Reed P. Warren

Circulating autoantibodies to neuronal and glial filament proteins in autism

Autoimmunity may be a pathogenic factor in autism, a behavioral disorder of early childhood onset. Circulating autoantibodies are produced in organ-specific autoimmunity; therefore, we investigated them in the plasma of autistic subjects, mentally retarded (MR) subjects, and healthy controls. Autoantibodies (IgG isotype) to neuron-axon filament protein (anti-NAFP) and glial fibrillary acidic protein (anti-GFAP) were analyzed by the Western immunoblotting technique. We found a significant increase in incidence of anti-NAFP (P = .004) and anti-GFAP (P = .002) in autistic subjects, but not in MR subjects. Clinically, these autoantibodies may be related to autoimmune pathology in autism.

Immune abnormalities in patients with autism

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.

Echinacea-induced cytokine production by human macrophages

Echinacea purpurea, a plant originally used by native Americans to treat respiratory infections, was evaluated for its ability to stimulate the production of cytokines by normal human peripheral blood macrophages in vitro. Commercial preparations of echinacea fresh pressed juice and dried juice were tested at concentrations ranging from 10 micrograms/ml to 0.012 microgram/ml and compared to endotoxin stimulated and unstimulated controls. Cytokine production was measured by ELISA after 18 h of incubation for IL-1 and 36 and 72 h for TNF-alpha, IL-6, and IL-10. Macrophages cultured in concentrations of echinacea as low as 0.012 microgram/ml produced significantly higher levels of IL-1, TNF-alpha, IL-6 and IL-10 (P < 0.05) than unstimulated cells. The high levels of IL-1, TNF-alpha, and IL-10 induced by very low levels of echinacea are consistent with an immune activated antiviral effect. Echinacea induced lower levels of IL-6 in comparison to the other cytokines measured. These results demonstrate the immune stimulatory ability of the unpurified fresh pressed juice of Echinacea purpurea and offer some insight into the nature of the resulting immune response as compared to endotoxin.

Strong association of the third hypervariable region of HLA-DRβ1 with autism

We reported that the major histocompatibility complex (MHC) including the null allele of the C4B gene and the extended haplotype B44-C30-DR4 is associated with autism. We report now that the third hypervariable region (HVR-3) of certain DR β l alleles have very strong association with autism. The HVR-3 of DRβ1∗ 0401 or the shared HVR-3 alleles DR01∗ 0404 and DRβ1∗ 0101, was expressed on extended haplotypes in 23 of 50 (46%) autistic subjects as compared to only 6 of 79 (7.5%) normal subjects. Another HVR-3 sequence, the DRβ1∗ 0701 allele, was carried on extended haplotypes in 16 (32.0%) of the autistic subjects as compared to 8 (10.1 %) of the normal subjects.

Increased frequency of the null allele at the complement C4b locus in autism

Associations between C4 deficiency and autoimmune disorders have been found over the past several years. Since autism has several autoimmune features, the frequencies of null (no protein produced) alleles at the C4A and C4B loci were studied in 19 subjects with autism and their family members. The autistic subjects and their mothers had significantly increased phenotypic frequencies of the C4B null allele (58% in both the autistic subjects and mothers, compared with 27% in control subjects). The siblings of the autistic subjects also had an increased frequency of the C4B null allele, but this increase was not significant. The fathers had normal frequencies of this null allele. All family members had normal frequencies of the C4A null allele, all normal C4A and C4B alleles and all BF and C2 alleles.

Hyperserotoninemia and serotonin receptor antibodies in children with autism but not mental retardation

Autism is a biological disorder manifesting psychiatric symptoms in children. The etiology is not known, although diverse factors may contribute to the cause of autism. The major symptoms are impaired social interaction, communication, and language skills. Apparently, they are related to abnormal development of certain centers within the brain, in particular cerebellum, brain stem, and limbic region (Bauman 1991). Hyperserotoninemia is seen in 30-66% of autistic children (Yuwiler et al 1992; Laszlo et al 1994), and some also have autoantibodies to brain serotonin receptor (Todd and Ciaranello 1985; Yuwiler et al 1992). Since up to 60% of autistic children show mental retardation, we measured serotonin levels and brain serotonin receptor antibodies in the plasmas of autistic, mentally retarded, and normal children. The present study describes hyperserotoninemia and serotonin receptor antibodies in autistic children but not in mentally retarded children.

Immunogenetic studies in autism and related disorders

The major histocompatibility complex comprises a number of genes that control the function and regulation of the immune system. One of these genes, the C4B gene, encodes a product that is involved in eliminating pathogens such as viruses and bacteria from the body. We previously reported that a deficient form of the C4B gene, termed the C4B null allele (no C4B protein produced) had an increased frequency in autism. In this study we attempted to confirm the increased incidence of the C4B null allele in autism and investigated the presence of a C4B null allele in two other childhood disorders, attention-deficit hyperactivity disorder and dyslexia (reading disability). In addition, we explored the relationship of autism to the DRβ1 gene, a gene located close to the C4B in autism. We confirmed the finding of an increased frequency of the C4B null allele in autism and found that the related disorders also had an increased frequency of this null allele. In addition, two alleles of the DRβ1 gene also had significantly increased representation in the autistic subjects.

Deficiency of suppressor-inducer (CD4+CD45RA+) T cells in autism

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.

Increased frequency of the extended or ancestral haplotype B44-SC30-DR4 in autism

Autism likely results from several different etiologies or a combination of pathological mechanisms. Recent studies suggest that this disorder may be associated with immune abnormalities, pathogen-autoimmune processes and perhaps the major histocompatibility complex (MHC). In a preliminary study we found that 22 autistic subjects had an increased frequency of the extended or ancestral MHC haplotype B44-SC30-DR4. The current study attempted to confirm this observation by studying 23 additional randomly chosen autistic subjects, most of their parents and 64 unrelated normal subjects. In agreement with earlier findings B44-SC30-DR4 was associated with autism. In combining the data from the original and current studies, B44-SC30-DR4 or a substantial fragment of this extended haplotype was represented in 40% of the autistic subjects and/or their mothers as compared to about 2% of the unrelated subjects. It is concluded that one or more genes of the MHC is (are) involved in the development of some cases of autism.

DR-positive T cells in autism: association with decreased plasma levels of the complement C4B protein.

Autism is a developmental disorder characterized by severe communication, social and behavioral abnormalities. Over the past several years a fair amount of evidence has accumulated suggesting that some cases of autism may be associated with immune abnormalities and with products of the HLA complex including the C4B gene located in the class III region of HLA. This study sought additional evidence for an association of autoimmune processes with autism by investigating the presence of activated T cells in 26 autistic subjects. Fourteen of the autistic subjects had DR+ T cells, an indicator of activated T cells, but none of the autistic subjects had T cells expressing the interleukin-2 receptor, another indicator of T cell activation. Similar findings of incomplete or partial T cell activation have been reported in autoimmune disorders and in a recent study of autism. In the current investigation, the DR+ T cells were not found to be associated with age of the autistic patients but were inversely correlated with a decreased plasma level of the C4B protein. In conclusion, this study provides additional evidence for the involvement of an autoimmune mechanism in autism.