2003 Progress Report: Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core

EPA Grant Number: R829388C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R829388
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism
Center Director: Pessah, Isaac N.
Title: Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
Investigators: Van de Water, Judith , Gershwin, M. Eric , Korvatska, Elena
Current Investigators: Van de Water, Judith , Ashwood, Paul
Institution: University of California - Davis
EPA Project Officer: Hahn, Intaek
Project Period: September 30, 2001 through September 29, 2002
Project Period Covered by this Report: September 30, 2002 through September 29, 2003
RFA: Centers for Children's Environmental Health and Disease Prevention Research (2001) RFA Text |  Recipients Lists
Research Category: Health , Children's Health , Health Effects

Objective:

Profile serologic samples from autistic and matched control children collected from Research Projects I (Environmental Epidemiology), from Project II (Animal Models), and tissue culture experiments performed in Project III (Molecular/Cell Mechanisms).

  • To define the blood levels of key neurotrophins and neuropeptides by ELISA or recycling immunoaffinity chromatography
  • To ascertain what tissue-specific antibodies are found in sera of patients with autism using a variety of neuronal antigens by immunoblot. Using patient sera, also perform immunohistochemical analysis of brain tissue from autistic patients and controls. Similar studies will be performed during the generation of animal models in Project II.
  • Using our brain tissue from autistic patients solicited through the M.I.N.D. Institute’s collaboration with the Autism Tissue Program of the National Alliance for Autism Research, screen for the presence of differentially expressed antigens as recognized by patient sera. Also of importance is the determination of existing in situ deposits of antibody in the brain of patients (when available).
  • To develop a protein array derived from a brain cDNA library for screening with patient sera for any unknown antigens or receptors found bound by antibodies from patients with autism.

Progress Summary:

We are currently setting up an ELISA based system to profile the neurotrophins and neuropeptides in plasma. In addition, we are developing an antibody microarray system for this analysis that will allow us greater sensitivity and more efficient sample use.

We have access to protein extracts from different human brain regions as well as fetal brain and whole adult brain. We are using these extracts to perform Western blot analysis. We are have analyzed sera from mothers of children with autism (AU) from the AGRE tissue bank for reactivity against fetal and adult brain tissue. Thus far, our study contains samples from 23 unrelated subjects provided by AGRE including12 mothers with 2 consecutive AU/PDD children, 10 mothers with 3 consecutive AU/PDD children and 1 mother with 4 consecutive Au children. Our control population thus far consists of 14 unrelated women, including 4 mothers with 1 typically developing (TD) child, 7 mothers with 2 TD children, 2 mothers with 3 TD children and 1 mother with 4 TD children. This very preliminary data suggests that, as expected, there is very little difference in the staining pattern of placenta between control mothers (Figure 1A.) and mothers of AU patients (Figure 1B). However, there appeared to be a difference in both the frequency and location of bands in the mothers of the AU group than in the control mother group (Figures 2A and 2B). In addition, there were some bands that appeared to be recognized only by the mothers of children with AU. For example, one band, at approximately 175 kD, was present in nearly 60% of the samples from mothers with autisic children and none of the control maternal sera. The dark staining band present in all samples at approximately 56 kD represents the IgG reference band. The positive control (PND) is a sample from a patient with paraneoplastic disorder, which are known to present with autoantibodies to neural tissue. Clearly, further studies are needed to determine frequency of reactivity to fetal brain tissue as well as adult brain. Similar analysis is currently underway for each region of the brain for both mothers and children with autism as well as TD controls. To date, our pediatric data suggests that while plasma from children with autism does not contain significant antibodies to fetal brain, several subjects to show reactivity against various regions of the brain. Data from the Western blot studies will enhance our immunohistochemical studies by allowing us to determine which regions of the brain contain potential autoantigens. The immunohistochemical staining results will then help us to determine which cells contain the putative autoantigenic determinants.

Thus, we are continuing to use our existing bank of sera from patients and controls with autism to examine brain sections from Rhesus macaques for antibodies to neural tissue and will expand this study using the newly acquired CHARGE samples.

Analysis cellular activation, including cytokine production, of samples obtained from autistic and matched control children through Research Project I- (Environmental Epidemiology), and from Research Project II (Animal Models). To evaluate the effects of these activation products on cultured neuronal tissue.

We have begun to measure cytokine expression in peripheral blood mononuclear cells (PBMC) from CHARGE samples as described in Research Project II. This includes the cytokine profile of PBMC following stimulation with the MMR antigen as well as various antigen controls. The experimental design permits determination of possible synergistic effects of xenobiotics on this response. We are analyzing the cytokine profile of mice exposed to thimerosal being conducted in Project II. The analysis of DCs is being undertaken in close collaboration with Project III (see Project III report). These studies will be supplemented by measurement of the gene transcription using a focused cytokine gene array system performed by Core III (Molecular Biomarkers).

We have begun our stimulation assays and are using the newly developed multi-analyte bead system from BD to determine cytokine production by FACS. This system allows us to measure six cytokines simultaneously for less cost per sample. The advantages of this are twofold. First, we can measure more cytokines using fewer cells per assay. This helps tremendously when the sample volume is limited by the age of the child and allows us to add additional antigens into the test system. Second, the lower cost will allow us to assay more samples than we initially planned.

Future Activities:

Our future goals are to provide research support for each of the three Center research projects as we now obtain patient samples. The administration of sample cataloguing and dissemination is very labor intensive. Thus, we have begun to further refine this process in an attempt to streamline this procedure. Finally, as additional data is acquired from our Western blot studies on mothers and children, we will further identify any significant autoantigens detected.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this subproject

Supplemental Keywords:

Autism, antibodies, autoantibodies, immunohistochemical, antigens, thimerosal,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Health Risk Assessment, Chemistry, Risk Assessments, Susceptibility/Sensitive Population/Genetic Susceptibility, Disease & Cumulative Effects, Physical Processes, Children's Health, genetic susceptability, Biology, Risk Assessment, neurotoxic, chemical exposure, biomarkers, epidemiology, xenobiotics, exposure, gene-environment interaction, pesticides, neurodevelopment, halogenated aromatics, children, neurobehavioral, neurodevelopmental, neurotoxicity, etiology, human exposure, susceptibility, autism, neurobehavioral effects, biological markers, mechanisms, exposure assessment, human health risk, synergistic interactions, biomarker

Progress and Final Reports:

Original Abstract
  • Final

  • Main Center Abstract and Reports:

    R829388    CECEHDPR - University of California at Davis Center for the Study of Environmental Factors in the Etiology of Autism

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R829388C001 Environmental Factors in the Etiology of Autism; Analytic Biomakers (xenobiotic) Core
    R829388C002 Environmental Factors in the Etiology of Autism; Cell Activation/Signaling Core
    R829388C003 Environmental Factors in the Etiology of Autism; Molecular Biomakers Core
    R829388C004 Environmental Factors in the Etiology of Autism; Childhood Autism Risks from Genetics and the Environment (The CHARGE Study)
    R829388C005 Environmental Factors in the Etiology of Autism; Animal Models of Autism
    R829388C006 Environmental Factors in the Etiology of Autism; Molecular and Cellular Mechanisms of Autism