Amanda Enstrom

Differential monocyte responses to TLR ligands in children with autism spectrum disorders

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1beta, IL-6, IL-8, TNFalpha, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1beta, IL-6, and TNFalpha responses following TLR 2, and IL-1beta response following TLR 4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1beta, IL-6, GM-CSF, and TNFalpha responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

Decreased transforming growth factor beta1 in autism: A potential link between immune dysregulation and impairment in clinical behavioral outcomes

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. There is evidence of both immune dysregulation and autoimmune phenomena in autism. We examined the regulatory cytokine transforming growth factor beta-1 (TGF beta 1) because of its role in controlling immune responses. Plasma levels of active TGF beta 1 were evaluated in 75 children with ASD compared with 68 controls. Children with ASD had significantly lower plasma TGF beta 1 levels compared with typically developing controls (p=0.0017) and compared with children with developmental disabilities other than ASD (p=0.0037), after adjusting for age and gender. In addition, there were significant correlations between psychological measures and TGF beta 1 levels, such that lower TGF beta 1 levels were associated with lower adaptive behaviors and worse behavioral symptoms. The data suggest that immune responses in autism may be inappropriately regulated due to reductions in TGF beta 1. Such immune dysregulation may predispose to the development of possible autoimmune responses and/or adverse neuroimmune interactions during critical windows in development.

Altered Gene Expression and Function of Peripheral Blood Natural Killer Cells in Children with Autism

Immune related abnormalities have repeatedly been reported in autism spectrum disorders (ASD), including evidence of immune dysregulation and autoimmune phenomena. NK cells may play an important role in neurodevelopmental disorders such as ASD. Here we performed a gene expression screen and cellular functional analysis on peripheral blood obtained from 52 children with ASD and 27 typically developing control children enrolled in the case-control CHARGE study. RNA expression of NK cell receptors and effector molecules were significantly upregulated in ASD. Flow cytometric analysis of NK cells demonstrated increased production of perforin, granzyme B, and interferon gamma (IFNgamma) under resting conditions in children with ASD (p<0.01). Following NK cell stimulation in the presence of K562 target cells, the cytotoxicity of NK cells was significantly reduced in ASD compared with controls (p<0.02). Furthermore, under similar stimulation conditions the presence of perforin, granzyme B, and IFNgamma in NK cells from ASD children was significantly lower compared with controls (p<0.001). These findings suggest possible dysfunction of NK cells in children with ASD. Abnormalities in NK cells may represent a susceptibility factor in ASD and may predispose to the development of autoimmunity and/or adverse neuroimmune interactions during critical periods of development.

Increased IgG4 levels in children with autism disorder

Accumulating evidence indicates that immune dysfunction is associated with autism disorders in a significant subset of children. Previous reports have shown abnormal immunoglobulin (Ig) levels, including an increased presence of autoreactive antibodies in the circulation of individuals with autism. As IgG is the predominant antibody isotype in circulation, we expected that an altered immune response could result in an abnormal IgG subclass profile in children with autism. We examined circulating plasma levels of IgG1, IgG2, IgG3, and IgG4 in 241 children from the CHARGE (Childhood Autism Risks from Genetics and the Environment) study, a large epidemiologic case-control investigation, including 114 children who meet full criteria for autism disorder (AU), 96 typically developing control children (TD) from a randomly selected sample of the general population, and 31 children with developmental delays (DD). We report significantly increased levels of the IgG4 subclass in children with AU compared with TD control children (p=0.016) and compared with DD controls (p=0.041). These results may suggest an underlying immunological abnormality in AU subjects resulting in elevated IgG4 production. Further investigation is necessary to elucidate the relationship between immunological findings and behavioral impairments in autism.

Decreased cellular IL-23 but not IL-17 production in children with autism spectrum disorders

A potential role for T(H)17 cells has been suggested in a number of conditions including neurodevelopmental disorders such as autism spectrum disorders (ASD). In the current study, we investigated cellular release of IL-17 and IL-23 following an in-vitro immunological challenge of peripheral blood mononuclear cells (PBMC) from children with ASD compared to age-matched typically developing controls. Following stimulation, the concentration of IL-23, but not IL-17, was significantly reduced (p=0.021) in ASD compared to controls. Decreased cellular IL-23 production in ASD warrants further research to determine its role on the generation and survival of T(H)17 cells, a cell subset important in neuroinflammatory conditions that may include ASD.

Synthesis and Screening of “One-Bead One-Compound” Combinatorial Peptide Libraries

Publisher Summary In the “one-bead one-compound” (OBOC) combinatorial library method, compound beads are prepared by a “split-mix” synthesis approach that results in the display of many copies of the same compound on one single bead. This chapter focuses on the methods used for preparing peptide libraries, and the various screening methods that can be applied to both peptide and small molecule libraries. TentaGel resin is a good choice for OBOC peptide libraries due to its uniformity in size as well as its nonstickiness. This resin can be swollen in a wide range of solvents from water to toluene. In principle, either the Fmoc/t-But (9-fluorenylmethoxycarbonyl/tertbutyl) or t-Boc/Bn (tert-butyloxycarbonyl/benzyl) amino acid protection strategies could be used for library synthesis on TentaGel. The chapter also describes the procedure that uses N,N’-diisopropylcarbodiimide (DIC) as an activating reagent and 1-hydroxybenzotriazole (HOBt) as an additive to synthesize peptide libraries. Regarding the OBOC combinatorial libraries screening, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with the target molecule will be identified and then isolated for structure determination. The enzyme-linked colorimetric assay screening method is discussed that can be used to screen both peptide and small molecule OBOC libraries.

Age-related gene expression in Tourette syndrome

Because infection and immune responses have been implicated in the pathogenesis of Tourette syndrome (TS), we hypothesized that children with TS would have altered gene expression in blood compared to controls. In addition, because TS symptoms in childhood vary with age, we tested whether gene expression changes that occur with age in TS differ from normal control children. Whole blood was obtained from 30 children and adolescents with TS and 28 healthy children and adolescents matched for age, race, and gender. Gene expression (RNA) was assessed using whole genome Affymetrix microarrays. Age was analyzed as a continuous covariate and also stratified into three groups: 5-9 (common age for tic onset), 10-12 (when tics often peak), and 13-16 (tics may begin to wane). No global differences were found between TS and controls. However, expression of many genes and multiple pathways differed between TS and controls within each age group (5-9, 10-12, and 13-16), including genes involved in the immune-synapse, and proteasome- and ubiquitin-mediated proteolysis pathways. Notably, across age strata, expression of interferon response, viral processing, natural killer and cytotoxic T-lymphocyte cell genes differed. Our findings suggest age-related interferon, immune and protein degradation gene expression differences between TS and controls.

CD8+ T cell function in children with autism

389 Psychosocial and life style influences on the genetics of inflammaging in older humans L. Coe , G. Love , J.A. Morozink , E.M. Friedman , C.D. Ryff b a University of Wisconsin, Harlow Center for Biological Psychology, Madison, WI 53715, United States b University of Wisconsin, Institute on Aging, Madison, WI 53706, United States c University of Wisconsin, Population Health, Madison, WI 53726, United

Development and Characterization of Potent Peptide Inhibitors of p60c-src PTK Using Pseudosubstrate-Based Inhibitor Design Approach

The cytoplasmic protein p60c-src is a ubiquitous non-receptor protein tyrosine kinase (PTK) that is over-expressed and/or activated in several cancer types making it an important anti-cancer target [1]. In addition, c-src knockout mice have a phenotype consistent with osteopetrosis suggest that inhibitors against this enzyme may be therapeutic for osteoporosis. In the past few years, we have developed a pseudo-substrate-based inhibitor strategy [2,3] to target the peptide-substrate binding site of p60c-src PTK. This strategy has led to the identification of highly selective and potent inhibitors of p60c-src PTK [2]. By screening a random “one-bead one-compound” combinatorial peptide-bead library with a functional phosphorylation assay [4], we identified MIYKYYF as a very inefficient peptide substrate for this enzyme. However, MIYKYYF was found to be a moderately potent inhibitor (IC50 = 6 μM) of p60c-src PTK using YIYGSFK (Km = 55 μM) as a substrate. In this study, we describe the development and characterization of potent pseudosubstrate-based inhibitors (IC50 = 0.14–0.6 μM) of p60c-src PTK, using MIYKYYF as the template.

Development of Peptidomimetic Substrates and Inhibitors that Bind to the Peptide Binding Pocket of the Catalytic Site of p60c-src Protein Tyrosine Kinase

Src family protein tyrosine kinases (PTKs) are excellent targets for anti-cancer drug discovery due to their association with cell transformation and carcinogenesis. In the past few years, we have developed a pseudosubstrate-based inhibitor strategy to target the active site of p60c-src PTK. This strategy has led to the identification of highly selective and potent peptide inhibitors of p60c-src PTK [1,2] that bind to the peptide substrate binding pocket and not the ATP binding pocket. Despite their high inhibitory activity against p60c-src PTK in a cell free protein kinase assay, the peptide inhibitors did not show any significant biological effect on intact v-src transfected 3T3 cells. This was probably due to their inability to penetrate the cell membrane and their susceptibility to proteolysis. Previous work in our laboratory on peptide substrates [3,4] and inhibitors [1,2] led us to believe that the dipeptide motif, -Ile-Tyr- (-I-Y-), is critical for binding to the active site of p60c-src PTK. In this study, we describe the development of cell permeable peptidomimetic substrates and inhibitors of p60c-src PTK using -Ile-Tyr- as a core structure.