance it was shown that about 15% of individuals in one population displayed a marked sensitivity to mercury exposure in their porphyrin physiology, again supporting the concept of a genetically susceptible population that is more sensitive to mercury than the general population. This observation on porphyrin aberrancies brings into consideration other possible effects of mercury toxicity that are secondary to porphyrin depletion. Porphyrins are the precursors to heme synthesis. Heme is the oxygen binding prosthetic group in hemoglobin and depletion of heme would affect oxygen delivery to the mitochondria and decrease energy production. Also, heme is a component of the electron transport system of mitochon-dria and a prosthetic group in the P450 enzymes which are fundamental in the detox of the body from many organic toxicants including pesticides and PCBs. Just recently, a report was released implying that lack of heme was the major reason why ß-amyloid plaques build up in the brains of Alz-heimer’s diseased subjects. It seems that heme attaches to ß-amyloid helping it remain soluble and excretable. Without adequate heme one of the major pathological diagnostic hallmarks of Alzheimer’s disease appears. It is well known that mercury rapidly disrupts the normal polymerization of tubulin into microtubulin in brain tissue and aberrant tubulin polymerization is a consistent factor observed in Alzheimer’s diseased brain. Therefore, it is the multiple inhibitions of mercury that can cause various neurological and systemic problems and many of these are secondary to the primary site of mercury binding.

Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal. Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC. Mol Psychiatry. 2004 Apr;9(4):358-70.77

Methylation events play a critical role in the ability of growth factors to promote normal development. Neuro- developmental toxins, such as ethanol and heavy metals, interrupt growth factor signaling, raising the possibility that they might exert adverse effects on methylation. We found that insulin-like growth factor-1 (IGF-1)- and dopamine-stimulated methionine synthase (MS) activity and folate-dependent methylation of phospholipids in SH- SY5Y human neuroblastoma cells, via a PI3-kinase- and MAP-kinase-dependent mechanism. The stimulation of this pathway increased DNA methylation, while its inhibition increased methylation-sensitive gene expression. Ethanol potently interfered with IGF-1 activation of MS and blocked its effect on DNA methylation, whereas it did not inhibit the effects of dopamine. Metal ions potently affected IGF-1 and dopamine-stimulated MS activity, as well as folate-dependent phospholipid methylation: Cu(2+) promoted enzyme activity and methylation, while Cu(+), Pb(2+), Hg(2+) and Al(3+) were inhibitory. The ethylmercury-containing preservative thimerosal inhibited both IGF-1- and dopamine-stimulated methylation with an IC(50) of 1 nM and eliminated MS activity. Our findings outline a novel growth factor signaling pathway that regulates MS activity and thereby modulates methylation reactions, including DNA methylation. The potent inhibition of this pathway by ethanol, lead, mercury, aluminum and thimerosal suggests that it may be an important target of neurodevelopmental toxins.

5. Biomarkører Legemiddelmyndighetene i USA (FDA) har følgende definisjon på en biomarkør: En karakteristikk som måles objektivt som indikator på en normal biologisk eller patogen prosess eller farmakologisk respons på terapeutisk intervensjon (FDA Office of Biostatistics, 2006).

En biomarkør som kan måles både i blod, urin og avføring vil ofte være en mer pålitelig biomarkør enn om den bare kan måles i en av kildene. Måling i alle de tre kildene er ikke tilgjengelig for alle markører. Dette skyldes

– 30 – delvis markøren, delvis på grunn av kostnaden ved slike målinger og ikke minst at betydningen for behandling av ASD ikke er klarlagt.

5.1. Blodbilde Dette bør undersøkes som et minstekrav for barn under utredning for ASD.

5.1.1. Røde og hvite blodplater med differensiealtelling For å utelukke infeksjoner,allergier, leukemi etc.

5.1.2. Metabolsk panel Hypothyreose er kjent som bidragsyter til nedsatt mental funksjonsevne og hjerneutvikling.

5.2. Lever funksjon Leverfunksjon er vanlig screening ved indremedisinske sykdommer. Ved ASD kan de være nødvendig med tilleggsprøver.78

5.3. Aminosyrer Avvik i aminosyreprofiler på grunn av mistanke om inborn errors of metabolism eller abnorm tarm metabolisme og næringsopptak. Kan måles i blod og urin.

5.4. Tarmfunksjon og metabolisme. Dipeptidylpeptidase-IV (DPP4) er enzymer som er viktige ved nedbrytning av “di-proline” peptider som proteinene i gluten, soya og kasein. Disse enzymene produseres i tarmslimhinnen, men har også funksjoner i hjernen og i lungevevet. I hjernen er det bundet til et dopamin-GMP-signalsystem, og noe av den giftige effekten kvikk- sølv har kan medieres gjennom dysfunksjonelle dopaminsignaler ved binding til dette systemet. Det er kjent at DPP IV-funksjonen påvirkes negativt av kvikksølv og at DPP IV ofte rammes av autoimmun aktivitet/autoimmune angrep (Vojdani et al. 2003).24

Det er stor genetisk variasjon i peptidasene, spesielt når det gjelder pyro-glu-transpeptidaser, endogene peptidaser, som blant annet bryter ned proteiner fra gluten. Nedbryting av gluten ved hjelp av peptidaser fra buk- spyttkjertelen og andre peptidaser lager korte peptider som er like i struktur og funksjon som eksorfiner (Abassi 1992)79, (Watanabe et al 1993).80 Noen av disse peptidene har opioidliknende (eksorfiner) funksjon (Hole 1979, Sun 1999).81, 82

Årsaksforhold til eller en sekundær effekt av dette kan være skader på slimhinnen i tynntarmen, som vi ser ved cøliaki eller autistisk enterokolitt (Ashwood et al. 2004).35 Denne skaden vil redusere eller kanskje hindre pro- duksjonen av fordøyelsesenzymer i tarmveggen. Følgene av dette kan bli nedsatt fordøyelse og/eller nedbrytning, samt økt absorbering av biologisk/farmakologisk aktive peptider og andre immunologisk aktive og antigene ma- kromolekyler (D’Eufemia et al. 1996).83 Nedsatt fordøyelse kan gi nedsatt absorpsjon av essensielle næringsstoffer.

Vojdani 200324 viser til defekt produksjon av enzymer fra pankreas og tarmslimhinnen kombinert med at funksjonen til DPP4 blokkeres av kvikksølv eller autoimmunitet samt endret mikrobiell status og økt intestinal permeabilitet synes å forårsake høye systemiske konsentrasjoner av eksorfin hos pasienter med ASD.

5.4.1. Peptidmarkører i urin Ved ASD kan enkelte biologisk aktive peptider som hovedsakelig er derivert av gluten og/eller kasein forekomme i mye større mengder enn normalt i blod. Forbindelse utskilles i urinen over en viss terskel slik en ser ved fenyl- ketonuri. Dermed reflekterer peptidinnholdet i urin til en viss grad innhold i blod (Watanabe 1993)80.

– 31 – 5.4.2. Permeabilitet i tarmen Unormal absorpsjon av laktulose og mannitol kan brukes for å påvise endret permeabilitet i tarm. Videre vil måling av hyperpeptiduri gi et bilde av permeabiliteten. Dette kan måles ved peptidanalyser i urin, og til en viss grad også i serum83.

5.4.3. Mikrobielle produkter urin Det er en rekke mikrobielle metabolitter, hovedsakelig fra tarmfloraen, som kan påvises i urin men nytten av slike målinger hos barn med ASD er ikke klarlagt. I det følgende omtales noen undersøkelser som i noen tilfeller kan tenkes å være av en viss verdi. IAG Indolyl Acryloyl Glycin er en tryptofan- metabolitt som kan finnes i urin ved ASD.84, 85 Kan påvises ved Urinpeptidanalyse. Betydning ikke klarlagt

H2S produseres av mange tarmbakterier. Det foreligger nå en ny test for å påvise H2S i urin, men nytteverdien er uklar. Falske positive resultater bør utelukkes.

5.4.4. Mikrober og mikrobielle produkter i avføring Undersøkelse av avføringsprøver kan foretas ved mange kommersielle laboratorier for å påvise forekomst av parasitter, sopp og visse bakteriearter. Forekomst av visse parasitter og overvekst av sopp kan være av en visse terapeutisk betydning. Ved diaré vil det kunne være av betydning å dyrke på forekomst av såkalte tarmpatogene bakterier. Dyrkning for påvisning av endret antall normalt forekommende bakterier, f.eks E.coli, enterokokker, klostridier mfl er av liten eller ingen verdi og koster uforholdsmessig mye.

Nye og forbedrede molekylære metoder vil muligens kunne endre på dette.

5.4.5. Mikrobielle metabolitter Korte frie fettsyrer. I avføring kan det foreligge 8 ulike bakterielt produserte korte frie fettsyrer. Den relative mengden av disse fettsyrene er klart endret ved visse tilstander, for eksempel atopisk eksem og cøliaki. Ved cøliaki bevares “cøliaki-mønsteret”, selv etter at barnet er satt på en glutenfri diett (Tjellstrøm et. Al 2007).49 Tilsvarende undersøkelser burde gjøres på barn med ADS.

En bakterielt produsert kort fri fettsyre, propionsyre, kan framkalle ASD-liknende symptomer hos forsøksdyr (MacFabe).48 Mengde propionsyre i avføring er hos barn med ADS er ikke kjent.

Nedbrytning av mucin, tarmslim. Tarmen produseres daglig store mengder slim, mucin som inneholder mye svovel og dette blir normalt nedbrutt av tarmbakterier. Denne bakterielle funksjonen etableres vanligvis i løpet av 2. levehalvår. Forekomst av mucin i avføring kan måles med en enkel elektroforetisk metode, men dette er ikke undersøkt på barn med ADS.

Kolesterol/koprostanol. Noen barn med ASD har lavt serumkolesterol og årsakene til dette er ikke klarlagt. Ko- lesterolet gjennomgår en enterohepatisk sirkulasjon, og i tarmen kan en viss del omdannes til koprostanol. Dette kan ikke absorberes men utskilles i avføringen. Lavt serumkolesterol kan indikere økt bakteriell tarmaktivitet, men dette er ikke undersøkt hos barn med ASD

Tryptisk aktivitet. Trypsin er tarmens viktigst peptidase og mesteparten blir inaktivert av tarmbakterier. Ved visse tarmsykdommer finnes økt mengde tryptisk aktivitet i avføring. Dett kan måles med en enkel metode, men dette er ikke undersøkt hos barn med ASD.

5.5. Tungmetaller Noen av de kliniske symptomene sett ved ASD tilsvarer symptomene ved kvikksølvforgiftning.

Hos nydiagnostiserte barn med ASD finner man mindre kvikksølv i serum, hår og urin enn hos jevnaldrende. Nyere studier som er referert tidligere viser at barn med ASD er dårligere utskillere av tungmetaller enn nevrotypiske barn.

– 32 – Målinger av tungmetaller hos barn med ASD er signifikant høyere enn hos nevrotypiske barn. Det foreligger nå klare og entydige bevis for at barn med ASD har høyere in vivo toksisk belastning enn nevrotypiske jevnaldrende.75, 86

5.5.1. Tungmetallmarkører i blod Tungmetallnivåer (spesielt av kvikksølv, bly og arsenikk) i erytrocytter gjenspeiler pågående eksponering eller rask omsetning fra vev. Spesielt bly kan lagres i bein og utløses ved raske vekstperioder uten fornyet eksponering.

5.5.2. Tungmetallmarkører i urin Påvisning av porfyriner i urin er en metode for å vurdere nivået av visse tungmetaller87

Provokasjonstest med kjente kelatorer. Etter provokasjon samles urin over 6 til 24 timer og man måler mengde tungmetaller. Verdien av slike undersøkelser øker om de blir gjentatt (Adams et al. 2009).86, 88

5.6. Oksidativt stress

5.6.1. Markører for oksidativt stress i blod Redusert glutation (GSH), det motsatte av oksidert glutation (GSSG), kan måles direkte, men dette er ikke kom- mersielt tilgjengelig i vidt omfang.

Nivåene i serum av transferrin og ceruloplasmin er en indikasjon på oksidativt stress. Begge er signifikant redusert hos autistiske barn sammenliknet med utviklingsmessig normale ikke-autistiske søsken.60

5.6.2. Markører for oksidativt stress i urin 8-hydroksyguanin (8-OHG) er en markør for oksidering av RNA i mitrokondrier og i cellenes cytoplasma. Det er en lett tilgjengelig markør for intracellulært oksidativt stress.89 DNA-markøren for oksidativt stress (8-dOHG) er i de fleste tilfeller ikke forhøyet. Isoprostaner viser oksidering av fettsyrer og gjenspeiler stress i cellemembranene.90

5.7. Immun systemet 5.7.1. Autoantistoffer til endovaskulatur Dette er vanskelige undersøkelser som fremdeles er på forskninsgsstadiet

5.7.2. Neopterin og biopterin Neopterin er en antatt metabolitt av biopterin og indikerer graden av cellemediert immunaktivering. Biopterin brukes for å måle kroppens forsøk på å nedregulere immunaktiveringen.91

5.8. Neopterin og Biopterin markører urin Økt nivåer av neopterin er forbundet med aktivering av det cellulære immunsystemet. Redusert biopterin er nødvendig for nevrotransmittersyntesen (Messahel 1998).92

5.8.1. ASO (AST) og anti-DNase B Markører for streptokokkbakterier, Dersom de er forhøyet antyder det en nylig eller pågående streptokokkinfek- sjon med mulig negativ implikasjon. Ved positiv test hos personer forbundet med endringer i tvangsatferd eller tics, er det hevdet at endringer i disse markørene kan forutsi respons til immunterapi og mulig bedring. Dette er også kjent som PANDAS.93

5.8.2. Immunoglobulinklasser: IgG (1–4), IgM, IgA og IgE Markørene kan være av en viss betydning hos barn med nedsatt helse eller mage tarm problematikk. Betydnin- gen ved ADS er noe usikker.

– 33 – 5.8.3. Antistoffer i hjernen Antistoffer til en lang rekke hjerneproteiner er påvist ved ASD, bl.a. antistoffer til myelinprotein (MBP).Dette er vanskelige tester som fremdeles er på forskningsstadiet.94

5.8.4. Vaksinetiter Vaksinetiter kan brukes for å vise immunrespons fra vaksiner og kan gjenspeile respons til IVIG. Det er også nyttig for å vite om det er behov for å gi ytterligere vaksiner. Høye spesifikke IgG-nivåer kan bare tolkes som immunitet til valgte agens.

5.8.5. Kalprotectin Kalprotektin og eosinofilt protein X og i avføringsprøver er markører for allergier og immunsystemet i tarmen (Wagner 2008).95 Betydningen ved ADS er usikker.

5.9. Metylering og transulfatering Biokjemien for metylering, transulfatering og sulfatering er svært kompleks. Prosessene som er avhengig av dette omfatter bl.a. mikrobiell balanse, immunfunksjon, tarmintegritet, avgiftning, utskillelse av tungmetaller og cel- lulær metabolisme.

Metylering involverer og påvirker bl.a. transkripsjon av gener og epigenetiske forhold, nevrotransmitternes funksjon, metabolisme av fettsyrer, allergisk respons, myelinering, celleenergi, cellemembranens og proteiners struktur og funksjon.

Transulfatering omfatter dannelsen av glutation, cystein og sulfat. Det er en viktig del av kroppens avgiftningssystem. Metylering er en viktig del av forløpet for transulfatering. Sulfatering (sulfation); et svovelatom eller gruppe bindes til et annet molekyl. Det er også navnet på et fase II- avgiftningssystem i leveren. Svovel er viktig i syntese av enzymer, proteiner og kroppsvev, dannelsen av gallesyrer, fordøyelse, avgiftning og cellerespirasjon. Forskning har vist at mellom 73 % og 92 % av alle med ASD kan ha forstyrrelser i svovelkjemien.96, 97

5.9.1. Metylerings- og transulfateringskapasitet, markører i blod Transulfatering omfatter syntese av cystein, en aminosye som er lite tilgjengelig i vev , fra homocystein via to B6- avhengige enzymer, cystationin-beta-syntase og cystationin-lyase. Forholdet Homocystein/Cystein (Hcys/Cys) er en pålitelig markør for den metabolske reaksjonsveien. Følgende er ofte lave ved autisme: – Fastende cystein i plasma eller cystine, sulfat og glutation. – Fastende metionin i plasma. Metionin er hovedmetyldonoren via kofaktoren S-adenosylmetionin (SAM eller SAMe) (Waring 1997).101

5.9.2. Metylerings- og transulfateringskapasitet, markører i urin Oksidativt stress eller toksisk effekt av kvikksølv kan svekke metyleringen i hjernen. Homocystein i plasma, som leveres via aktiviteten av to lever enzymer (står for 70 % av kroppens behov), er mest sannsynlig ikke en tilstrek- kelig følsom markør for dette. Markører for svovelmetabolisme i urin kan gi mer innsikt.

5.10. Fettsyrer Membranprofil for fettsyrer

Erythrocyte membran fettsyrer kan gi et bilde av fettsyresammensetning i cerebralt vev og kan måles. Sammen- setningen i cellemembranen av omega 3 fettsyrer – målt før og under behandling kan gi en indikasjon på om behandlingen virker.

– 34 – 6. Aktuelle reaksjonsveier 6.1. Metylering Metylering er grunnleggende og en av de mest essentielle prosesser involvert i dannelsen av DNA.

Metyleringsmønstret etableres i løpet av den tidlige embryoutviklingen der cellene kan differensiere og bidra til å stabilisere kromosomstrukturen, noe som fører til færre mutasjoner. I tillegg til DNA (og RNA, som overfører genetiske opplysninger) er vitaminer, hormoner, nevrotransmittere som dopamin og serotonin, enzymer og antistoffer alle avhengige av metylering.

Vi siterer én empirisk studie og en review som støtter standpunktet om at metylering kan være nedsatt hos barn med ASD.

Low plasma methionine, cysteine and glutathione levels are associated with increased frequency of common polymorphisms affecting methylation and glutathione pathways in children with autism., James SJ et al. Experimental Biology 2005;19:A51-A5298 The 10-fold increase in the prevalence of autism in the last 15 years is a major public health concern. Although abnormal thiol metabolism has been associated with other neurologic diseases, these pathways and related polymorphisms have not been evaluated in autistic children. Plasma levels of metabolites in methionine transmethylation and transsulfuration pathways were measured in 90 autistic and 45 control children using HPLC with elec- trochemical detection. Polymorphic variants in transcobalamin II (TCII), methylene- tetrahydrofolate reductase (MTHFR), methionine synthase reductase (MTRR), catecholamine-O-methyltransferase (COMT), and glutathione-S-transferase (GST) M1/T1 were evaluated in 233 autistic children and 183 controls. The results indicated that mean levels of methionine, cysteine, total glutathione, and the ratio of oxidized to reduced glutathione were significantly decreased among the autistic children. The frequency of MTHFR 677CT/1298AG heterozygosity, TCII 776GG, COMT 1947GG, and the GST M1/T1 double null genotype was increased in the autistic children relative to controls. We hypothesize that an increased vulnerability to oxidative stress (environmental and/or intracellular) may contribute to the development and clinical manifestations of autism.

How environmental and genetic factors combine to cause autism: A redox/methylation hypothesis Deth R, Muratore C, Benzecry J, Power-Charnitsky VA, Waly M. Neurotoxicology. 2008 Jan;29(1):190-201. Epub 2007 Oct 1399. Recently higher rates of autism diagnosis suggest involvement of environmental factors in causing this developmental disorder, in concert with genetic risk factors. Autistic children exhibit evidence of oxidative stress and impaired methylation, which may reflect effects of toxic exposure on sulfur metabolism. We review the metabolic relationship between oxidative stress and methylation, with particular emphasis on adaptive responses that limit activity of cobalamin and folate-dependent methionine synthase. Methionine synthase activity is required for dopamine-stimulated phospholipid methylation, a unique membrane-delimited signaling process mediated by the D4 dopamine receptor that promotes neuronal synchronization and attention, and synchrony is impaired in autism. Genetic polymorphisms adversely affecting sulfur metabolism, methylation, detoxification, dopamine signaling and the formation of neuronal networks occur more frequently in autistic subjects. On the basis of these observations, a “redox/methylation hypothesis of autism” is described, in which oxidative stress, initiated by environment factors in genetically vulnerable individuals, leads to impaired methylation and neurological deficits secondary to reductions in the capacity for synchronizing neural networks.

6.2. Transulfatering Transulfatering omfatter trinnene i det biomedisinske reaksjonsmønsteret etter homocystein, så som cystein, taurin, sulfat og glutation. Det er utført omfattende forsking på transulfatering ved autisme og Down syndrom. Barn med autisme har “signifikant lavere plasmakonsentrasjoner av metionin, SAM, homocystein, cystationin, cystein og total glutation ved baseline og signifikant høyere konsentrasjoner av SAH, adenosin og oksidert glutation” (James 2004) 100 Disse resultatene viser at barn med autisme ikke bare har dårlig metylering men også dårlig transulfatering.

– 35 – Når metylering og transulfatering virker som de skal, går cystein bl.a videre til å bli glutation. Metionin er forlø- peren til cystein og den hastighetsbegrensende aminosyren ved glutationsyntese. Fordi metionin styrer hvor mye cystein og glutation som kan dannes, er lave metioninnivåer forbundet med lavt cystein og lavt glutation. Sulfatering er evnen til å utnytte sulfat i en lang rekke biokjemiske prosesser. Hos individer med autisme skilles store mengder sulfat ut i urin101 uavhengig av unormalt lave sulfatnivåer i plasma. Dette funnet er på linje med tilstanden som oppstår i Nas1-knockout-musen utviklet av Daniel Markovich.102 Hans arbeid viser blant annet hvorfor sulfateringsproblematikken ved autisme kan endre måten oksalat håndteres av kroppen på, ved at oksalat transporteres til steder det normalt ikke ville forekomme.

Vi siterer to teoretiske publikasjoner om transulfateringsreaksjoner og ett om funn hos barn med autisme og deres foreldre.

Abnormal Transmethylation/transsulfuration Metabolism and DNA Hypomethylation Among Parents of Children with Autism., Jill James S, Melnyk S, Jernigan S, Hubanks A, Rose S, Gaylor DW., J Autism Dev Disord. 2008 May 30.103 An integrated metabolic profile reflects the combined influence of genetic, epigenetic, and environmental factors that affect the candidate pathway of interest. Recent evidence suggests that some autistic children may have reduced detoxification capacity and may be under chronic oxidative stress. Based on reports of abnormal methionine and glutathione metabolism in autistic children, it was of interest to examine the same metabolic profile in the parents. The results indicated that parents share similar metabolic deficits in methylation capacity and glutathione-dependent antioxidant/detoxification capacity observed in many autistic children. Studies are underway to determine whether the abnormal profile in parents reflects linked genetic polymorphisms in these pathways or whether it simply reflects the chronic stress of coping with an autistic child.

The metabolism of homocysteine: pathways and regulation. Finkelstein JD. Eur J Pediatr. 1998 Apr;157 Suppl 2:S40-4. Review.104 Two pathways, the methionine cycle and transsulfuration, account for virtually all methionine metabolism in mammals. Every tissue possesses the methionine cycle. Therefore, each can synthesize AdoMet, employ it for transmethylation, hydrolyze AdoHcy, and remethylate homocysteine. Transsulfuration, which occurs only in liver, kidney, small intestine and pancreas, is the means for catabolizing homocysteine. Liver has a unique iso- enzyme of MAT that allows the utilization of excess methionine for the continued synthesis of AdoMet. Meta- bolic regulation is based on the distribution of available homocysteine between remethylation and conversion to cystathionine. The tissue content of the enzymes and their inherent kinetic properties provide the basis for the regulatory mechanism. The effector properties of the metabolites AdoMet, AdoHcy and methylTHF are of particular relevance.

Markovich D, Aronson PS. Specificity and regulation of renal sulfate transporters. Annu Rev Physiol. 2007; 69:361-75.102 Sulfate is essential for normal cellular function. The kidney plays a major role in sulfate homeostasis. Sulfate is freely filtered and then undergoes net reabsorption in the proximal tubule. The apical membrane Na(+)/sulfate cotransporter NaS1 (SLC13A1) has a major role in mediating proximal tubule sulfate reabsorption, as demonstrated by the findings of hyposulfatemia and hypersulfaturia in Nas1-null mice. The anion exchanger SAT1 (SLC26A1), the founding member of the SLC26 sulfate transporter family, mediates sulfate exit across the basolateral membrane to complete the process of transtubular sulfate reabsorption. Another member of this family, CFEX (SLC26A6), is present at the apical membrane of proximal tubular cells. It also can transport sulfate by anion exchange, which probably mediates backflux of sulfate into the lumen. Knockout mouse studies have demonstrated a major role of CFEX as an apical membrane Cl(‑)/oxalate exchanger that contributes to NaCl reabsorption in the proximal tubule. Several additional SLC26 family members mediate sulfate transport and show some level of renal expression (e.g., SLC26A2, SLC26A7, SLC26A11). Their roles in mediating renal tubular sulfate transport are presently unknown. This paper reviews current data available on the function and regulation of three sulfate transporters (NaS1, SAT1, and CFEX) and their physiological roles in the kidney.

– 36 – Oversikt over metylerings- og transulfateringsrelaterte reaksjonsveier

Metylerings- og transulfateringsrelaterte reaksjonsveier. En representasjon av sykluser og reaksjonsveier som om- handler metylering og transulfatering og deres forbindelser til folatsyklusen, glykolysen og ureasyklusen. Genene markert med blått er identifisert i litteraturbaserte søk i metodeavsnittet i artikkel dette er hentet fra. De hele linjene betegner direkte og de stiplede linjene indirekte (flertrinns) interaksjon/omdannelse. Sharma et al. Lipids in Health and Disease, 2006 5:1 doi:10.1186/1476-511X-5-1.105

Forkortelser: THF: tetrahydrofolat; MS: metioninsyntase; BHMT: betainhomocysteinmetyltransferase; MAT: metioninadenosyltransferase; SAM: S-adenosylmetionin; SAH: S-adenosylhomocystein; SAHH: SAH-hydrolase; ADA: adenosindeaminase; AK: adenosinkinase; CBS: cystationinbetasyntase.

7. Biomedisinske behandlinger 7.1. Vitaminer og mineraler generelt Nyere forskning antyder at ubalanser eller mangler av vitaminer og mineraler er vanlig hos barn med ASD. Det er antydet at disse manglene kan føre til mange av de biokjemiske ubalansene og symptomene som ses hos barn med ASD (Zinn et al. 2009).106

Forskning viser at vitamin- og mineraltilskudd som bl.a. B6, magnesium og sink kan bidra til å normalisere en del biokjemiske ubalanser og forbedre funksjonen både hos mange barn med normal utvikling og barn med ASD.

– 37 – Vi siterer syv studier som støtter dette standpunktet. Når det gjelder ASD spesielt siterer vi en studie som støtter standpunktet om ubalanser i mineraler (Zinn et al. 2009)106 og en studie som støtter kosttilskudd av B6 og magnesium (Martineau et al. 1985).107

The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders. Faber S, Zinn GM, Kern Ii JC, Skip Kingston HM.Biomarkers. 2009 Mar 11:1-10. [Epub ahead of print]106 The frequency of zinc deficiency, copper toxicity and low zinc/copper in children with autism spectrum disorders (ASDs) may indicate decrement in metallothionein system functioning. A retrospective review of plasma zinc, serum copper and zinc/copper was performed on data from 230 children with autistic disorder, pervasive developmental disorder-NOS and Asperger’s syndrome. The entire cohort’s mean zinc level was 77.2 mug dl(- 1), mean copper level was 131.5 mug dl(-1), and mean Zn/Cu was 0.608, which was below the 0.7 cut-off of the lowest 2.5% of healthy children. The plasma zinc/serum copper ratio may be a biomarker of heavy metal, particularly mercury, toxicity in children with ASDs.

Vitamin B6, magnesium, and combined B6-Mg: Therapeutic effects in childhood autism. Martineau J, Barthelemy C, Garreau B, Lelord G. Biological Psychiatry 1985;20:467-8107 This article reports the behavioral, biochemical, and electrophysiological effects of four therapeutic crossed- sequential double-blind trials with 60 autistic children: Trial A--vitamin B6 plus magnesium/magnesium; Trial B--vitamin B6 plus magnesium; Trial C--magnesium; and Trial D--vitamin B6. Therapeutic effects were controlled using behavior rating scales, urinary excretion of homovanillic acid (HVA), and evoked potential (EP) recor- dings. The behavioral improvement observed with the combination vitamin B6-magnesium was associated with significant modifications of both biochemical and electrophysiological parameters: the urinary HVA excretion decreased, and EP amplitude and morphology seemed to be normalized. These changes were not observed when either vitamin B6 or magnesium was administered alone.

Rational dosages of nutrients have a prolonged effect on learning disabilities. Carlton R et al.Alternative Therapies 2000; 6:8591.108 CONTEXT: Reports that administration of nutrients has increased the academic performance of learning-disabled children exist in the literature. OBJECTIVE: To document the effects of nutrients on learning-disabled children in a controlled study. DESIGN: A randomized, double-blind, placebo-controlled crossover trial, which followed 1 year of open-label nutrients. Children who improved in the open-label trial were eligible to enter the controlled phase of the study. SETTING: Subjects were enrolled from the general community through adverti- sements. PATIENTS OR OTHER PARTICIPANTS: Twenty children met the criteria for being learning disabled. INTERVENTION: Each child was tried out on some (but not necessarily all) of the B vitamins and minerals used in this study. These were administered semi-blinded for the first year; double-blinded in crossover rotations during the second year; and open-label in the ensuing years. MAIN OUTCOME MEASURES: At various time points, school-certified psychologists administered psychoeducational tests. School report cards were evaluated at baseline and for all subsequent periods. RESULTS: Twenty learning-disabled children entered the study, but 1 dropped out because of nausea. The remaining 19 children showed significant academic and behavioral improvements within a few weeks or months of open-label treatment with nutrient supplements. Some children gained 3 to 5 years in reading comprehension within the first year of treatment; and all children in special education classes became mainstreamed, and their grades rose significantly. Twelve of the children completed the 1-year double-blind phase, after which approximately half of the children chose to remain on the nutrients for at least 2 additional years. For those who discontinued, it took at least 1 year to begin to see the first indications of decline in academic performance, and another year for their grades to drop significantly. In contrast, for children who remained on nutrients, the gains continued the upward trend; at the end of year 4, the difference in scores between the 2 groups had reached statistical significance (P < .01). CONCLUSION: The overall results of this study tentatively support the concept that learning disabilities may in some cases be a nutrient-responsive disorder.

– 38 – The effect of vitamin-mineral supplementation on the intelligence of American schoolchildren: a randomized, double-blind placebo-controlled trial. Schoenthaler SJ, Bier ID, Young K, Nichols D, Jansenns S., Journal of Alternative and Complementary Medicine 2000;6:1929.109 CONTEXT: Many medical, nutrition, and education professionals have long suspected that poor diet impairs the academic performance of Western schoolchildren; academic performance often improves after improved diet. However, others have suggested that such academic gains may be due to psychologic effects rather than nutrition. To resolve this issue, two independent research teams conducted randomized trials in which children were given placebos or low-dose vitamin-mineral tablets designed to raise nutrient intake to the equivalent of a well- balanced diet. Both teams reported significantly greater gains in nonverbal intelligence among the supplemented groups. The findings were important because of the apparent inadequacy of diet they revealed and the magnitude of the potential for increased intelligence. However, none of the ten subsequent replications, or the two origi- nal trials, were without limitations leaving this issue in controversy. Objectives: To determine if schoolchildren who consume low-dose vitamin-mineral tablets will have a significantly larger increase in nonverbal intelligence than children who consume placebos in a study that overcomes the primary criticisms directed at the previous 12 controlled trials. Design: A double-blind, placebo-controlled trial using stratified randomization within each teacher’s class based on preintervention nonverbal intelligence. Settings and subjects: Two “working class,” primarily Hispanic, elementary schools in Phoenix, Arizona, participated in the study. Slightly more than half the teachers in each school distributed the tablets daily to 245 schoolchildren aged 6 to 12 years. Intervention: Daily vitamin-mineral supplementation at 50% of the U.S. daily recommended allowance (RDA) for 3 months versus placebo. Outcome measures: Post-test nonverbal IQ, as measured by the Wechsler Intelligence Scale for Children-Revised (WISC-R), while controlling for pretest nonverbal IQ as a covariate. Four main results: First, a significant difference of 2.5 IQ points (95% CI: 1.85-3.15) was found between 125 children given active tablets and 120 children given placebo tablets (p = 0.038). Second, this finding is consistent with the mean 3.2 IQ point net gain found in the 12 similar but less rigorous studies. Third, a significantly higher proportion of children in the active group gained 15 or more IQ points when compared to the placebo group (p < 0.01). Fourth, although 81 matched pairs produced no difference at all in nonverbal IQ gain, the modest 2.5 IQ point net gain for the entire sample can be explained by the remaining 24 children who took active tablets, and had a 16 point higher net gain in IQ than the remaining 19 placebo controls. CONCLUSIONS: This study confirms that vitamin-mineral supplementation modestly raised the nonverbal intelligence of some groups of Western schoolchildren by 2 to 3 points but not that of most Western schoolchildren, presumably because the majority were already adequately nourished. This study also confirms that vitamin-mineral supplementation markedly raises the non-verbal intelligence of a minority of Western schoolchildren, presumably because they were too poorly nourished before supplementation for optimal brain function. Because nonverbal intelligence is closely associated with academic performance, it follows that schools with children who consume substandard diets should find it difficult to produce academic performance equal to those schools with children who consume diets that come closer to providing the nutrients suggested in the U.S. RDA. The parents of schoolchildren whose academic performance is substandard would be well advised to seek a nutritionally oriented physician for assessment of their children’s nutritional status as a possible etiology.

Magnesium,Vitamin B6 intake reduces central nervous system hyperexcitability in children. Mousain- BoBosc M et al. Jam Coll Nutr 2004;23:545S-548S110 OBJECTIVE: Ionic magnesium (Mg(2+)) depletion has long been known to cause hyperexcitability with convul- sive seizures in rodents, effects that have been reversed by treatment with magnesium (Mg). Metabolic disorders and genetic alterations are suspected in this pathology, in which Mg(2+) transport and intracellular distribution may be reduced without change in serum Mg(2+) concentrations. We evaluated the effects of Mg(2+)/vitamin B6 regimen on the behavior of 52 hyperexcitable children (under 15 years of age) and their families. METHODS: To assess intracellular Mg(2+), we measured intra-erthrocyte Mg(2+) levels (ERC-Mg). Our reference values for normal subjects were 2.46 to 2.72 mmol/L. In 30 of the 52 hyperactive children, there were low ERC-Mg values: 2.041 +/- 0.279 mmol/L). Combined Mg(2+)/vitamin B6 intake (100 mg/day) for 3 to 24 weeks restored normal ERC-Mg values (2.329 +/- 0.386 mmol/L). RESULTS: In all patients, symptoms of hyperexcitability (physical aggressivity, instability, scholar attention, hypertony, spasm, myoclony) were reduced after 1 to 6 months treatment. Other family members shared similar symptoms, had low ERC-Mg values, and also responded clinically to increased Mg(2+)/vitamin B6 intakes. Two typical families are described. CONCLUSION: This open study

– 39 – indicates that hyperexcitable children have low ERC-Mg with normal serum Mg(2+) values, and that Mg(2+)/ vitamin B6 supplementation can restore normal ERC-Mg levels and improve their abnormal behavior.

Vitamin B6 versus mianserin and placebo in acute neuroleptic-induced akathisia: a randomized, double-blind, controlled study. Miodownik C, Lerner V, Statsenko N, Dwolatzky T, Nemets B, Berzak E, Bergman J. Clinical Neuropharmacology,Vol.29, No.2,March/April 2006, 68-72111 Treatment strategies against acute neuroleptic-induced akathisia (NIA) include anticholinergic (antimuscari- nic) agents, dopamine agonists, GABAergic agents, beta-blockers, benzodiazepines, and serotonin antagonists. However, many patients who have acute akathisia fail to respond. In previous studies, mianserin and vitamin B6 were found to be effective in the treatment of acute akathisia. The purpose of this study was to compare the efficacy of B(6), mianserin and placebo in the treatment of acute NIA. Sixty schizophrenia and schizoaffective inpati- ents who have NIA were randomly divided to receive vitamin B(6) 1,200 mg/d, mianserin 15 mg/d, or placebo for 5 days, in a double-blind design. The Barnes Akathisia Rating Scale, Brief Psychiatric Rating Scale, and Clinical Global Impression were used to assess the severity of NIA and psychotic symptoms. The assessment was made at baseline and daily for the duration of the study. Compared with the placebo group, the vitamin B(6)-treated and mianserin-treated patients showed a significant improvement in the subjective (P < 0.0001), subjective distress (P < 0.0001), and global (P < 0.0001) subscales. The objective subscale did not show significant positive results (P = 0.056), but there was a trend toward symptom amelioration in both groups. A reduction of at least 2 points on the Barnes Akathisia Rating Scale global subscale was noted in the vitamin B(6) group (13/23, 56%) as well as in the mianserin groups (13/20, 65%), and in only one patient in the placebo group (1/17, 6%; P < 0.0005). Our results indicate that high doses of B(6) and a low dose of mianserin may be a useful addition to current treatments of NIA. The efficacy of vitamin B(6) and mianserin suggests that the pathophysiology of acute NIA is heterogeneous with the various subtypes of acute NIA responding differently to the various pharmacological approaches.

Does zinc moderate essential fatty acid and amphetamine treatment of attention-deficit/hyperactivity disorder?Arnold LE, Pinkham SM, Votolato N. J Child Adolesc Psykopharmacol 2000;10:111-7112 Zinc is an important co-factor for metabolism relevant to neurotransmitters, fatty acids, prostaglandins, and melatonin, and indirectly affects dopamine metabolism, believed intimately involved in attention-deficit/hyperactivity disorder (ADHD). To explore the relationship of zinc nutrition to essential fatty acid supplement and stimulant effects in treatment of ADHD, we re-analyzed data from an 18-subject double-blind, placebo-controlled crossover treatment comparison of d-amphetamine and Efamol (evening primrose oil, rich in gamma-linolenic acid). Subjects were categorized as zinc-adequate (n = 5), borderline zinc (n = 5), and zinc-deficient (n = 8) by hair, red cell, and urine zinc levels; for each category, placebo-active difference means were calculated on teachers’ ratings. Placebo-controlled d-amphetamine response appeared linear with zinc nutrition, but the relationship of Efamol response to zinc appeared U-shaped; Efamol benefit was evident only with borderline zinc. Placebo-controlled effect size (Cohen’s d) for both treatments ranged up to 1.5 for borderline zinc and dropped to 0.3-0.7 with mild zinc deficiency. If upheld by prospective research, this post-hoc exploration suggests that zinc nutrition may be important for treatment of ADHD even by pharmacotherapy, and if Efamol benefits ADHD, it likely does so by improving or compensating for borderline zinc nutrition.

7.2. Glutenfri og kaseinfri diett uten hvetestivelse Opioide peptider avledet av proteiner i mat (eksorfiner) er funnet i urinen til autistiske pasienter. Reichelt et al. 2003114 argumenterte med at eksorfiner og faktorer som stimulerer serotoninopptaket kan forklare mange av symptomene ved autistiske forstyrrelser. Reichelt et al. argumenterte videre med at de individuelle symptomene burde kunne forklares ved de egenskape- ne og atferdseffektene av peptidene som er funnet. De foreslår at eksorfiner og modulatorer av serotoninopptak er nøkkelmediatorer ved utviklingen av autisme.

En gluten- og/eller kaseinfri diett ble allerede introdusert for å redusere autistisk atferd tidlig på 80-tallet. Det er helt opplagt at nesten alle foreldre har sett at kostholdet påvirker symptomene. Dette åpner for en mekanistisk biokjemisk evaluering.

– 40 – Nyere forskning har påvist en forbindelse mellom peptider og cystein opptaks transportøren EAAT3. Det kan se ut som om peptidene hindrer EAAT3. EAAT3 er den eneste kilden til cysteinopptak for nevronene og er også til stede i såkalte “crypt cells” i tarmslimhinnen. Dette bidrar til å forklare fordelene med GFCF-diett og kan være den forbindelsen mellom tarmen og hjernen som har vært etterspurt som støtte for dietten. Artikkelen om dette funnet er under skriving og har fått arbeidstittel: “Casein and gluten-derived opiate peptides inhibit cysteine uptake in human neuronal glial cells”. Forfattere: M.Trivedi, N.Hodgson og R. Deth.

Vi siterer to publiserte studier som støtter standpunktet om at glutenfri og kaseinfri diett kan forbedre funksjonene hos barn med ASD.

Reports on dietary intervention in autistic disorders. Knivsber AM, Reichelt KL, Nødland M. Reichelt KL, Knivsberg AM,Nutr Neurosci. 2001;4(1):25-37.113 Gluten and/or casein free diet has been implemented to reduce autistic behaviour, in addition to special education, since early in the eighties. Over the last twelve years various studies on this dietary intervention have been published in addition to anecdotal, parental reports. The scientific studies include both groups of participants as well as single cases, and beneficial results are reported in all, but one study. While some studies are based on urinary peptide abnormalities, others are not. The reported results are, however, more or less identical; reduction of autistic behaviour, increased social and communicative skills, and reappearance of autistic traits after the diet has been broken.

A randomised, controlled study of dietary intervention in autistic syndromes. Knivsberg AM, Reichelt KL, Høien T, Nødland M. Nutr Neurosci 2002;5:251-61.114 Impaired social interaction, communication and imaginative skills characterize autistic syndromes. In these syndromes urinary peptide abnormalities, derived from gluten, gliadin, and casein, are reported. They reflect processes with opioid effect. The aim of this single blind study was to evaluate effect of gluten and casein-free diet for children with autistic syndromes and urinary peptide abnormalities. A randomly selected diet and control group with 10 children in each group participated. Observations and tests were done before and after a period of 1 year. The development for the group of children on diet was significantly better than for the controls.

7.3. Essensielle fettsyrer Det er flere faktorer som bidrar til en mangel på essensielle fettsyrer hos mange personer. Det finnes to familier av essensielle flerumettede fettsyrer (PUFA), n-6 og n-3. Sett i lys av den viktige rollen fettsyrene spiller for nerve- membranenes funksjon og cellenes homeostase, kan mangler i fettsyremetabolismen ha viktige biologiske følger ved autisme (Bu, 2006).115

Det er påvist at barn med autisme som gruppe har større mangler av essensielle fettsyrer enn individer med psykisk utviklingshemming (Vancassel 2001).119 Nylig sammenliknet Wiest 2009117 friske personer med personer med autisme og viste at dokosaheksaensyre (DHA, 22:6n-3) var signifikant nedsatt i fosfatidyletanolamin i autismegruppen. Dette var den andre studien som målte dimetylacetaler hos barn med autisme, og dette antyder at funksjonen til peroksisomer og enzymene i peroksisomene i fettsyremetabolismen kan være påvirket. Evan 2008116 beskriver funn av endring i lipidmetabolisme hos autistiske individer.

PUFA kan spille en viktig rolle i reduksjonen av autistiske trekk. Vi siterer tre studier som støtter dette.

Plasma fatty acid profiles in autism: a case-control study. Wiest MM, German JB, Harvey DJ, Watkins SM, Hertz-Picciotto I. Prostaglandins Leukot Essent Fatty Acids. 2009 Apr;80(4):221-7. 117 Increasing evidence is mounting in support of fatty acid metabolism playing a role in neurodevelopmental disorders such as autism. In order to definitely determine whether fatty acid concentrations were associated with autism, we quantitatively measured 30 fatty acids from seven lipid classes in plasma from a large subset of subjects enrolled in the Childhood Autism Risk from Genetics and the Environment (CHARGE) study. The CHAR- GE study is a large, population-based case-control study on children aged 2-5 born in California. Our subset

– 41 – consisted of 153 children with autism and 97 developmentally normal controls. Results showed that docosahexaenoic acid (DHA, 22:6n-3) was significantly decreased in phosphatidylethanolamine. Dimethyl acetals were significantly decreased in phosphatidylethanolamine and phosphatidylcholine as well. These results are consistent with the only other study to measure dimethyl acetals in children with autism, and suggest that the function of peroxisomes and the enzymes of the peroxisome involved with fatty acid metabolism may be affected in autism.

Role of polyunsaturated fatty acids in the management of Egyptian children with autism. Meguid NA, Atta HM, Gouda AS, Khalil RO. Clin Biochem. 2008 Sep;41(13):1044-8.118 OBJECTIVE: Estimation of free polyunsaturated fatty acids (PUFAs) in blood and evaluation of behavior of autistic children before and after taking fish oil (Efalex) were performed. DESIGN AND METHODS: 30 autistic children (18 males and 12 females) aged 3-11 years and 30 healthy children as control group were included in this study. Tandem mass spectrometry and CARS were used to estimate the free PUFAs from dried blood spot and to evaluate the autistic behavior respectively. RESULTS: Before taking Efalex, linolenic acid showed a significant reduction (71%), followed by docosahexaenoic acid (65%) and arachidonic acid (45%), while linoleic acid was the least affected PUFA (32%). After taking Efalex, 66% of autistic children showed clinical and biochemical improvement, linolenic acid and docosahexaenoic acid showed the highest levels after Efalex supplementation. CONCLUSION: PUFA supplementation may play an important role in ameliorating the autistic behavior.

Plasma fatty acid levels in autistic children.Vancassel S, Durand G, Barthélémy C, Lejeune B, Martineau J, Guilloteau D, Andrès C, Chalon S. Prostaglandins Leukot Essent Fatty Acids 2001;65: 1-7.119 Phospholipid fatty acids are major structural components of neuronal cell membranes, which modulate membrane fluidity and hence function. Evidence from clinical and biochemical sources have indicated changes in the metabolism of fatty acids in several psychiatric disorders. We examined the phospholipid fatty acids in the plasma of a population of autistic subjects compared to mentally retarded controls. Our results showed a marked reduction in the levels of 22: 6n-3 (23%) in the autistic subjects, resulting in significantly lower levels of total (n-3) polyunsaturated fatty acids (PUFA) (20%), without significant reduction in the (n-6) PUFA series, and con- sequently a significant increase in the (n-6)/(n-3) ratio (25%). These variations are discussed in terms of potential differences in PUFA dietary intake, metabolism, or incorporation into cellular membranes between the two groups of subjects. These results open up interesting perspectives for the investigation of new biological indices in autism. Moreover, this might have new therapeutic implications in terms of child nutrition.

7.4. Metyleringssyklusene Autistiske barn har ofte påviselige tegn på oksidativt stress og nedsatt metylering, noe som kan gjenspeile effekten av toksisk påvirkning på svovelmetabolismen (Deeth 2008)99. James 200867 henviser til unormal metionin- og glutation metabolisme hos autistiske barn. Det å støtte disse syklusene kan være svært viktig for det enkelte barn. Cystein kan gis som direkte tilskudd med NAC, enten intravenøst eller som tabletter. Metyl-B12 kan gis som injeksjoner og/eller nesespray – muligens også transdermalt, men her er ikke opptaket bestemt. Metionin kan gis til måltider, men doseringen bør ikke være for høy, ellers kan kroppens lager av andre aminosyrer tømmes kuns- tig. Folat, leukovorin eller metylfolat oralt kan hjelpe. Tilskudd av B6, andre B-vitaminer og magnesium samt det å avhjelpe oksidativt stress bidrar også til at disse reaksjonsveiene fungerer bedre. TMG kan være en alternativ metode for å støtte metylering. Det foreligger foreløpig lite forskning om nasal administrering av oxytocin. James studie viser positive resultater av slik behandling.

7.5. Oksidativt stress Redusering av oksidativt stress kan være en nøkkelfaktor for å forbedre utviklingen og klinisk manifestasjon hos barn med autisme. Det kan være viktig å normalisere cystein nivåene ved å støtte reaksjonsveiene som omhand- ler folat, metylering og transulfatering. Mitokondriene kan støttes. Her brukes acetyl-L-karnitin, ubikinon eller liposomal co-Q10 samt D-ribose. Melatonin bidrar også til å redusere oksidativt stress. Oksalater i urin ved bruk av høydose-vitamin C kan også gi nyttig informasjon. Flere typer bioflavonoider er svært gode antioksidanter.

– 42 – Med unntak av James 200867 finnes det ingen publiserte peer review-studier som går direkte på oksidativt stress og som har evaluert klinisk effekt hos barn med autisme.

7.6. Fordøyelse ASD involverer også mage-tarm, primært samt sekundært. Mat intoleranse og lekk tarm er vanlig. Det komplekse samspillet mellom IM og barnet, kan ha blitt påvirket av mange faktorer som virusinfeksjoner, bruk av antibiotika osv. Dette kan i sin tur ha ført til blant annet produksjon av mikrobielle toksiner og giftige dipeptider, samt endringer i grunnleggende intestinale fysiologiske funksjoner påvirket av IM. Vi nevner her endringer i tarmens svovel metabolisme og produksjon av korte frie fettsyrer.

Standard mage-tarm behandling omfatter diett intervensjoner (gluten og kasein fri diett ). Dette er ofte til stor hjelp for undergrupper av barn med ASD. Noen forskere har registrert forbedringer ved bruk av probiotika og fordøyelsesenzymer120. “Probiotika” omfatter produkter som har et stort spekter av forskjellige effekter og bør derfor bare brukes på individuelt nivå.

7.7. Tungmetaller Målet er å redusere tungmetallnivået i kroppen som er påvist. Dette kan delvis måles ved at unormale porfyrin- verdier normaliseres ved hjelp av kelering. Videre kan direkte måling av tungmetaller i urin ved kelatering også benyttes. Dersom tungmetallbelastning er påvist, er det rimelig å utføre standard keleringsregimer. Adams et al. 200987 har vist positiv effekt på glutationnivåer ved bruk av DMSA.

7.8. Hyperbar oksygen behandling (HBOT) Hyperbar oksygenbehandling (HBOT) er både blitt kalt en effektiv behandlingsmetode og en metode som søker etter sykdommer den kan behandle. Det er sagt at moderne bruk av hyperbar oksygen i klinisk medisin startet på 1960-tallet med arbeidet til Borema og Churchill-Davidson. Behandlingen hadde stor suksess ved forskjellige medisinske tilstander og forskere var ivrige etter å behandle en rekke andre tilstander i hyperbare kammere. Studier av den fysiologiske påvirkningen av oksygen under trykk har gitt oss mye mer informasjon de siste tretti årene når det gjelder virkningsmekanismen til HBOT. Forskning på effekten av HBOTpå cellenivå har gitt oss tilstrekkelige data til at mange leger anser HBOT som en verdifull behandling ved bestemte tilstander. Innen akuttmedisinen er det tre hovedindikasjoner for HBOT: kullosforgiftning, cyanidforgiftning eller hydrogensulfi- dforgiftning. Bruk av HBOT som tilleggsbehandling ved autisme har oppstått de siste ti år.

Ut av flere siterer vi den siste studien som støtter standpunktet om at HBOT kan ha positiv påvirkning hos grup- per av barn med autisme.

Hyperbaric treatment for children with autism: a multicenter, randomized, double-blind, controlled trial. Rossignol DA, Rossignol LW, Smith S, Schneider C, Logerquist S, Usman A, Neubrander J, Madren EM, Hintz G, Grushkin B, Mumper EA.BMC Pediatr. 2009 Mar 13;9(1):21.121 ABSTRACT: BACKGROUND: Several uncontrolled studies of hyperbaric treatment in children with autism have reported clinical improvements; however, this treatment has not been evaluated to date with a controlled study. We performed a multicenter, randomized, double-blind, controlled trial to assess the efficacy of hyperbaric treatment in children with autism. METHODS: 62 children with autism recruited from 6 centers, ages 2-7 years (mean 4.92+/-1.21), were randomly assigned to 40 hourly treatments of either hyperbaric treatment at 1.3 atmosphere (atm) and 24% oxygen (“treatment group”, n=33) or slightly pressurized room air at 1.03 atm and 21% oxygen (“control group”, n=29). Outcome measures included Clinical Global Impression (CGI) scale, Aberrant Behavior Checklist (ABC), and Autism Treatment Evaluation Checklist (ATEC). RESULTS: After 40 sessions, mean physician CGI scores significantly improved in the treatment group compared to controls in overall functioning (p=0.0008), receptive language (p<0.0001), social interaction (p=0.0473), and eye contact (p=0.0102); 9/30 children (30%) in the treatment group were rated as “very much improved” or “much improved” compared to 2/26 (8%) of controls (p=0.0471); 24/30 (80%) in the treatment group improved compared to 10/26 (38%) of controls (p=0.0024). Mean parental CGI scores significantly improved in the treatment group compared to controls in overall functioning (p=0.0336), receptive language (p=0.0168), and eye contact (p=0.0322). On

– 43 – the ABC, significant improvements were observed in the treatment group in total score, irritability, stereotypy, hyperactivity, and speech (p<0.03 for each), but not in the control group. In the treatment group compared to the control group, mean changes on the ABC total score and subscales were similar except a greater number of children improved in irritability (p=0.0311). On the ATEC, sensory/cognitive awareness significantly improved (p=0.0367) in the treatment group compared to the control group. Post-hoc analysis indicated that children over age 5 and children with lower initial autism severity had the most robust improvements. Hyperbaric treatment was safe and well-tolerated. CONCLUSIONS: Children with autism who received hyperbaric treatment at 1.3 atm and 24% oxygen for 40 hourly sessions had significant improvements in overall functioning, receptive language, social interaction, eye contact, and sensory/cognitive awareness compared to children who received slightly pressurized room air.

– 44 – Konklusjoner Formålet med denne litteraturgjennomgangen har vært å belyse det brede spekteret av etiopatologiske mekanismer som de siste årene er blitt presentert som mulig delaktige i det kliniske uttrykket av autisme.

Gjennomgangen belyser nye testmetoder og behandlingsmetoder for personer med autisme.

Dersom resultatene i alle de forskjellige studiene som er sitert her var enstydige og pekte i samme retning, ville denne gjennomgangen vært unødvendig, ettersom det i så fall ville vært gullstandard. Denne gjennomgangen ville heller ikke vært nødvendig dersom alle studiene påviste en årsakssammenheng som var logisk i henhold til medisinsk teori og praksis.

Ved avslutning av søk på PubMed var det mer enn 14.000 artikler om ASD. Antall nye hver måned var nærmere 100.

Nyere og pågående forskning på autisme avdekker stadig nye funn innenfor mage–tarm, immunsystemet og nervesystemet.

Styrken til denne gjennomgangen er fokuset på områder av medisinen som tradisjonelt sett ikke er blitt forbundet med atferdsforstyrrelser.

Avvik og interaksjoner i bestemte biokjemiske reaksjonsveier som metylering, transulfatering og dannelse av fettsyrer og peptider er for tiden i fokus. Forskningen avdekker stadig mer av den komplekse samhandlingen mellom verten, vertens tarmflora og alle typer eksterne påvirkninger som kosthold, miljøfaktorer osv. Hvert en- keltmenneske representerer summen av enorme mengder samspill mellom vert/mikrobe eller mikrobe/mikrobe som i sin tur påvirker mange forskjellige biokjemiske, immunologiske og også atferdsmessige parametre hos verten og dermed bidrar til å forme personens atferd. Betennelser i tarm, tungmetallforgiftninger og forstyrrelser i immunsystemet ved autisme bør være sterke pådrivere for videre forskning.

Hovedbudskapet er at vi nå kan påvirke enkelte av faktorene som fører til unormal atferd. Med økt innsats kan vi forbedre helsetilstanden til et betydelig høyere antall enkeltpersoner med atferdsforstyrrelser, herunder autisme.

Denne holdningen står i kontrast til det mer tradisjonelle synspunktet som mange fagpersoner har, nemlig at autisme er en genetisk hjerneforstyrrelse som ikke kan behandles. Dette synet bør revurderes,

Autisme er ikke en hjerneforstyrrelse, det er en forstyrrelse som påvirker hjernen.

– 45 – Referanser 1 Martha R. Herbert,Clinical Neuropsychiatry(2005) 2,354-379

2 Strandberg K, Sedvall G, Midtvedt T, Gustafsson B, Effect of some biologically active amines on the cecum wall of germfree rats.Proc Soc Exp Biol Med. 1966 Mar;121(3):699-702.

3 Gustafsson BE, Midtvedt T, Strandberg K.Effects of microbial contamination on the cecum enlargement of germfree rats.Scand J Gastroenterol. 1970;5(4):309-14.

4 Veenstra-Vanderweele et al.,Annual Review of Genomics and Human Genetics 5, 379-405, 2004

5 Baird G, Simonoff E, Pickles A, Chandler S, Loucas T, Meldrum D, Charman T. Prevalence of disorders of the autism spectrum in a population cohort of children in South Thames: the Special Needs and Autism Project (SNAP). Lancet 2006 Jul 15;368:210-5.

6 Morbidity and Mortality Weekly Report. Mental Health in the United States: Parental Report of Diagnosed Autism in Children Aged 4–17 Years – United States, 2003–2004. May 5, 2006 / 55(17);481-486

7 Chakrabarti, S. Fombonne, E. Pervasive Developmental Disorders in Preschool children: Confirmation of High Prevalence. Am J Psychiatry, 167, June 2005 1133-1141.

8 Hem KG., Husum TL. A7249 Prevalens av autisme.2. September 2008, SINTEF, Norway, http://www.sintef. no/upload/Helse/Levekår%20og%20tjenester/Forekomst%20av%20autisme%20sluttrapp.pdf

9 Fombonne, E. Epidemiological studies of Pervasive Developmental Disorders. Chapter in Handbook of Autism and Pervasive Developmental Disorders. Editors: Volkmar, F. Rhea, P. Ami, K. Donald, C. Third Edition. John Wiley & Sons, Inc. 2005.

10 Valicenti-McDermott M, McVicar K, Rapin I, Wershil BK, Cohen H, Shinnar S. Frequency of gastrointestinal symptoms in children with autistic spectrum disorders and association with family history of autoimmune disease. J Dev Behav Pediatr. 2006 Apr;27(2 Suppl):S128-36.

11 Pardo CA, Vargas DL, Zimmerman AW Immunity, neuroglia and neuroinflammation in autism. Int Rev Psychiatry. 2005 Dec;17(6):485-95. Review.

12 Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA. Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol. 2005 Jan;57(1):67-81. Erratum in: Ann Neurol. 2005 Feb;57(2):304.

13 Jyonouchi H, Geng L, Ruby A, Reddy C, Zimmerman-Bier B. Evaluation of an association between gastrointestinal symptoms and cytokine production against common dietary proteins in children with autism spectrum disorders. J Pediatr. 2005 May;146(5):605-10.

14 Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. Dysregulated innate immune responses in young children with autism spectrum disorders: their relationship to gastrointestinal symptoms and dietary intervention. Neuropsychobiology. 2005;51(2):77-85.

15 Hamilton RG, Franklin Adkinson N Jr. In vitro assays for the diagnosis of IgE-mediated disorders. J Allergy Clin Immunol. 2004 Aug;114(2):213-25; quiz 226. Review.

16 Gupta S, Aggarwal S, Rashanravan B, Lee T. Th1- and Th2-like cytokines in CD4+ and CD8+ T cells in autism. J Neuroimmunol. 1998 May 1;85(1):106-9.

17 Hornig M, Weissenböck H, Horscroft N, Lipkin WI. An infection-based model of neurodevelopmental damage. Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):12102-7.

– 46 – 18 Messahel S, Pheasant AE, Pall H, Ahmed-Choudhury J, Sungum-Paliwal RS, Vostanis P. Urinary levels of neopterin and biopterin in autism. Neurosci Lett. 1998 Jan 23;241(1):17-20.

19 Jyonouchi H, Sun S, Le H. Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression. 1: J Neuroimmunol. 2001 Nov 1;120(1-2):170-9.

20 Warren RP, Yonk LJ, et al.Burger RA, Cole P, Odell JD, Warren WL, White E, Singh VK Deficiency of suppressor-inducer (CD4+CD45RA+) T cells in autism. Immunol Invest. 1990 Jun;19(3):245-51.

21 Warren RP, Margaretten NC, Pace NC, Foster A. Immune abnormalities in patients with autism. J Autism Dev Disord. 1986 Jun;16(2):189-97.

22 Li, X., et al., Elevated immune response in the brain of autistic patients, J. Neuroimmunol. (2009), doi:10.1016/j. jneuroim.2008.12.002

23 van Gent T, Heijnen CJ, Treffers PD. Autism and the immune system. J Child Psychol Psychiatry. 1997 Mar;38 (3):337-49. Review.

24 Vojdani A, Pangborn JB, Vojdani E, Cooper EL. Infections, toxic chemicals and dietary peptides binding to lymphocyte receptors and tissue enzymes are major instigators of autoimmunity in autism. Int J Immunopathol Pharmacol. 2003 Sep-Dec;16(3):189-99.

25 El-Fawal HA, Waterman SJ, De Feo A, Shamy MY. Neuroimmunotoxicology: humoral assessment of neurotoxicity and autoimmune mechanisms. Environ Health Perspect. 1999 Oct;107 Suppl 5:767-75. Review.

26 Singh VK, Lin SX, Newell E, Nelson C. Abnormal measles-mumps-rubella antibodies and CNS autoimmunity in children with autism. J Biomed Sci. 2002 Jul-Aug;9(4):359-64.

27 Chez MG, Dowling T, et al. Elevation of tumor necrosis factor-alpha in cerebrospinal fluid of autistic children. Pediatr Neurol. 2007 Jun;36(6):361-5.

28 Warren RP, Foster A, Margaretten NC. Reduced natural killer cell activity in autism. J Am Acad Child Adolesc Psychiatry. 1987 May;26(3):333-5.

29 Denney DR, Frei BW, Gaffney GR.,Lymphocyte subsets and interleukin-2 receptors in autistic children., J Autism Dev Disord. 1996 Feb;26(1):87-97.

30 Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, Malik M, Berelowitz M, Dhillon AP, Thomson MA, Harvey P, Valentine A, Davies SE, Walker-Smith JA. Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children.Lancet. 1998 Feb 28;351(9103):637-41. Partial retraction in: Murch SH, Anthony A, Casson DH, Malik M, Berelowitz M, Dhillon AP, Thomson MA, Valentine A, Davies SE, Walker-Smith JA. Lancet. 2004 Mar 6;363(9411):750.

31 Horvath K, Papadimitriou JC, Rabsztyn A, Drachenberg C, Tildon JT. Gastrointestinal abnormalities in children with autistic disorder. J Pediatr. 1999 Nov;135(5):559-63.

32 Wakefield AJ, Anthony A, Murch SH, Thomson M, Montgomery SM, Davies S, O’Leary JJ, Berelowitz M, Walker-Smith JA. Enterocolitis in children with developmental disorders.Am J Gastroenterol. 2000 Sep;95(9):2285-95.

– 47 – 33 Furlano RI, Anthony A, Day R, Brown A, McGarvey L, Thomson MA, Davies SE, Berelowitz M, Forbes A, Wakefield AJ, Walker-Smith JA, Murch SH. Colonic CD8 and gamma delta T-cell infiltration with epithelial damage in children with autism. J Pediatr. 2001 Mar;138(3):366-72.

34 Ashwood P, Anthony A, Pellicer AA, Torrente F, Walker-Smith JA, Wakefield AJ. Intestinal lymphocyte populations in children with regressive autism: evidence for extensive mucosal immunopathology. Clin Immunol. 2003 Nov;23(6):504-17.

35 Ashwood P, Anthony A, Torrente F, Wakefield AJ. Spontaneous mucosal lymphocyte cytokine profiles in children with autism and gastrointestinal symptoms: mucosal immune activation andreduced counter regulatory interleukin-10. J Clin Immunol. 2004 Nov; 24(6):664-73

36 Vojdani A, O’Bryan T, Green JA, Mccandless J, Woeller KN, Vojdani E, Nourian AA, Cooper EL. Immune response to dietary proteins, gliadin and cerebellar peptides in children with autism. Nutr Neurosci. 2004 Jun;7(3):151-61.

37 Horvath K, Perman JA. Autism and gastrointestinal symptoms., Curr Gastroenterol Rep. 2002 Jun;4(3):251-8.

38 Wakefield AJ, Ashwood P, Limb K, Anthony A. The significance of ileo-colonic lymphoid nodular hyperplasia in children with autistic spectrum disorder. Eur J Gastroenterol Hepatol. 2005 Aug;17(8):827-36.

39 Hisashi Kawashima, Takayuki Mori, Yasuyo Kashiwagi, Kouji Takekuma. Digestive Diseases and Sciences, 2000 Apr;45(4):723-9Detection and Sequencing of Measles Virus from Peripheral Mononuclear Cells from Patients with Inflammatory Bowel Disease and Autism.

40 Torrente F, Ashwood P, Day R, Machado N, Furlano RI, Anthony A, Davies SE, Wakefield AJ, Thomson MA, Walker-Smith JA, Murch SH.Small intestinal enteropathy with epithelial IgG and complement deposition in children with regressive autism., Mol Psychiatry. 2002;7(4):375-82, 334

41 Torrente F, Anthony A, Heuschkel RB, Thomson MA, Ashwood P, Murch SH.Focal-enhanced gastritis in regressive autism with features distinct from Crohn’s and Helicobacter pylori gastritis. Am J Gastroenterol. 2004 Apr;99(4):598-605.

42 Sudo N, Chida Y, Aiba Y, Sonoda J, Oyama N, Yu XN, Kubo C, Koga Y. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J Physiol. 2004 Jul 1;558(Pt 1):263-75. Epub 2004 May 7.

43 Bolte ER Autism and Clostridium tetani .Med Hypotheses. 1998 Aug;51(2):133-44.

44 Finegold SM, Molitoris D, Song Y, Liu C, Vaisanen ML, Bolte E, McTeague M, Sandler R, Wexler H, Marlowe EM, Collins MD, Lawson PA, Summanen P, Baysallar M, Tomzynski TJ, Read E, Johnson E, Rolfe R, Nasir P, Shah H, Haake DA, Manning P, Kaul A. Gastrointestinal microflora studies in late-onset autism. Clin Infect Dis. 2002 Sep 1;35(Suppl 1):S6-S16.

45 Martirosian G. Anaerobic intestinal microflora in pathogenesis of autism? Postepy Hig Med Dosw (Online). 2004;58:349-51.

46 Parracho HM, Bingham MO, Gibson GR, McCartney AL. Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. J Med Microbiol. 2005 Oct;54(Pt 10):987-91

47 Finegold SM. Therapy and epidemiology of autism – clostridial spores as key elements. Med Hypotheses. 2008;70(3):508-11. Epub 2007 Sep 29.

– 48 – 48 Shultz SR, MacFabe DF, Ossenkopp KP, Scratch S, Whelan J, Taylor R, Cain DP. Intracerebroventricular injection of propionic acid, an enteric bacterial metabolic end-product, impairs social behavior in the rat: implications for an animal model of autism. Neuropharmacology. 2008 May;54(6):901-11. Epub 2008 Feb 13.

49 Tjellström B, Stenhammar L, Högberg L, Fälth-Magnusson K, Magnusson KE, Midtvedt T, Sundqvist T, Houlston R, Popat S, Norin E. Gut microflora associated characteristics in first-degree relatives of children with celiac disease. Scand J Gastroenterol. 2007 Oct;42(10):1204-8.

50 Doganavsargil B, Akil I, Sen S, Mir S, Basdemir G. Autopsy findings of a case with oxalosis. Pediatr Dev Pathol. 2009 May-Jun;12(3):229-32.

51 Cochat P, Schärer K. Should liver transplantation be performed before advanced renal insufficiency in primary hyperoxaluria type 1? Pediatr Nephrol. 1993 Apr;7(2):212-8; discussion 218-9.

52 Duncan SH, Richardson AJ, Kaul P, Holmes RP, Allison MJ, Stewart CS. Oxalobacter formigenes and Oxalobacter formigenes and its potential role in human health. Appl Environ Microbiol. 2002 Aug;68(8):3841-7.

53 Faber S, Zinn GM, Kern Ii JC, Skip Kingston HM The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders. Biomarkers. 2009 Mar 11:1-10. [Epub ahead of print]

54 Nishijima S, Sugaya K, Hokama S, Oshiro Y, Uchida A, Morozumi M, Ogawa Y.Effect of vitamin B6 deficiency on glyoxylate metabolism in rats with or without glyoxylate overload. Biomed Res. 2006 Jun;27(3):93-8.

55 Holmes RP, Assimos DG. Glyoxylate synthesis, and its modulation and influence on oxalate synthesis. J Urol. 1998 Nov;160(5):1617-24

56 Baker PW, Bais R, Rofe AM, Biochemical Journal 1994 Sep 15;302 ( Pt 3):753-7. Formation of the L-cysteine- glyoxylate adduct is the mechanism by which L-cysteine decreases oxalate production from glycollate in rat hepatocytes.

57 Sharma V, Schwille PO. Biochem Int. 1992 Jul;27(3):431-8. Oxalate production from glyoxylate by lactate dehydrogenase in vitro: inhibition by reduced glutathione, cysteine, cysteamine.

58 Yao Y, Walsh WJ, McGinnis WR, Pratico D. Altered vascular phenotype in autism: correlation with oxidative stress. Arch Neurol. 2006 Aug;63(8):1161-4.

59 James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW, Neubrander JA. Metabolic biomarkers of increased oxidative stress and impaired methylation capacity in children with autism. Am J Clin Nutr. 2004 Dec;80(6):1611-7.

60 Chauhan A, Chauhan,Oxidative Stress in Autism, Pathophysiology. 2006 Aug;13(3):171-81. Epub 2006 Jun 12

61 Herbert, Martha,Large Brains in Autism: The Challenge of Pervasive Abnormality. Neuroscientist. 2005 Oct;11(5):417-40

62 Kern JK, Jones AM.J Evidence of Toxicity, Oxidative Stress, and Neuronal Insult in Autism Toxicol Environ Health B Crit Rev. 2006 Nov-Dec;9(6):485-99

63 Uhlmann V, Martin CM, Sheils O, Pilkington L, Silva I, Killalea A, Murch SB, Walker-Smith J, Thomson M, Wakefield AJ, O’Leary. Potential viral pathogenic mechanism for new variant inflammatory bowel disease. JJ. Mol Pathol. 2002 Apr;55 (2):84-90.

– 49 – 64 Bradstreet JJ., El Dahr J., Anthony A., Kartzinel J.,Wakefield AJ. Detection of Measles Virus Genomic RNA in Cerebrospinal Fluid of Children with Regressive Autism: a Report of Three Cases. Journal of American Physicians and Surgeons 2004 9:39-45.

65 TaqMan RT-PCR Detection of Measles Virus Genomic RNA in Cerebrospinal Fluid in Children with Regressive Autism. IMFAR May 2004.

66 Warren RP, Odell JD, Warren WL, et al. Strong association of the third hypervariable region of HLA-DR beta 1 with autism. J Neuroimmunol 1996;67:97–102.

67 James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan S.Thimerosal Neurotoxicity is Associated with Glutathione Depletion: Protection with Glutathione Precursors. Neurotoxicology, Jan 2005 Neurotoxicology. 2005 Jan;26(1):1-8.

68 Goth SR, Chu RA, Gregg JP, Cherednichenko G, Pessah IN.Uncoupling of ATP-mediated calcium signaling and dysregulated interleukin-6 secretion in dendritic cells by nanomolar thimerosal. Environmental Health Perspectives, 2006 July;114(7):1083-91

69 Petrik MS, Wong MC, Tabata RC, Garry RF, Shaw CA. Aluminum adjuvant linked to Gulf War illness induces motor neuron death in mice. Neuromolecular Med. 2007;9(1):83-100

70 Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC Activation of Methionine Synthase by Insulin-like Growth Factor-1 and Dopamine: a Target for Neurodevelopmental Toxins and Thimerosal. Mol Psychiatry. 2004 Apr;9(4):358-70.

71 Hornig M, Chian D, Lipkin WI.Neurotoxic Effects of Postnatal Thimerosal are Mouse Strain Dependent., , Mol Psychiatry. 2004 Sep;9(9):833-45.

72 Baskin DS, Ngo H, Didenko VV.Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured hu man neurons and fibroblasts. Toxicological Science, Aug;74(2):361-8. Epub 2003 May 28

73 Havarinasab S, Hultman P. Autoimmun Organic mercury compounds and autoimmunity., Rev. 2005 Jun;4(5):270-5. Epub 2005 Jan 5. Review.

74 Leong CC, Syed NI, Lorscheider FL.Retrograde degeneration of neurite membrane structural integrity of nerve growth cones following in vitro exposure to mercury. Neuroreport. 2001 Mar 26;12(4):733-7

75 Holmes AS, Blaxill MF, Haley BE. Reduced levels of mercury in first baby haircuts of autistic children. International Journal of Toxicology 2003 Jul-Aug;22(4):277-85

76 Haley,B.E., and Small,T.Biomarkers supporting mercury toxicity as the major exacerbator of neurological illness, recent evidence via the urinary porphyrin tests. Medical Veritas 3 (2006) 1-14

77 Waly M, Olteanu H, Banerjee R, Choi SW, Mason JB, Parker BS, Sukumar S, Shim S, Sharma A, Benzecry JM, Power-Charnitsky VA, Deth RC.Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal. Mol Psychiatry. 2004 Apr;9(4):358-70.

78 Edelson SB, Cantor DS. Autism: xenobiotic influences. Toxicol Ind Health. 1998 Jul-Aug;14(4):553-63.

79 Abassi Z et al Neutral endopeptidase inhibition increases urinary excretion and plasma level of endothelin. Metabolism 1992 41:683-685

80 Watanabe Y et al .Increased secretion of proline-containing peptides in dipeptidyl peptidase IV deficient rats. Res Comm Chem Pathol. Pharmacol. 1993 81:323 -350

– 50 – 81 Hole K, Bergslien H, Jørgensen HA, Berge OG, Reichelt KL, Trygstad OE. A peptide-containing fraction in the urine of schizophrenic patients which stimulates opiate receptors and inhibits dopamine uptake. Neuroscience. 1979;4(12):1883-93.

82 Sun Z and Cade Jr. A peptide found in schizophrenia and autism causes behavioral changes in rats. Autism 3, 1999 :85 – 95

83 D’Eufemia P, Celli M, Finocchiaro R, Pacifico L, Viozzi L, Zaccagnini M, Cardi E, Giardini O.Abnormal intestinal permeability in children with autism. Acta Paediatr. 1996 Sep;85(9):1076-9.

84 Anderson RJ, Bendell DJ, Garnett I, Groundwater PW, Lough WJ, Mills MJ, Savery D, Shattock PE. J Pharm Pharmacol. 2002 Feb;54(2):295-8. Identification of indolyl-3-acryloylglycine in the urine of people with autism.

85 Bull G, Shattock P, Whiteley P, Anderson R, Groundwater PW, Lough JW, Lees G. Med Sci Monit. 2003 Oct;9(10):CR422-5, Indolyl-3-acryloylglycine (IAG) is a putative diagnostic urinary marker for autism spectrum disorders.

86 J. B. Adams et al The Severity of Autism Is Associated with ToxicMetal Body Burden and Red Blood Cell Glutathione Levels. Journal of Toxicology Volume 2009.

87 Bradstreet J, Geier DA, Kartzinel JJ, Adams JB, Geier MR.A Case-Control Study of Mercury Burden in Children with Autistic Spectrum Disorders.Journal of American Physicians and Surgeons 8 (3) Summer 2003 76-79.

88 Nataf R, Skorupka C, Amet L, Lam A, Springbett A, Lathe R. Porphyrinuria in childhood autistic disorder: implications for environmental toxicity. Toxicol Appl Pharmacol. 2006 Jul 15;214(2):99-108. Epub 2006 Jun 16.

89 Svoboda P, Maekawa M, Kawai K, Tominaga T, Savela K, Kasai H. Urinary 8-hydroxyguanine may be a better marker of oxidative stress than 8-hydroxydeoxyguanosine in relation to the life spans of various species. Antioxid Redox Signal. 2006 May-Jun;8(5-6):985-92.

90 Ming X, Stein TP, Brimacombe M, Johnson WG, Lambert GH, Wagner GC. Increased excretion of a lipid peroxidation biomarker in autism. Prostaglandins Leukot Essent Fatty Acids. 2005 Nov;73(5):379-84.

91 Sweeten TL, Posey DJ, McDougle CJ. blood monocyte counts and neopterin levels in children with autistic disorder. Am J Psychiatry. 2003 Sep;160(9):1691-3. & Messahel S, Pheasant AE, Pall H, Ahmed-Choudhury J, Sungum-Paliwal RS, Vostanis P. Urinary levels of neopterin and biopterin in autism. Neurosci Lett. 1998 Jan 23;241(1):17-20.

92 Messahel S et al. Urinary levels of neopterin and biopterin in Autism. Neurosci Lett Jan/1998 241(1):17-320

93 Kim SW, Grant JE, Kim SI, Swanson TA, Bernstein GA, Jaszcz WB, Williams KA, Schlievert PM. A possible association of recurrent streptococcal infections and acute onset of obsessive-compulsive disorder. J Neuropsychiatry Clin Neurosci. 2004 Summer;16(3):252-60. Review.

94 Singer HS, Morris CM, Williams PN, Yoon DY, Hong JJ, Zimmerman AW. Antibrain antibodies in children with autism and their unaffected siblings. J Neuroimmunol. 2006 Jul 12;

95 Wagner M, Peterson CG, Ridefelt P, Sangfelt P, Carlson M. Fecal markers of inflammation used as surrogate markers for treatment outcome in relapsing inflammatory bowel disease. World J Gastroenterol. 2008 Sep 28;14(36):5584-9; discussion 5588.

96 Waring RH et al. Biochemical parameters in autistic children. Dev Brain Dysfunction. 1997;10:40-43

– 51 – 97 Alberti A, Pirrone P, Elia M, Waring RH, Romano C. Sulphation deficit in “low-functioning” autistic children: a pilot study. Biol Psychiatry. 1999 Aug 1;46(3):420-4.

98 James SJ et al Low plasma methionine, cysteine and glutathione levels are associated with increased frequency of common polymorphisms affecting methylation and glutathione pathways in children with autism.,. Experimental Biology 2005;19:A51-A52

99 Deth R, Muratore C, Benzecry J, Power-Charnitsky VA, Waly M.How environmental and genetic factors combine to cause autism: A redox/methylation hypothesis Neurotoxicology. 2008 Jan;29(1):190-201. Epub 2007 Oct 13.

100 James SJ, Melnyk S, Jernigan S, Cleves MA, Halsted CH, Wong DH, Cutler P, Bock K, Boris M, Bradstreet JJ, Baker SM, Gaylor DW. Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism. Am J Med Genet B Neuropsychiatr Genet. 2006 Aug 17; [Epub ahead of print]

101 Waring, R.H.; Klovrza, L.V. Sulphur Metabolism in Autism .Journal of Nutritional & Environmental Medicine, Volume 10, Number 1, 1 March 2000 , pp. 25-32(8)

102 Markovich D, Aronson PS., Specificity and regulation of renal sulfate transporters. Annu Rev Physiol. 2007;69:361-75. Review.

103 Jill James S, Melnyk S, Jernigan S, Hubanks A, Rose S, Gaylor DW.,Abnormal Transmethylation/ transsulfuration Metabolism and DNA Hypomethylation Among Parents of Children with Autism., J Autism Dev Disord. 2008 May 30.

104 The metabolism of homocysteine: pathways and regulation. Finkelstein JD. Eur J Pediatr. 1998 Apr;157 Suppl 2:S40-4. Review.

105 Sharma et al. Lipids in Health and Disease 2006 5:1 doi:10.1186/1476-511X-5-1

106 Faber S, Zinn GM, Kern Ii JC, Skip Kingston HM The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders. Biomarkers. 2009 Mar 11:1-10. [Epub ahead of print]

107 Martineau J, Barthelemy C, Garreau B, Lelord G.Vitamin B6, magnesium, and combined B6-Mg: Therapeutic effects in childhood autism.Martineau J, Biological Psychiatry 1985;20:467-8

108 Carlton R et al.Rational dosages of nutrients have a prolonged effect on learning disabilities. Alternative Therapies 2000; 6:8591.

109 Schoenthaler SJ, Bier ID, Young K, Nichols D, Jansenns S.,The effect of vitamin-mineral supplementation on the intelligence of American schoolchildren: a randomized, double-blind placebo-controlled trial. Journal of Alternative and Complementary Medicine 2000;6:1929.

110 Mousain-BoBosc M et al. Jam Coll Magnesium,Vitamin B6 intake reduces central nervous system hyperexcitability in children. Nutr 2004;23:545S-548S

111 Miodownik C, Lerner V, Statsenko N, Dwolatzky T, Nemets B, Berzak E, Bergman Vitamin B6 versus mianserin and placebo in acute neuroleptic-induced akathisia: a randomized, double-blind, controlled study. J. Clinical Neuropharmacology,Vol.29, No.2,March/April 2006, 68-72

112 Arnold LE, Pinkham SM, Votolato N.Does zinc moderate essential fatty acid and amphetamine treatment of attention-deficit/hyperactivity disorder? J Child Adolesc Psykopharmacol 2000;10:111-7

113 Knivsber AM, Reichelt KL, Nødland M. Reports on dietary intervention in autistic disorders.,Nutr Neurosci. 2001;4(1):25-37.

– 52 – 114 Knivsberg AM, Reichelt KL, Høien T, Nødland M A randomised, controlled study of dietary intervention in autistic syndromes. Nutr Neurosci 2002;5:251-61

115 Bu B, Ashwood P, Harvey D, King IB, Water JV, Jin LW. Fatty acid compositions of red blood cell phospholipids in children with autism. Prostaglandins Leukot Essent Fatty Acids. 2006 Apr;74(4):215-21.

116 Evans, T et al. The Autistic Phenotype Exhibits a Remarkably Localized Modification of Brain Protein by Products of Free Radical-Induced Lipid Oxidation, American Journal of Biotechnology and Biochemistry 4 (2): 61-72, 2008

117 Wiest MM, German JB, Harvey DJ, Watkins SM, Hertz-Picciotto I. Prostaglandins Leukot Essent Fatty Acids. 2009 Apr;80(4):221-7.

118 Meguid NA, Atta HM, Gouda AS, Khalil RO. Role of polyunsaturated fatty acids in the management of Egyptian children with autism. Clin Biochem. 2008 Sep;41(13):1044-8

119 Vancassel S, Durand G, Barthélémy C, Lejeune B, Martineau J, Guilloteau D, Andrès C, Chalon S. Plasma fatty acid levels in autistic children.Prostaglandins Leukot Essent Fatty Acids 2001;65: 1-7

120 Brudnak MA, Rimland B, Kerry RE, Dailey M, Taylor R, Stayton B, Waickman F, Waickman M, Pangborn J, Buchholz I. Enzyme-based therapy for autism spectrum disorders -- is it worth another look? Med Hypotheses. 2002 May;58(5):422-8.

121 Rossignol DA, Rossignol LW, Smith S, Schneider C, Logerquist S, Usman A, Neubrander J, Madren EM, Hintz G, Grushkin B, Mumper EA. Hyperbaric treatment for children with autism: a multicenter, randomized, double-blind, controlled trial. BMC Pediatr. 2009 Mar 13;9(1):21.

– 53 – Vedlegg: Forkortelser Forkortelser benyttet i kunnskapsoppsummeringen: ”Biokjemiske ubalanse og biomedisinsk behandling av autisme: Gjennomgang av litteraturen”, mars 2010.

ADA: adenosine deaminase IAG: indolyl acryloyl glycin ADHD: attention deficit hyperactivity disorder ICDRC: International Child Development Resource AGT: glyoxylate aminotransferase Center AK: adenosine kinase IFN: interferon APA: American Psychological Association IgA: immunoglobulin A ARI: Autism Research Institute IgE: immunoglobulin E ASD: autism spectrum disorder IgG: immunoglobulin G ASO: antistreptolysin IgM: immunoglobulin M AST: aspartate aminotransferase IL-2: interleukin -2 ATEC: Autism Treatment Evaluation IL-4: interleukin -4 BHMT: betaine-homocysteine methyltransferase IL-10: interleukin -10 B6: pyridoxine IM: intestinal microbiota GF: gluten free IV: intravenous CBC: complete blood count IVIG: intravenous immune globulin CBS: cystathionine beta synthase MAT: methionine adenosyltransferase CD3: cluster of differentiation 3 MBP: myelin basic protein CD8: cluster of differentiation 8 MMR: Measles,Mumps,Rubella CF: casein free MS: methionine synthase CoQ 10: ubiquione NAC: n-acetyl cysteine CYS: cysteine NAFP: neuron-axon filament protein DHA: docosahexaneoic acid NK: natural killer DMSA: dimercaptosuccinic acid PANDAS: Pediatric Autoimmune Neuropsychiatric DNA: deoxy ribonucleic acid Disorders Associated with Streptococcal infections. DNASE B: deoxyribonuclease B PPA: propionic acid DP: dietary proteins PUFA: polyunsaturated fatty acid DPPIV: dipeptidyl peptidase IV RNA: ribonucleic acid DSM_IV: Diagnostic and Statistical Manual of Mental ROS: reactive oxygen species disorders IV SAH: s-adenosylhomocysteine EAAT3: excitatory amino acid transporter 3 SAHH: SAH hydrolase FDA: Federal Drug Administration SAM: S-adenosylmethione GI: gastro intestinal T-cells: T lymhocytes GMP: guanosine monophosphate Th: T helper cells GSH: reduced glutathione THF: tetrahydrofolate GSSG: oxidized glutathione TMG: trimethylglycine Hcys: homocystein TNF-alfa: tumornekrosefaktor alfa HBOT: hyperbaric oxygen therapy 8-dOHG: dihydroxyguanine HHV6: human herpesvirus six 8-OHG 8: hydroxyguanine HPA: hypothalamic-pituary-adrenal

– 54 – Vedlegg: Søkeord Søkeord benyttet i kunnskapsoppsummeringen: ”Biokjemiske ubalanse og biomedisinsk behandling av autisme: Gjennomgang av litteraturen”, mars 2010.

Søk i databasene MEDLINE og The Cochrane Library per august 2009.

Child development pervasive disorder, PDD; Autis* og/eller annen relevant diagnose innen ASD i kombinasjon med: aluminium intestinal permeability antifungal lead antiviral lymph nodular hyperplasia autoimmunity malnutrition bacteria melatonin biochemical imbalance mercury biomarker methylation biomedical microbial metabolism brain antibodies microbial metabolites casein microflora chelation minerals cytokines neurology detoxification neurotoxin dietary organic acid dysbiosis oxalate environmental oxidative stress fatty acid pcb food allergies porphyrins gastrointestinal regression gluten restlessness glutathione sulfate heavy metals sulphur homocysteine Th1/Th2 hormone treatment thimerosal hyperactivity toxic hyperbaric oxygen transsulfuration immune dysregulation urinary peptides immune response vaccine immunology viral infections inflammatory A63 viral titers inflammation vitamins insomnia yeast

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