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New Biomarkers for Autism

New Biomarkers for Autism

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Inflammation, autoimmunity, and neurochemistry signaling pathways have been implicated in the etiology and severity of autism spectrum disorder (ASD). This small cross-sectional study found that inflammation plays a role in neurochemical dysregulation in young autism patients. The authors suggest that gamma-aminobutyric acid, oxytocin, and an interferon-induced protein can be used as biochemical correlates to social and cognitive impairment, and may possibly be used to flag infants at risk for autism who may benefit from early behavioral intervention.
Inflammation, autoimmunity, and neurochemistry signaling pathways have been implicated in the etiology and severity of autism spectrum disorder (ASD). This small cross-sectional study found that inflammation plays a role in neurochemical dysregulation in young autism patients. The authors suggest that gamma-aminobutyric acid, oxytocin, and an interferon-induced protein can be used as biochemical correlates to social and cognitive impairment, and may possibly be used to flag infants at risk for autism who may benefit from early behavioral intervention.

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Published by: Children Of Vietnam Veterans Health Alliance on Feb 26, 2014
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RESEARCH Open Access
Association of social and cognitive impairmentand biomarkers in autism spectrum disorders
Altaf Alabdali
, Laila Al-Ayadhi
and Afaf El-Ansary
 The neurological basis for autism is still not fully understood, and the role of the interaction betweenneuro-inflammation and neurotransmission impairment needs to be clearer. This study aims to test the possibleassociation between impaired levels of gamma aminobutyric acid (GABA), serotonin, dopamine, oxytocin, andinterferon-
-induced protein-16 (IFI16) and the severity of social and cognitive dysfunctions in individuals with autismspectrum disorders.
Materials and methods:
 GABA, serotonin, dopamine, oxytocin, and IFI16 as biochemical parameters related toneurochemistry and inflammation were determined in the plasma of 52 Saudi autistic male patients, categorized asmild-moderate and severe as indicated by their Childhood Autism Rating Scale (CARS) or social responsiveness scale(SRS), and compared to 30 age- and gender-matched control samples.
 The data indicated that Saudi patients with autism have remarkably impaired plasma levels of the measuredparameters compared to age and gender-matched controls. While serotonin in platelet-free plasma and dopamine didnot correlated with the severity in social and cognitive dysfunction, GABA, oxytocin, and IFI16 were remarkablyassociated with the severity of both tested scores (SRS and CARS).
 The relationship between the selected parameters confirms the role of impaired neurochemistryand neuro-inflammation in the etiology of autism spectrum disorders and the possibility of using GABA, oxytocin,and IFI16 as markers of autism severity. Receiver operating characteristic analysis together with predictiveness dia-grams proved that the measured parameters could be used as predictive biomarkers of clinical symptoms and pro-vide significant guidance for future therapeutic strategy to re-establish physiological homeostasis.
 Autism spectrum disorders, Childhood autism rating scale, Interferon-
-induced protein-16,Neuroinflammation, Neurotransmitters, Oxytocin, Social responsiveness scale
Autism spectrum disorders (ASDs) are a behaviorally de-fined group of disorders characterized by impaired socialskills, impaired language, and restricted areas of interest[1]. Additional features may include poor eye contact[2], repetitive behavior [3], sensory modulatory dysfunc- tion [4], and varying levels of cognition and motor dis-turbances [5,6]. Clinical signs are usually present at the age of 3 years, but prospective studies of infants at riskhave demonstrated that deficits in social responsiveness,communication, and play could be present early, at theage of 6
12 months [7]. However, the condition is oftenmissed and not diagnosed until later in a child
s life, espe-cially when the condition is mild or even moderate in se- verity. There has been increasing interest in developingeffective interventions for young children with autismsince the evidence suggests that early intervention pro-grams are indeed beneficial for children with autism, oftenimproving developmental functioning and decreasing mal-adaptive behaviors and symptom severity and can also im-prove outcomes in later years for many individuals [8].Abnormalities in neurotransmitter systems have fre-quently been reported in autism. Clinical observations in-clude altered levels of various neurotransmitters comparedto controls, including alterations in dopamine metabolism
* Correspondence: elansary@ksu.edu.sa
Biochemistry Department, Science College, King Saud University, P.O box22452, Zip code 11495 Riyadh, Saudi Arabia
Autism Research and Treatment Center, King Saud University, Riyadh, SaudiArabiaFull list of author information is available at the end of the article
© 2014 Alabdali et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedicationwaiver (http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwisestated.
 et al. Journal of Neuroinflammation
[9] and dysregulations of serotonergic systems [10-13]. Expression of several types of gamma aminobutyric acid(GABA) receptors is altered in the brains of subjects withautism, with levels being significantly reduced in autismcompared to controls [14-16]. In addition, impaired vaso- pressin/oxytocin metabolism was also reported in patientswith autism compared to controls [17]. Moreover, studieshave revealed increased levels of pro-inflammatory cyto-kines in brain, cerebrospinal fluid (CSF), and blood fromchildren with ASDs [18].Recently, there has been increasing interest in the roleof chronic inflammation in neurological disorders. Theprogressive increase of clinical and experimental researchinto inflammation has been initiated by knowledge thatinflammation-induced changes in brain disorders are notlimited to specific neurotransmitter abnormalities but re-flect multifunctional changes in the oxidative/antioxidantstatus, and immune, endocrine, and neurotransmittercircuits in the brain [19]. Neuroinflammation was firstidentified in a study of post-mortem samples from 11 in-dividuals with autism aged 5
44, in which activated as-trocytes and microglial cells together with abnormalinflammatory cytokines were found [20,21]. Additional support for the presence of tissue neuroinflammatory-based changes in brains of people with autism comesfrom the various studies showing reduction in Purkinjecell numbers, possibly due to inflammation-inducedoxidative stress leading to an increased excitation/inhib-ition ratio that could potentially be due to imbalancedglutamatergic and GABAergic systems [13,14]. Al- though it is accepted that neuroinflammation can alterneurotransmitter function and its transporters [22] pro-ducing cognitive, behavioral, and psychiatric impair-ments, and that excess central nervous system (CNS)glutamate levels can induce neurotoxicity-mediated in-flammation [23-26], the interaction between these two pathologic mechanisms have not been clarified in pa-tients with autism.A number of studies have shown that inflammatory cytokines, including tumor necrosis factor (TNF)-
, in-terferon (IFN)-
, IL6, IL8, and IL12, are elevated inblood mononuclear cells, plasma, serum, CSF, and brainof autistic subjects [27-30]. Among these cytokines, IFN-
 orchestrates the trafficking of specific immunecells to sites of inflammation through up-regulatingprotein mediators
 expression such as IFN-
-inducedprotein-16 (IFI16). Additionally, TNF-
 synergistically regulates the expression of these in adhesion and che-mokine molecules [31].This work is an attempt to understand the interactionbetween GABA, serotonin, dopamine, and oxytocin as im-portant neurotransmitters and IFI16 as an inflammatory mediator that has not been extensively studied in ASDcompared to other cytokines. Moreover, a biochemicalcorrelate to the social responsiveness scale (SRS) and theChildhood Autism Rating Scale (CARS) as measures of social and cognitive impairments in patients with autismwas screened among the measured parameters.
Materials and methods
This cross-sectional study was conducted on 52 male chil-dren who had ASD, recruited from the Autism Researchand Treatment Center, Faculty of Medicine, King SaudUniversity, Riyadh, Saudi Arabia; 40 were non-verbal and12 were verbal. Their ages ranged between 3 and 12 years(mean SD=7.0±2.34 years). The control group com-prised 30 age and sex-matched apparently healthy childrenwith mean age 7.2±2.14 years. The patients met the diag-nostic criteria of ASD according to the 4
edition of theDiagnostic and Statistical Manual of Mental Disorders [1].The controls were normally developing, healthy children,unrelated to the autistic subjects and without any of theexclusion criteria. These children were the healthy oldersiblings of healthy infants who were attending the WellBaby Clinic at King Khalid University Hospital for rou-tine check-up of their growth parameters. They had noclinical indications of infectious disease or neuropsy-chiatric disorders. All participants had normal resultsfor urine analysis and sedimentation rate. The localEthical Committee of the Faculty of Medicine, KingSaud University, Riyadh, Saudi Arabia, approved thisstudy. In addition, an informed written consent of par-ticipation in the study was signed by the parents or thelegal guardians of the investigated subjects according tothe Helsinki principles.The CARS score was completed as a further measure-ment of the severity of disease. CARS rates the child ona scale from one to four in each of 15 areas (relating topeople; emotional response; imitation; body use; objectuse; listening response; fear or nervousness; verbal com-munication; non-verbal communication; activity level;level and reliability of intellectual response; adaptationto change; visual response; taste, smell and touch re-sponse; and general impressions). According to the scale,children who have scored 30
36 have mild to moderateautism (n = 23), while those with scores ranging be-tween 37 and 60 points have severe autism (n = 27) [32].Regarding the SRS, a questionnaire was completed in15 to 20 minutes. A total score of 76 or higher is con-sidered severe and strongly associated with a clinicaldiagnosis of autistic disorder. A score of 60
75 is inter-preted as falling in the mild to moderate range of socialimpairment [33]. Participants were excluded from thestudy if they had a diagnosis of fragile X syndrome, epi-leptic seizures, obsessive-compulsive disorder, affectivedisorders, or any additional psychiatric or neurologicaldiagnoses.
 et al. Journal of Neuroinflammation
:4 Page 2 of 14http://www.jneuroinflammation.com/content/11/1/4
Sample collection
After overnight fast, 10 mL blood samples were collectedfrom both groups in test tubes containing sodium heparin asanticoagulant. Tubes were centrifuged at 3,500 rpm at roomtemperature for 15 minutes. Plasma and red blood cells wereobtained and deep frozen (at
80°C) until analysis time.
Biochemical analysis
Determination of serotonin
The ELISA diagnostic kit used for serotonin was a productof IBL International, Germany. The assay procedure fol-lows the basic principle of competitive ELISA wherebthere is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody bindingsites. The amount of biotinylated antigen bound to theantibody is inversely proportional to the analyte concentra-tion in the sample. When the system is in equilibrium, thefree biotinylated antigen is removed by a washing step andthe antibody-bound biotinylated antigen is determined by use of anti-biotin alkaline phosphatase as marker and p-nitro phenyl phosphate as a substrate. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a response curve prepared by usingknown standards.
Determination of dopamine
The quantitative determination of dopamine in humanplasma was measured using ELISA diagnostic kit, a prod-uct of American Laboratory Products Company (ALPCO),United States.Dopamine was extracted by using a cis-diol-specificaffinity gel, acylated and then derivatized enzymatically.The competitive ELISA kit used the microtiter plateformat. The antigen is bound to the solid phase of themicrotiter plate. The derivatized standards, controls, andsamples and the solid phase-bound analytes competedfor a fixed number of antiserum binding sites. After thesystem was in equilibrium, free antigen and free antigen-antiserum complexes were removed by washing. Theantibody bound to the solid phase was detected by ananti-rabbit IgG-peroxidase conjugate using tetramethyl-benzidine as a substrate. The reaction was monitored at450 nm. Quantification of unknown samples was achievedby comparing their absorbance with a reference curve pre-pared with known standard concentrations.
Determination of gamma aminobutyric acid (GABA)
The quantitative determination of GABA in human plasmawas measured using ELISA diagnostic kit, a product of Immunodiagnostic AG, Germany. This assay was based onthe method of competitive enzyme linked immunoassays.The sample preparation included the addition of a derivati-zation reagent for GABA. Afterwards, the treated sampleswere incubated in wells of a microtiter plate coated with apolyclonal antibody against GABA-derivative, together withassay reagent containing GABA-derivative (tracer). Duringthe incubation period, the target GABA in the sample com-peted with the tracer for the binding of the polyclonal anti-bodies on the wall of the microtiter wells. GABA in thesample displaced the tracer out of the binding to the anti-bodies. Therefore, the concentration of antibody-boundtracer was inversely proportional to the GABA concentra-tion in the sample.During the second incubation step, a peroxidase conju-gate was added to each microtiter well to detect the tracer.After being washed away, the unbound components tetra-methylbenzidine were added as a peroxidase substrate. Fi-nally, the enzymatic reaction was terminated by an acidicstop solution. The color changed from blue to yellow, andthe absorbance was measured at 450 nm using a spec-trophotometer. The intensity of the yellow color was in- versely proportional to the GABA concentration in thesample; therefore, a high GABA concentration in the sam-ple reduced the concentration of antibody-bound tracerand lowered the photometric signal. A dose responsecurve of absorbance unit (optical density at 450 nm) vs.concentration was generated using the values obtainedfrom the standards. GABA present in the patient samples(EDTA plasma) was determined directly from this curve.
Determination of oxytocin
The ELISA assay kit was a product of BioSource, USA. Thisimmunoassay kit allows the
 in vitro
 quantitative determin-ation of human oxytocin concentrations in plasma. The mi-crotiter plate provided in this kit had been pre-coated withan antibody specific to oxytocin. Standards or samples werethen added to the appropriate microtiter plate wells with ahorseradish peroxidase (HRP)-conjugated antibody and in-cubated. Substrate solutions were then added to each well.The enzyme-substrate reaction was terminated by the ad-dition of a sulfuric acid solution and the color change wasmeasured spectrophotometrically at 450 nm. The concen-tration of oxytocin in the samples was then determinedby comparing the optical density of the samples to thestandard curve.
Determination of interferon-
-inducible protein 16 (IFI16)
The human IFI16 ELISA kit was provided with by Bio-Source. This immunoassay kit allows for the
 in vitro
quantitative determination of human IFI16 concentra-tions in plasma. The IFI16 ELISA kit applied the quanti-tative sandwich enzyme immunoassay technique. Themicrotiter plate had been pre-coated with a monoclonalantibody specific for IFI16. Standards or samples werethen added to the microtiter plate wells and IFI16, if present, bound to the antibody pre-coated wells. Inorder to quantitatively determine the amount of IFI16present in the sample, a standardized preparation of 
 et al. Journal of Neuroinflammation
:4 Page 3 of 14http://www.jneuroinflammation.com/content/11/1/4

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