Further characterization of autoantibodies to GABAergic neurons in the central nervous system produced by a subset of children with autism
- Sharifia Wills†1, 5,
- Christy C Rossi†2, 4,
- Jeffrey Bennett2, 4,
- Veronica Martinez-Cerdeño2, 4,
- Paul Ashwood3, 4, 5,
- David G Amaral2, 4 and
- Judy Van de Water1, 4, 5Email author
© Wills et al; licensee BioMed Central Ltd. 2011
Received: 8 November 2010
Accepted: 26 April 2011
Published: 26 April 2011
Autism is a neurodevelopmental disorder characterized by impairments in social interaction and deficits in verbal and nonverbal communication, together with the presence of repetitive behaviors or a limited repertoire of activities and interests. The causes of autism are currently unclear. In a previous study, we determined that 21% of children with autism have plasma autoantibodies that are immunoreactive with a population of neurons in the cerebellum that appear to be Golgi cells, which are GABAergic interneurons.
We have extended this analysis by examining plasma immunoreactivity in the remainder of the brain. To determine cell specificity, double-labeling studies that included one of the calcium-binding proteins that are commonly colocalized in GABAergic neurons (calbindin, parvalbumin or calretinin) were also carried out to determine which GABAergic neurons are immunoreactive. Coronal sections through the rostrocaudal extent of the macaque monkey brain were reacted with plasma from each of seven individuals with autism who had previously demonstrated positive Golgi cell staining, as well as six negative controls. In addition, brain sections from adult male mice were similarly examined.
In each case, specific staining was observed for neurons that had the morphological appearance of interneurons. By double-labeling sections with plasma and with antibodies directed against γ-aminobutyric acid (GABA), we determined that all autoantibody-positive neurons were GABAergic. However, not all GABAergic neurons were autoantibody-positive. Calbindin was colabeled in several of the autoantibody-labeled cells, while parvalbumin colabeling was less frequently observed. Autoantibody-positive cells rarely expressed calretinin. Sections from the mouse brain processed similarly to the primate sections also demonstrated immunoreactivity to interneurons distributed throughout the neocortex and many subcortical regions. Some cell populations stained in the primate (such as the Golgi neurons in the cerebellum) were not as robustly immunoreactive in the mouse brain.
These results suggest that the earlier report of autoantibody immunoreactivity to specific cells in the cerebellum extend to other regions of the brain. Further, these findings confirm the autoantibody-targeted cells to be a subpopulation of GABAergic interneurons. The potential impact of these autoantibodies on GABAergic disruption with respect to the etiology of autism is discussed herein.
Autism is a lifelong neurodevelopmental disorder that is diagnosed in early childhood and is characterized by a core deficit in social interaction with impairments in communication, stereotypical movements and restricted behaviors . Converging evidence over the past 40 years indicates that immune dysfunction may be an important factor contributing to the development of a subset of cases of autism [2–4]. Several studies have shown peripheral immune abnormalities in patients with autism [5–7]. There is provocative evidence for an ongoing inflammatory response in some individuals with autism [8, 9].
The possibility has been raised that some forms of autism may be due to an autoimmune process . There are a number of precedents for autoimmune diseases of the central nervous system. The best known among these are multiple sclerosis [11, 12] and Sydenham's chorea [13, 14]. The potential for an autoimmune etiology with respect to psychiatric disorders, including the pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections, remains intriguing but controversial .
A number of reports have identified antibodies in individuals with autism that are directed against several central nervous system proteins. These include glial and neuron-axon filament proteins  (but see ), myelin basic protein  (but see ), serotonin receptor , nerve growth factor , cerebellar peptides , brain-derived neurotrophic factor , brain endothelial cells  and the caudate nucleus . We have described an increased incidence of autoantibodies to brain proteins in autism compared with controls .
In a previous study , we used Western blot analysis and tissue immunohistochemistry to investigate the presence of autoantibodies to cerebellar tissue. Using Western blot analysis, we found that the plasma of 21% of individuals with autism demonstrated immunoreactivity to a protein of approximately 52 kDa from the human cerebellum that was present in only 2% of typically developing controls. When these plasma samples were then used as primary antibody sources for tissue immunohistochemistry using sections from the macaque monkey brain, 21% of the samples from children with autism spectrum disorder (ASD) compared to 0% of typically developing controls demonstrated intense immunoreactivity to what were morphologically determined to be Golgi cells of the cerebellum. This study utilized sections from the cerebellum based on early data that the cerebellum was preferentially involved in autism. However, it is now clear that autism affects many brain regions , and the question arises whether the autoantibodies present in autistic individuals identify a broader class of neurons that are distributed throughout the brain. Thus, to build upon our previous findings, in the current study we used plasma from the same individuals who demonstrated positive cerebellar Golgi cell immunoreactivity to stain tissue sections from the full rostrocaudal extent of the macaque monkey brain. To evaluate conservation of the target antigens, we also examined brain sections from adult male mice. We were particularly interested in determining whether the autoantibody immunoreactivity would identify neurons from disparate brain regions and whether the identified neurons would have any unifying morphological features.
Participants and sample collection
The study protocol followed the ethical guidelines of the most recent Declaration of Helsinki  and was approved by the Institutional Review Board of the University of California-Davis School of Medicine. All participants enrolled in the study had written informed consent provided by their parents and provided consent to participate if developmentally able. Subjects for this study were enrolled through the Medical Investigations of Neurodevelopmental Disorders (M.I.N.D.) Institute clinic. The current study was designed to be a detailed follow-up of our previous study that described the presence of Golgi cell immunoreactivity in the plasma of a subpopulation of children with autism . Our current study population consisted of a subgroup of children previously diagnosed on the autism spectrum (n = 7) (age range, 2.5 to 7 years) who showed reactivity to an approximately 52-kDa protein in the cerebellum as determined by Western blot analysis as well as on the basis of intense immunoreactivity to Golgi cells of the cerebellum in the cynomolgus monkey (Macaca fascicularis). We also examined seven participants from our previous study who were diagnosed on the autism spectrum but did not show immunoreactivity to monkey brain sections. Our control population consisted of typically developing children with an absence of reactivity to both the 52-kDa protein and to neurons of the cynomolgus monkey brain (n = 6) (age range, 2.5 to 8 years). Any participants in the control group who were above criteria in either the Autism Diagnostic Interview-Revised (ADI-R) or the Autism Diagnostic Observation Schedule (ADOS) [1, 29–31] were excluded. Samples from children with ASD with an absence of reactivity detected by both Western blot analysis and immunohistochemical staining were also examined. A diagnosis of ASD was confirmed in all participants on the basis of the ADI-R and ADOS score. The final ASD status is defined as meeting criteria on the communication, social interaction and repetitive behavior domains of the ADI-R with onset prior to 36 months of age and scoring at or above the social plus communication cutoff for autism on the ADOS module 1 or 2. The Social Communication Questionnaire  was used to screen for characteristics of ASD among the typically developing controls. Children who scored above the screening cutoff were fully assessed using the ADI-R and ADOS.
Animals and fixation
Nonhuman primate tissue
We choose nonhuman primate tissue as our antigen source because of the highly conserved nature of autoantigens as well as the similar but compressed neurodevelopment of rhesus monkeys compared with humans . All procedures were carried out under an approved University of California-Davis Institutional Animal Care and Use Protocol and strictly adhered to National Institutes of Health policies on primate animal subjects. The brain sections were obtained from two different male adult cynomolgus macaques (Macaca fascicularis): one was 9 years old and the other was 15 years, 4 months old at the time they were killed. The brains from these animals had been used for neural tract tracing studies, and library sections were used for the present study. No animals were killed expressly for these studies. The sections were obtained from animals for which no health problems were reported.
Tissue fixation and histological processing were performed according to procedures described previously . Briefly, the animals were deeply anesthetized and perfused intracardially with 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C at a rate of 250 mL/minute for 2 minutes, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C at a rate of 250 mL/minute for 10 minutes. The flow rate was then reduced to 100 mL/minute for 50 minutes. The animal was then perfused with 5% sucrose in 0.1 M phosphate buffer (pH 7.4) to remove as much fixative as possible.
The brain was blocked stereotaxically, extracted from the skull and cryoprotected in a solution containing 10% glycerol and 2% dimethyl sulfoxide (DMSO) for 1 day, followed by 20% glycerol and 2% DMSO for 3 days. The fixed brain was then frozen using the isopentane method  and stored at -70°C until sectioning. Frozen sections were cut in the coronal plane at a thickness of 30 μm with a sliding microtome and placed into a cryoprotectant tissue-collecting solution (30% ethylene glycol, 25% glycerin in 0.05 M sodium phosphate buffer). The sections were stored at -80°C until they were used for immunohistochemistry.
Additionally, tissue from one adult rhesus macaque (Macaca mulatta) whose perfusion included glutaraldehyde was used for anti-γ-aminobutyric acid (anti-GABA) immunostaining. Briefly, the perfusion consisted of 0.9% sodium chloride at a rate of 250 mL/minute for 2 minutes, followed by 4% paraformaldehyde/0.1% glutaraldehyde at a rate of 250 mL/minute and finally 4% paraformaldehyde/0.1% glutaraldehyde at a rate of 100 mL/minute for 50 minutes. Tissue was blocked and sectioned in a manner similar to that described for the cynomolgus macaques.
Two adult male C57BL/6 mice were perfused according to the methods described above for the cynomolgus macaque tissue, with an initial flow rate of 6 mL/minute followed by a flow rate of 4.75 mL/minute to account for the animal's smaller size. Tissue was cryoprotected in 10% glycerol and 2% DMSO for 1 day, followed by 20% glycerol and 2% DMSO for 2 days, and was sectioned on a sliding microtome at 30 μm as previously described for the monkey tissue.
Free-floating macaque brain sections were processed using methods employed and described previously [34, 36, 37]. Serial sections were used for immunohistochemistry using samples from each subject individually. Briefly, 30-μm-thick coronal sections from the brain were taken from storage, placed into nets and then washed (3 × 10 minutes with 0.1 M phosphate-buffered saline (PBS)). The following steps were followed with agitation at room temperature, except for the primary incubation, which was performed at 4°C with agitation. Prior to primary incubation, the sections were pretreated with hydrogen peroxide (1.6% hydrogen peroxide in 0.1 M PBS (Fisher Scientific; Pittsburg, PA, USA) for 15 minutes to remove endogenous peroxidases and reduce background staining. During incubations, the tissue was maintained in solution at all times. The sections were blocked for 4 hours using 5% normal mouse serum (Pierce Biotechnology; Rockford, IL, USA) in 0.1 M PBS and 0.5% Triton X-100 (Fisher Scientific; Pittsburg, PA, USA). The nets were incubated with the primary antibody (subject plasma) at a dilution of 1:100 in 0.1 M PBS, 2% normal mouse serum and 0.3% Triton X-100 for 40 to 48 hours. The sections were then washed with 2% normal goat serum (Invitrogen; Carlsbad, CA, USA) in 0.1 M PBS (3 × 10 minutes) and incubated with biotinylated mouse anti-human monoclonal immunoglobulin G (IgG) (Zymed: Carlsbad, CA, USA) in 0.1 M PBS, 2% normal mouse serum and 0.3% Triton X-100 for 1 hour. Sections were then washed with 2% normal goat serum in 0.1 M PBS (3 × 10 minutes) followed by incubation for 45 minutes with avidin-biotin-complex (ABC) peroxidase (Biostain Super ABC/Peroxidase Basic Kit; Biomeda: Foster City, CA, USA) incubation in 0.1 M PBS. Following the first ABC peroxidase incubation, the sections were washed in 2% normal goat serum in 0.1 M PBS. The incubation with the secondary antibody (biotinylated mouse anti-human monoclonal antibody) was repeated for 45 minutes, followed by washes with 0.1 M PBS only. The second ABC peroxidase incubation was for 30 minutes, followed by washes with 0.05 M Tris buffer. Next, the sections were incubated with 3,3-diaminobenzidine (DAB) (Fisher Scientific) in 0.05 M Tris with 0.04% hydrogen peroxide for 30 minutes. The sections were then washed twice with 0.05 M Tris, once with 0.02 M potassium PBS (KPBS) and stored covered at 4°C in 0.02 M KPBS. Sections were then mounted from phosphate buffer onto gelatin-coated slides and dried with a fan, then placed in a 37°C incubator for 24 hours to dry. Mouse sections were processed in an identical manner, except for the substitution of 0.5% for 1.6% hydrogen peroxide and the additional use of an avidin and biotin blocking kit (Vector Laboratories; Burlingame, CA, USA) prior to incubation in the primary antibody solution.
Silver nitrate/gold chloride intensification
To increase the signal-to-noise ratio of the immunohistochemical reaction product, a silver nitrate/gold chloride intensification procedure was followed for immunohistochemical analysis for those sections stained with DAB. Intensification was not used for sections visualized by using fluorescence immunohistochemistry. Prior to intensification, the slides were defatted in a mixture of equal parts chloroform and 100% ethanol for a total of 4 hours. The sections were then hydrated through graded ethanols and placed in a 37°C incubator overnight. Following the overnight incubation, the slides were rinsed with running, deionized water for 10 minutes, followed by incubation in a 1% silver nitrate solution maintained at 56°C with the use of a water bath for 40 minutes. The slides were then rinsed with running, deionized water for 10 minutes and incubated for 10 minutes in a 0.2% gold chloride solution with agitation at room temperature. Following a second rinse, the slides were stabilized in a 5% sodium thiosulfate solution at room temperature for 15 minutes with agitation, followed by an additional rinse in running, deionized water for 10 minutes. Sections were then dehydrated through graded ethanols, followed by xylenes, and coverslipped using DPX mounting medium (Sigma Aldrich; St. Louis, MO, USA).
Slides were observed by bright-field microscopy on a Nikon Eclipse E600 (Nikon Americas; Melville, NY, USA) or a Leica Leitz DMRB microscope (Leica Microsystems; Buffalo Grove, IL, USA) and photographed using the Spot Diagnostic Instruments Digital Camera System and software (Spot Imaging Solutions; Sterling Heights, MO, USA). Preliminary identification of the various cell types was accomplished using morphological features such as their location, cell body size and dendritic configuration.
Since it appeared that the majority of neurons that demonstrated specific immunoreactivity when incubated with plasma from individuals with autism had the morphological characteristics of GABAergic neurons, we carried out analyses designed to confirm this finding. First, we compared the distribution of the plasma-stained neurons with libraries of sections stained immunohistochemically for the distribution of glutamic acid decarboxylase 65 (GAD65) and GAD67, GABA or in situ hybridization with probes to GABAergic neurons [34, 37]. Second, we carried out studies in which sections were double-labeled for the presence of plasma-identified interneurons and GABA immunoreactivity. Finally, we double-labeled additional sections for plasma-labeled interneurons and the calcium-binding proteins calbindin, parvalbumin and calretinin.
For double-labeling with plasma and anti-GABA, sections from the glutaraldehyde-fixed macaque brain were rinsed in PBS, then blocked in 0.2% gelatin, 1% Triton X-100 and 5% normal donkey serum in 0.1 M PBS for 2 hours, and incubated with plasma from an individual with autism as well as monoclonal mouse anti-GABA diluted 1:400 (a gift from Ismo Virtanen) at 4°C for 72 hours. Tissue was then rinsed and incubated in Texas Red-AffiniPure donkey anti-human IgG (1:100; Jackson ImmunoResearch; West Grove, PA, USA) and fluorescein isothiocyanate-conjugated horse anti-mouse IgG (1:500; Vector Laboratories) for 2 hours at 4°C. For double-labeling with plasma and antibodies directed to calcium-binding proteins (calbindin, parvalbumin or calretinin), a Tyramide Signal Amplification (TSA) kit (PerkinElmer; Waltham, MA, USA) was used to enhance the fluorescent signal obtained from labeling with plasma. Sections were first rinsed in 0.1 M PBS, pretreated with 1% hydrogen peroxide in PBS for 30 minutes, rinsed in 0.1 M PBS and then blocked with 0.5% Triton X-100 and 5% normal mouse serum in 0.1 M PBS for 4 hours. They were then incubated overnight with a 1:100 dilution of human plasma in 0.1% Triton X-100 and 1% normal mouse serum in PBS. Sections were subsequently rinsed in PBS and incubated in Tris-NaCl-Tween buffer (TNT) buffer for 15 minutes, followed by a 30-minute incubation in TNB buffer (Blocking buffer supplied with TSA kit; 0.1 M TRIS-HCl, pH 7.5. 0.15 M NaCl. 0.5% Blocking Reagent), and incubated in a 1:1,000 dilution of biotinylated anti-human IgG secondary (Zymed; Carlsbad, CA, USA) for 1 hour. The secondary antibody was washed away with TNT buffer, and sections were then incubated in streptavidin-horseradish peroxidase (included in the TSA kit) diluted 1:100 in TNB buffer for 30 minutes. After being washed with TNT buffer, sections were incubated in tetramethylrhodamine tyramide (PerkinElmer) diluted 1:50 in amplification diluent for 8 minutes at room temperature, washed in 0.1 M PBS and incubated overnight in a solution containing an antibody against calbindin (Swant; CH-1723 Marly 1, Switzerland), parvalbumin (Swant) or calretinin (Swant) diluted 1:1,000 in 0.02% gelatin, 0.1% Triton X-100 and 1% donkey serum in 0.1 M PBS. The next day sections were rinsed in 0.1 M PBS, incubated in secondary antibody (donkey anti-rabbit) diluted 1:200 in 0.1% Triton X-100 and 1% donkey serum in PBS for 2 hours, rinsed in PBS again and mounted onto slides and coverslipped with VECTASHIELD HardSet Mounting Medium (Vector Laboratories). Fluorescently labeled sections were examined and photographed using an Olympus FluoView confocal laser-scanning microscope, and analysis was performed using FluoView version 3.3 software (Olympus: Hamburg, Germany). For identification of labeled cells, we used the same criteria described for sections processed with diaminobenzidine.
General staining pattern
A series of approximately 10 coronal sections from the Macaca fascicularis brain were processed for each of the plasma samples evaluated in this study. Sections were selected to obtain a sample from each of the following regions: frontal lobe, striatum, amygdaloid complex, hippocampal formation, hypothalamus, thalamus, cerebellum and primary visual cortex (V1). Because the complete coronal sections were processed, many additional cortical and subcortical regions, such as the thalamus, were also available for analysis. Neurons immunoreactive for IgG autoantibodies from individuals with autism were observed in all brain regions. Regardless of the region studied, the neurons tended to be small and to have the appearance of GABAergic interneurons. To provide a description of the types of staining patterns we observed, we have selected a few regions to describe in some detail. In all cases, the staining that we noted was consistently observed for all of the plasma samples from children with autism that demonstrated staining of Golgi cells in the cerebellum. We did not see this pattern of staining in any of the plasma samples from children with autism who did not demonstrate Golgi cell staining or in plasma samples from age-matched typically developing children from this study population.
Cerebral cortex (area V1)
Cerebral cortex (posterior cingulate cortex)
We previously reported that plasma from children with autism contains autoantibodies that are reactive to cerebellar Golgi cells . The current study expands our earlier observations by exploring the pattern of immunohistochemistry in additional brain regions. We have demonstrated that the plasma from children with ASD contains antibodies that are reactive to neurons throughout the brain that are invariably GABAergic interneurons. We have also determined that not all GABAergic neurons are immunopositive, although we have not yet been able to determine in detail which categories of GABAergic neurons are positive for the plasma staining.
GABAergic cells form a heterogeneous population of neurons that are unevenly distributed in all layers of the monkey neocortex . At least 20% of the neurons in the primary visual cortex are GABAergic interneurons , and the distribution in the monkey brain is quite similar to that in the human brain . The plasma from children with autism allowed us to identify populations of cells in all layers of the cerebral cortex, with a slight preponderance in superficial layers. Colocalization studies using a monoclonal antibody to GABA revealed that the plasma from children with ASD appeared to recognize GABAergic interneurons in V1. There were fewer plasma-labeled cells in the deep layers of cortex relative to GABA staining. Double-labeling with plasma and antibodies toward the calcium-binding proteins calbindin, parvalbumin and calretinin revealed that plasma-labeled cells commonly expressed calbindin. The autoantibody-positive cells expressed parvalbumin less commonly and calretinin only rarely. The distribution of GABAergic interneurons that colocalize one of these calcium-binding proteins is variable from one cortical area to the next, and certain interneuron types can colocalize more than one of the proteins [44–47]. Defelipe  provided a detailed description of the correspondence of specific interneuron types and immunoreactivity for a calcium-binding protein in the neocortex. In general, parvalbumin is associated with chandelier and large basket cells; calretinin is associated with bipolar, double-bouquet and Cajal-Retzius cells; and calbindin is associated with double-bouquet, neurogliaform, Martinotti and, in rare cases, Cajal-Retzius cells. This implies that the plasma autoantibodies affect neurons harboring calbindin more than those colocalizing the other calcium-binding proteins. However, it is not possible at this time to predict the specific functional significance of targeting these specific classes of interneurons. The question why some GABAergic interneurons are identified by the plasma autoantibodies and others is not very difficult to speculate on, given that the antigen is not known. However, recent neuronal transcriptome studies [49, 50] have made it abundantly clear that tens to hundreds of proteins differentiate neuronal classes. Given the distinct morphological features of interneurons, it is highly likely that different classes of GABAergic neurons express different profiles of proteins and that one or more of these are being identified by the plasma autoantibodies.
Our finding that some children with autism produce autoantibodies directed against GABAergic neurons is all the more interesting, given the other indications that ASDs may be highly related to impaired GABAergic function. Previous studies have suggested that there is a depression of the GABAergic system, potentially due to altered GABA receptors and reduced GAD, in the etiology and maintenance of ASD [51–53]. It has also been proposed that suppression of GABAergic function in the brain of individuals with ASD may result in compensatory mechanisms and increased activation of additional GABAergic receptor subtypes . The potential imbalance of excitatory and inhibitory neurotransmission in autism could influence cortical networks that control social behaviors and neuromodulatory systems . Interestingly, a reduction in GABAergic activity is also capable of causing epilepsy, which has been reported in up to one-third of individuals with ASD [52, 56–60]. While the participants in the present study were not assessed for epileptic seizure activity, this would be of great interest for future longitudinal studies.
In a study by Fatemi and colleagues , GAD, which converts glutamate to GABA and is a marker commonly used to identify GABAergic neurons, was found to be reduced by 48% to 61% in the parietal cortex and cerebellum in five individuals with autism relative to eight controls. More recent studies by Fatemi and colleagues  have found downregulated expression of various combinations of GABAA receptor subunits in the parietal cortex, cerebellum and superior frontal cortex in the brains of individuals with autism. These investigators also reported alterations of GABAB receptor subunit expression in the same areas of the brain . Yip and colleagues [64, 65] reported increased levels of GAD67 mRNA levels in cerebellar interneurons but decreased levels in Purkinje cells. Blatt et al. examined the distribution and density of GABAergic, serotonergic, cholinergic and glutamatergic receptors in the hippocampus of individuals with ASD and typically developing controls. The only receptor system found to be significantly reduced in individuals with ASD was the GABAergic system . Decreased GABA levels have also been reported in the platelets of children with ASD .
While it is intriguing that some children with autism demonstrate autoantibodies that are immunoreactive to central nervous system GABAergic neurons, it is not at all clear whether these antibodies play a pathophysiological role in the etiology of autism or are an epiphenomenon indicative of some other pathological process. In order for the systemic antibodies to interact with central nervous system neurons, they must be able to reach their antigenic targets. As antibodies do not readily cross the blood-brain barrier, a breach would have to occur to allow access. Cytokines in the peripheral circulation or cytokines produced by central nervous system cells are capable of altering the blood-brain barrier through endothelial cell activation . Disturbances to the blood brain-barrier can be caused by a number of other factors, including extreme stress, subclinical infection and even nicotine or epinephrine exposure [69–71]. In studies by Diamond and colleagues [72, 73] in which an animal model of systemic lupus erythematosus (SLE) was explored, antibodies directed against the N-methyl-D-aspartate receptor were able to enter the brain following exposure to either lipopolysaccharide or norepinephrine. These antibodies not only produced neuropathological changes in the hippocampus or amygdala, respectively, but also led to impaired memory function or increased anxiety.
If we presume that the anti-GABAergic neuron antibodies produced by children with autism can gain entry to the brain, there are several possible mechanisms through which they may influence cell function. First, autoantibodies may act as an agonist or a receptor ligand, causing excessive receptor stimulation. This phenomenon has been demonstrated in approximately 27% of patients with epilepsy and 30% of individuals with SLE, as well as in some patients with encephalitis, in whom various autoantibodies to the glutamate receptor have been described . Further, both human and animal studies have demonstrated that the described glutamate receptor autoantibodies are pathologic, binding to neurons with a unique ability to activate their glutamate receptor antigen and leading to neuronal death, either through excitotoxicity or by complement fixation independent of receptor activation [74–76]. While a decreased number of neurons has been reported in the amygdala  and in the fusiform gyrus  of postmortem cases of autism, it is not yet clear whether there is a preferential loss of any particular category of neurons, such as GABAergic interneurons.
The second possibility for autoantibody-induced pathology is through antibody-mediated inhibition of a critical receptor, ligand or pathway. This would be similar to the pathologic mechanism through which the autoantibodies to thyroglobulin, thyroid peroxidase and/or the thyroid-stimulating hormone receptor function in Hashimoto's thyroiditis . Whether through excitation or inhibition, autoantibody-mediated changes are capable of resulting in a number of permanent changes, including inhibition-excitation imbalance, altered neural circuitry and changes in receptor numbers and distribution. Finally, it is possible that the autoantibody directly induces cell and tissue destruction through complement activation such as that seen in patients with SLE . Each of the above-discussed mechanisms, either individually or in concert, is a possibility with respect to the autoantibodies described herein. However, additional studies, including an animal model, are needed to determine the pathologic relationship between these autoantibodies and ASD.
It is of great interest that autoantibodies from a subset of children with ASD are directed against GABAergic neurons with identical patterns of staining distributed throughout the central nervous system. This study raises several issues, including the identity of the target antigen that these antibodies recognize and the reason for the selectivity of this response. Knowledge about what differentiates immunopositive from immunonegative GABAergic neurons may provide further clues regarding the characteristics of the autoantigen and proteomic studies currently underway to determine the identity of the target protein. One additional point to address is whether the presence of these GABAergic neuron-specific autoantibodies is pathologically significant or merely an epiphenomenon. Ultimately, the potential mechanisms and/or exposures that lead to the generation of these autoantibodies, the timing at which they occur, and their pathologic significance require further consideration.
This work was supported by National Institute of Environmental Health Sciences grant 1 P01 ES11269, the US Environmental Protection Agency through the Science to Achieve Results program (grant R829388), Cure Autism Now, National Institutes of Health grant MH41479, the National Alliance for Research on Schizophrenia and Depression, and the M.I.N.D. Institute. We also thank the study participants and their families for their contribution to this study.
- Lord C, Pickles A, McLennan J, Rutter M, Bregman J, Folstein S, Fombonne E, Leboyer M, Minshew N: Diagnosing autism: analyses of data from the Autism Diagnostic Interview. J Autism Dev Disord. 1997, 27: 501-517. 10.1023/A:1025873925661.View ArticlePubMedGoogle Scholar
- van Gent T, Heijnen CJ, Treffers PD: Autism and the immune system. J Child Psychol Psychiatry. 1997, 38: 337-349. 10.1111/j.1469-7610.1997.tb01518.x.View ArticlePubMedGoogle Scholar
- Warren RP, Foster A, Margaretten NC: Reduced natural killer cell activity in autism. J Am Acad Child Adolesc Psychiatry. 1987, 26: 333-335. 10.1097/00004583-198705000-00008.View ArticlePubMedGoogle Scholar
- Warren RP, Singh VK, Averett RE, Odell JD, Maciulis A, Burger RA, Daniels WW, Warren WL: Immunogenetic studies in autism and related disorders. Mol Chem Neuropathol. 1996, 28: 77-81. 10.1007/BF02815207.View ArticlePubMedGoogle Scholar
- Ashwood P, Van de Water J: A review of autism and the immune response. Clin Dev Immunol. 2004, 11: 165-174. 10.1080/10446670410001722096.PubMed CentralView ArticlePubMedGoogle Scholar
- Croonenberghs J, Wauters A, Devreese K, Verkerk R, Scharpe S, Bosmans E, Egyed B, Deboutte D, Maes M: Increased serum albumin, γ globulin, immunoglobulin IgG, and IgG2 and IgG4 in autism. Psychol Med. 2002, 32: 1457-1463.View ArticlePubMedGoogle Scholar
- Ashwood P, Wills S, Van de Water J: The immune response in autism: a new frontier for autism research. J Leukoc Biol. 2006, 80: 1-15. 10.1189/jlb.1205707.View ArticlePubMedGoogle Scholar
- Croonenberghs J, Bosmans E, Deboutte D, Kenis G, Maes M: Activation of the inflammatory response system in autism. Neuropsychobiology. 2002, 45: 1-6.View ArticlePubMedGoogle Scholar
- Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA: Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol. 2005, 57: 67-81. 10.1002/ana.20315.View ArticlePubMedGoogle Scholar
- Ashwood P, Van de Water J: Is autism an autoimmune disease?. Autoimmun Rev. 2004, 3: 557-562. 10.1016/j.autrev.2004.07.036.View ArticlePubMedGoogle Scholar
- Bar-Or A: The immunology of multiple sclerosis. Semin Neurol. 2008, 28: 29-45. 10.1055/s-2007-1019124.View ArticlePubMedGoogle Scholar
- Huizinga R, Linington C, Amor S: Resistance is futile: antineuronal autoimmunity in multiple sclerosis. Trends Immunol. 2008, 29: 54-60. 10.1016/j.it.2007.11.002.View ArticlePubMedGoogle Scholar
- Cardoso F: Sydenham's chorea. Curr Treat Options Neurol. 2008, 10: 230-235. 10.1007/s11940-008-0025-x.View ArticlePubMedGoogle Scholar
- Wolf DS, Singer HS: Pediatric movement disorders: an update. Curr Opin Neurol. 2008, 21: 491-496.View ArticlePubMedGoogle Scholar
- Singh VK, Warren R, Averett R, Ghaziuddin M: Circulating autoantibodies to neuronal and glial filament proteins in autism. Pediatr Neurol. 1997, 17: 88-90. 10.1016/S0887-8994(97)00045-3.View ArticlePubMedGoogle Scholar
- Kirkman NJ, Libbey JE, Sweeten TL, Coon HH, Miller JN, Stevenson EK, Lainhart JE, McMahon WM, Fujinami RS: How relevant are GFAP autoantibodies in autism and Tourette syndrome?. J Autism Dev Disord. 2008, 38: 333-341. 10.1007/s10803-007-0398-9.View ArticlePubMedGoogle Scholar
- Singh VK, Warren RP, Odell JD, Warren WL, Cole P: Antibodies to myelin basic protein in children with autistic behavior. Brain Behav Immun. 1993, 7: 97-103. 10.1006/brbi.1993.1010.View ArticlePubMedGoogle Scholar
- Libbey JE, Coon HH, Kirkman NJ, Sweeten TL, Miller JN, Stevenson EK, Lainhart JE, McMahon WM, Fujinami RS: Are there enhanced MBP autoantibodies in autism?. J Autism Dev Disord. 2008, 38: 324-332. 10.1007/s10803-007-0400-6.View ArticlePubMedGoogle Scholar
- Todd RD, Hickok JM, Anderson GM, Cohen DJ: Antibrain antibodies in infantile autism. Biol Psychiatry. 1988, 23: 644-647. 10.1016/0006-3223(88)90012-1.View ArticlePubMedGoogle Scholar
- Kozlovskaia GV, Kliushnik TP, Goriunova AV, Turkova IL, Kalinina MA, Sergienko NS: [Nerve growth factor auto-antibodies in children with various forms of mental dysontogenesis and in schizophrenia high risk group] [in Russian]. Zh Nevrol Psikhiatr Im S S Korsakova. 2000, 100: 50-52.PubMedGoogle Scholar
- Vojdani A, Bazargan M, Vojdani E, Samadi J, Nourian AA, Eghbalieh N, Cooper EL: Heat shock protein and gliadin peptide promote development of peptidase antibodies in children with autism and patients with autoimmune disease. Clin Diagn Lab Immunol. 2004, 11: 515-524.PubMed CentralPubMedGoogle Scholar
- Connolly AM, Chez M, Streif EM, Keeling RM, Golumbek PT, Kwon JM, Riviello JJ, Robinson RG, Neuman RJ, Deuel RM: Brain-derived neurotrophic factor and autoantibodies to neural antigens in sera of children with autistic spectrum disorders, Landau-Kleffner syndrome, and epilepsy. Biol Psychiatry. 2006, 59: 354-363. 10.1016/j.biopsych.2005.07.004.View ArticlePubMedGoogle Scholar
- Connolly AM, Chez MG, Pestronk A, Arnold ST, Mehta S, Deuel RK: Serum autoantibodies to brain in Landau-Kleffner variant, autism, and other neurologic disorders. J Pediatr. 1999, 134: 607-613. 10.1016/S0022-3476(99)70248-9.View ArticlePubMedGoogle Scholar
- Singh VK, Rivas WH: Prevalence of serum antibodies to caudate nucleus in autistic children. Neurosci Lett. 2004, 355: 53-56. 10.1016/j.neulet.2003.10.026.View ArticlePubMedGoogle Scholar
- Cabanlit M, Wills S, Goines P, Ashwood P, Van de Water J: Brain-specific autoantibodies in the plasma of subjects with autistic spectrum disorder. Ann N Y Acad Sci. 2007, 1107: 92-103. 10.1196/annals.1381.010.View ArticlePubMedGoogle Scholar
- Wills S, Cabanlit M, Bennett J, Ashwood P, Amaral DG, Van de Water J: Detection of autoantibodies to neural cells of the cerebellum in the plasma of subjects with autism spectrum disorders. Brain Behav Immun. 2009, 23: 64-74. 10.1016/j.bbi.2008.07.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Amaral DG, Schumann CM, Nordahl CW: Neuroanatomy of autism. Trends Neurosci. 2008, 31: 137-145. 10.1016/j.tins.2007.12.005.View ArticlePubMedGoogle Scholar
- Measles MMR and autism: the confusion continues [Editorial]. Lancet. 2000, 355: 1379-10.1016/S0140-6736(00)02129-2.
- DiLavore PC, Lord C, Rutter M: The pre-linguistic autism diagnostic observation schedule. J Autism Dev Disord. 1995, 25: 355-379. 10.1007/BF02179373.View ArticlePubMedGoogle Scholar
- Lord C, Leventhal BL, Cook EH: Quantifying the phenotype in autism spectrum disorders. Am J Med Genet. 2001, 105: 36-38. 10.1002/1096-8628(20010108)105:1<36::AID-AJMG1053>3.0.CO;2-4.View ArticlePubMedGoogle Scholar
- Joseph RM, Tager-Flusberg H, Lord C: Cognitive profiles and social-communicative functioning in children with autism spectrum disorder. J Child Psychol Psychiatry. 2002, 43: 807-821. 10.1111/1469-7610.00092.PubMed CentralView ArticlePubMedGoogle Scholar
- Berument SK, Rutter M, Lord C, Pickles A, Bailey A: Autism screening questionnaire: diagnostic validity. Br J Psychiatry. 1999, 175: 444-451. 10.1192/bjp.175.5.444.View ArticlePubMedGoogle Scholar
- Webb SJ, Monk CS, Nelson CA: Mechanisms of postnatal neurobiological development: implications for human development. Dev Neuropsychol. 2001, 19: 147-171. 10.1207/S15326942DN1902_2.View ArticlePubMedGoogle Scholar
- Pitkänen A, Amaral DG: The distribution of GABAergic cells, fibers, and terminals in the monkey amygdaloid complex: an immunohistochemical and in situ hybridization study. J Neurosci. 1994, 14: 2200-2224.PubMedGoogle Scholar
- Rosene DL, Roy NJ, Davis BJ: A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact. J Histochem Cytochem. 1986, 34: 1301-1315. 10.1177/34.10.3745909.View ArticlePubMedGoogle Scholar
- Jongen-Rêlo AL, Pitkänen A, Amaral DG: Distribution of GABAergic cells and fibers in the hippocampal formation of the macaque monkey: an immunohistochemical and in situ hybridization study. J Comp Neurol. 1999, 408: 237-271. 10.1002/(SICI)1096-9861(19990531)408:2<237::AID-CNE7>3.0.CO;2-1.View ArticlePubMedGoogle Scholar
- Pitkänen A, Amaral DG: Distribution of parvalbumin-immunoreactive cells and fibers in the monkey temporal lobe: the hippocampal formation. J Comp Neurol. 1993, 331: 37-74. 10.1002/cne.903310104.View ArticlePubMedGoogle Scholar
- Kobayashi Y, Amaral DG: Macaque monkey retrosplenial cortex: I. three-dimensional and cytoarchitectonic organization. J Comp Neurol. 2000, 426: 339-365. 10.1002/1096-9861(20001023)426:3<339::AID-CNE1>3.0.CO;2-8.View ArticlePubMedGoogle Scholar
- Butovsky O, Bukshpan S, Kunis G, Jung S, Schwartz M: Microglia can be induced by IFN-γ or IL-4 to express neural or dendritic-like markers. Mol Cell Neurosci. 2007, 35: 490-500. 10.1016/j.mcn.2007.04.009.View ArticlePubMedGoogle Scholar
- Kuhn SA, van Landeghem FK, Zacharias R, Farber K, Rappert A, Pavlovic S, Hoffmann A, Nolte C, Kettenmann H: Microglia express GABAB receptors to modulate interleukin release. Mol Cell Neurosci. 2004, 25: 312-322. 10.1016/j.mcn.2003.10.023.View ArticlePubMedGoogle Scholar
- Hendry SH, Schwark HD, Jones EG, Yan J: Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex. J Neurosci. 1987, 7: 1503-1519.PubMedGoogle Scholar
- Fitzpatrick D, Lund JS, Schmechel DE, Towles AC: Distribution of GABAergic neurons and axon terminals in the macaque striate cortex. J Comp Neurol. 1987, 264: 73-91. 10.1002/cne.902640107.View ArticlePubMedGoogle Scholar
- Ong WY, Garey LJ: Distribution of GABA and neuropeptides in the human cerebral cortex: a light and electron microscopic study. Anat Embryol (Berl). 1991, 183: 397-413.View ArticleGoogle Scholar
- DeFelipe J, González-Albo MC, Del Río MR, Elston GN: Distribution and patterns of connectivity of interneurons containing calbindin, calretinin, and parvalbumin in visual areas of the occipital and temporal lobes of the macaque monkey. J Comp Neurol. 1999, 412: 515-526. 10.1002/(SICI)1096-9861(19990927)412:3<515::AID-CNE10>3.0.CO;2-1.View ArticlePubMedGoogle Scholar
- Elston GN, González-Albo MC: Parvalbumin-, calbindin-, and calretinin-immunoreactive neurons in the prefrontal cortex of the owl monkey (Aotus trivirgatus): a standardized quantitative comparison with sensory and motor areas. Brain Behav Evol. 2003, 62: 19-30. 10.1159/000071957.View ArticlePubMedGoogle Scholar
- Goodchild AK, Martin PR: The distribution of calcium-binding proteins in the lateral geniculate nucleus and visual cortex of a New World monkey, the marmoset, Callithrix jacchus. Vis Neurosci. 1998, 15: 625-642.View ArticlePubMedGoogle Scholar
- Lewis DA, Lund JS: Heterogeneity of chandelier neurons in monkey neocortex: corticotropin-releasing factor- and parvalbumin-immunoreactive populations. J Comp Neurol. 1990, 293: 599-615. 10.1002/cne.902930406.View ArticlePubMedGoogle Scholar
- DeFelipe J: Types of neurons, synaptic connections and chemical characteristics of cells immunoreactive for calbindin-D28K, parvalbumin and calretinin in the neocortex. J Chem Neuroanat. 1997, 14: 1-19. 10.1016/S0891-0618(97)10013-8.View ArticlePubMedGoogle Scholar
- Nelson SB, Hempel C, Sugino K: Probing the transcriptome of neuronal cell types. Curr Opin Neurobiol. 2006, 16: 571-576. 10.1016/j.conb.2006.08.006.View ArticlePubMedGoogle Scholar
- Winden KD, Oldham MC, Mirnics K, Ebert PJ, Swan CH, Levitt P, Rubenstein JL, Horvath S, Geschwind DH: The organization of the transcriptional network in specific neuronal classes. Mol Syst Biol. 2009, 5: 291-PubMed CentralView ArticlePubMedGoogle Scholar
- Lam KS, Aman MG, Arnold LE: Neurochemical correlates of autistic disorder: a review of the literature. Res Dev Disabil. 2006, 27: 254-289. 10.1016/j.ridd.2005.03.003.View ArticlePubMedGoogle Scholar
- Hussman JP: Suppressed GABAergic inhibition as a common factor in suspected etiologies of autism. J Autism Dev Disord. 2001, 31: 247-248. 10.1023/A:1010715619091.View ArticlePubMedGoogle Scholar
- Krey JF, Dolmetsch RE: Molecular mechanisms of autism: a possible role for Ca2+ signaling. Curr Opin Neurobiol. 2007, 17: 112-119. 10.1016/j.conb.2007.01.010.View ArticlePubMedGoogle Scholar
- Dhossche D, Applegate H, Abraham A, Maertens P, Bland L, Bencsath A, Martinez J: Elevated plasma γ-aminobutyric acid (GABA) levels in autistic youngsters: stimulus for a GABA hypothesis of autism. Med Sci Monit. 2002, 8: PR1-6.PubMedGoogle Scholar
- Rubenstein JL, Merzenich MM: Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav. 2003, 2: 255-267. 10.1034/j.1601-183X.2003.00037.x.View ArticlePubMedGoogle Scholar
- Mouridsen SE, Rich B, Isager T: Epilepsy in disintegrative psychosis and infantile autism: a long-term validation study. Dev Med Child Neurol. 1999, 41: 110-114. 10.1017/S0012162299000213.View ArticlePubMedGoogle Scholar
- Wong V: Epilepsy in children with autistic spectrum disorder. J Child Neurol. 1993, 8: 316-322. 10.1177/088307389300800405.View ArticlePubMedGoogle Scholar
- Tuchman R, Rapin I: Epilepsy in autism. Lancet Neurol. 2002, 1: 352-358. 10.1016/S1474-4422(02)00160-6.View ArticlePubMedGoogle Scholar
- Levisohn PM: The autism-epilepsy connection. Epilepsia. 2007, 48 (Suppl 9): 33-35.View ArticlePubMedGoogle Scholar
- Danielsson S, Gillberg IC, Billstedt E, Gillberg C, Olsson I: Epilepsy in young adults with autism: a prospective population-based follow-up study of 120 individuals diagnosed in childhood. Epilepsia. 2005, 46: 918-923. 10.1111/j.1528-1167.2005.57504.x.View ArticlePubMedGoogle Scholar
- Fatemi SH, Halt AR, Stary JM, Kanodia R, Schulz SC, Realmuto GR: Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. Biol Psychiatry. 2002, 52: 805-810. 10.1016/S0006-3223(02)01430-0.View ArticlePubMedGoogle Scholar
- Fatemi SH, Reutiman TJ, Folsom TD, Thuras PD: GABAA receptor downregulation in brains of subjects with autism. J Autism Dev Disord. 2009, 39: 223-230. 10.1007/s10803-008-0646-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Fatemi SH: The hyperglutamatergic hypothesis of autism. Prog Neuropsychopharmacol Biol Psychiatry. 2008, 32: 911-913. 10.1016/j.pnpbp.2007.11.004.View ArticlePubMedGoogle Scholar
- Yip J, Soghomonian JJ, Blatt GJ: Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction. J Neurosci Res. 2008, 86: 525-530. 10.1002/jnr.21520.View ArticlePubMedGoogle Scholar
- Yip J, Soghomonian JJ, Blatt GJ: Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications. Acta Neuropathol. 2007, 113: 559-568. 10.1007/s00401-006-0176-3.View ArticlePubMedGoogle Scholar
- Blatt GJ, Fitzgerald CM, Guptill JT, Booker AB, Kemper TL, Bauman ML: Density and distribution of hippocampal neurotransmitter receptors in autism: an autoradiographic study. J Autism Dev Disord. 2001, 31: 537-543. 10.1023/A:1013238809666.View ArticlePubMedGoogle Scholar
- Rolf LH, Haarmann FY, Grotemeyer KH, Kehrer H: Serotonin and amino acid content in platelets of autistic children. Acta Psychiatr Scand. 1993, 87: 312-316. 10.1111/j.1600-0447.1993.tb03378.x.View ArticlePubMedGoogle Scholar
- Merrill JE, Benveniste EN: Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci. 1996, 19: 331-338. 10.1016/0166-2236(96)10047-3.View ArticlePubMedGoogle Scholar
- Theoharides TC, Konstantinidou AD: Corticotropin-releasing hormone and the blood-brain-barrier. Front Biosci. 2007, 12: 1615-1628. 10.2741/2174.View ArticlePubMedGoogle Scholar
- Hawkins BT, Abbruscato TJ, Egleton RD, Brown RC, Huber JD, Campos CR, Davis TP: Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution. Brain Res. 2004, 1027: 48-58. 10.1016/j.brainres.2004.08.043.View ArticlePubMedGoogle Scholar
- Kuang F, Wang BR, Zhang P, Fei LL, Jia Y, Duan XL, Wang X, Xu Z, Li GL, Jiao XY, Ju G: Extravasation of blood-borne immunoglobulin G through blood-brain barrier during adrenaline-induced transient hypertension in the rat. Int J Neurosci. 2004, 114: 575-591.View ArticlePubMedGoogle Scholar
- Kowal C, DeGiorgio LA, Nakaoka T, Hetherington H, Huerta PT, Diamond B, Volpe BT: Cognition and immunity; antibody impairs memory. Immunity. 2004, 21: 179-188. 10.1016/j.immuni.2004.07.011.View ArticlePubMedGoogle Scholar
- Huerta PT, Kowal C, DeGiorgio LA, Volpe BT, Diamond B: Immunity and behavior: antibodies alter emotion. Proc Natl Acad Sci USA. 2006, 103: 678-683. 10.1073/pnas.0510055103.PubMed CentralView ArticlePubMedGoogle Scholar
- Levite M, Ganor Y: Autoantibodies to glutamate receptors can damage the brain in epilepsy, systemic lupus erythematosus and encephalitis. Expert Rev Neurother. 2008, 8: 1141-1160. 10.1586/14737126.96.36.1991.View ArticlePubMedGoogle Scholar
- Gahring LC, Twyman RE, Greenlee JE, Rogers SW: Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation. Mol Med. 1995, 1: 245-253.PubMed CentralPubMedGoogle Scholar
- Levite M, Fleidervish IA, Schwarz A, Pelled D, Futerman AH: Autoantibodies to the glutamate receptor kill neurons via activation of the receptor ion channel. J Autoimmun. 1999, 13: 61-72. 10.1006/jaut.1999.0301.View ArticlePubMedGoogle Scholar
- Schumann CM, Amaral DG: Stereological analysis of amygdala neuron number in autism. J Neurosci. 2006, 26: 7674-7679. 10.1523/JNEUROSCI.1285-06.2006.View ArticlePubMedGoogle Scholar
- van Kooten IA, Palmen SJ, von Cappeln P, Steinbusch HW, Korr H, Heinsen H, Hof PR, van Engeland H, Schmitz C: Neurons in the fusiform gyrus are fewer and smaller in autism. Brain. 2008, 131: 987-999. 10.1093/brain/awn033.View ArticlePubMedGoogle Scholar
- McLachlan SM, Nagayama Y, Pichurin PN, Mizutori Y, Chen CR, Misharin A, Aliesky HA, Rapoport B: The link between Graves' disease and Hashimoto's thyroiditis: a role for regulatory T cells. Endocrinology. 2007, 148: 5724-5733. 10.1210/en.2007-1024.View ArticlePubMedGoogle Scholar
- Kozora E, Thompson LL, West SG, Kotzin BL: Analysis of cognitive and psychological deficits in systemic lupus erythematosus patients without overt central nervous system disease. Arthritis Rheum. 1996, 39: 2035-2045. 10.1002/art.1780391213.View ArticlePubMedGoogle Scholar
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