Adenosine-to-inosine (A-to-I) RNA editing is a neurodevelopmentally-regulated epigenetic modification shown to modulate complex behavior in animals. Little is known about human A-to-I editing, but it is thought to constitute one of many molecular mechanisms connecting environmental stimuli and behavioral outputs. Thus, comprehensive exploration of A-to-I RNA editing in human brains may shed light on gene-environment interactions underlying complex behavior in health and disease. Synaptic function is a main target of A-to-I editing, which can selectively recode key amino acids in synaptic genes, directly altering synaptic strength and duration in response to environmental signals. Here we performed a high-resolution survey of synaptic A-to-I RNA editing in a human population, and examined how it varies in autism, a neurodevelopmental disorder in which synaptic abnormalities are a common finding. Using ultra-deep (>1000×) sequencing, we quantified the levels of A-to-I editing of 10 synaptic genes in postmortem cerebella from 14 neurotypical and 11 autistic individuals. A high dynamic range of editing levels was detected across individuals and editing sites, from 99.6% to below detection limits. In most sites, the extreme ends of the population editing distributions were individuals with autism. Editing was correlated with isoform usage, clusters of correlated sites were identified, and differential editing patterns examined. Finally, a dysfunctional form of the editing enzyme ADARB1 was found more commonly in postmortem cerebella from individuals with autism. These results provide a population-level, high-resolution view of A-to-I RNA editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism.
RNA editing; neurodevelopment; autism; epigenetics; human cerebellum; A-to-I
MHC-I proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8+ T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. We show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 310 helix, that flips from an exposed “open” position in the PR transition state, to a “closed” position with buried hydrophobic side chains in the peptide-loaded (PL) mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2Ld MHC-I heavy chain, complexed with mAb 64-3-7 demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature PL conformation, chaperone dissociation, and antigen presentation.
Advances in sequencing technology are making genomic data more accessible within the healthcare environment. Published pharmacogenetic guidelines attempt to provide a clinical context for specific genomic variants; however, the actual implementation to convert genomic data into a clinical report integrated within an electronic medical record system is a major challenge for any hospital. We created a two-part solution that integrates with the medical record system and converts genetic variant results into an interpreted clinical report based on published guidelines. We successfully developed a scalable infrastructure to support TPMT genetic testing and are currently testing approximately two individuals per week in our production version. We plan to release an online variant to clinical interpretation reporting system in order to facilitate translation of pharmacogenetic information into clinical practice.
Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.
Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.
Natural killer (NK) cells provide an initial host immune response to infection by many viral pathogens. Consequently, the viruses have evolved mechanisms to attenuate the host response, leading to improved viral fitness. One mechanism employed by members of the β-herpesvirus family, which includes the cytomegaloviruses, is to modulate the expression of cell surface ligands recognized by NK cell activation molecules. A novel set of cytomegalovirus (CMV) genes, exemplified by the mouse m145 family, encode molecules that have structural and functional features similar to those of host major histocompatibility-encoded (MHC) class I molecules, some of which are known to contribute to immune evasion. In this review, we explore the function, structure, and evolution of MHC-I-like molecules of the CMVs and speculate on the dynamic development of novel immunoevasive functions based on the MHC-I protein fold.
Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 in order to avoid NK cell control in vivo. So far it is unclear if there is a direct interaction between m152 and RAE-1, and if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152, and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (Kd < 5 μM), and with 1:1 stoichiometry. The binding is quantitatively different depending on particular RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1β, although contributing to its affinity for m152, does not influence the affinity of RAE-1 γ or δ, suggesting that other differences contribute to the RAE-1/m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.
Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.
Angry, agitated outbursts (AAOs) are a common precipitant of children’s psychiatric hospitalization. In the hospital, AAOs present both management and diagnostic challenges, e.g., while they have recently been described as manic “rages”, older studies suggest that they may be exacerbated temper tantrums. Factor analyses of 109 AAOs had by 46 hospitalized 4 to 12 year olds yielded 3 subsets of behaviors expressing different intensities of anger and 2 subsets expressing different intensities of distress (sadness). Cluster analysis of behavior time course supported the anger-distress distinction; the former behaviors are most probable at AAO onset and then decline while the latter are more evenly distributed across the outburst. Age trends, factor structure, and temporal organization of AAOs all indicate that they are exacerbated tantrums. The AAOs of children with anxiety or PDD diagnoses showed significantly more distress relative to anger. AAOs have clinical implications; their particular characteristics may have diagnostic significance.
anger; anxiety; disruptive behavior; emotion dysregulation; sadness
To examine the safety and efficacy of liquid risperidone to reduce duration of rages in children with severe mood dysregulation (SMD) or possible bipolar disorder (BP).
There were 151 admissions of 5–12 year old children to a psychiatric inpatient unit. Diagnostic information and history of prior rages were obtained at admission. In hospital, a first rage outburst was treated with seclusion. If a 2nd rage occurred, the child was offered liquid risperidone to help him/her regain control. Risperidone dose was increased by 0.02 mg/kg for any successive rages. Duration of unmedicated and last medicated rage were compared. Rage frequency in children with SMD and several definitions of BP were compared.
Although 82 of 151 admissions were prompted by rages, they occurred during only 49 hospitalizations, and occurred more than once in only 24. In 16 multiply-medicated children duration of rages dropped from a baseline of 44.4± 20.2 minutes to 25.6 ± 12.5 minutes at the child’s last dose. Neither SMD nor any definition of BP influenced rage response in this small sample. The average liquid risperidone dose was 0.02 mg/kg. All but 2 children also took atypical antipsychotics daily. No adverse events were observed.
Liquid risperidone may be a safe and effective way to shorten the duration of rage episodes regardless of diagnosis. However, definitive conclusions cannot be drawn in the absence of a placebo control as children were also receiving other behavioral and psychopharmacologic treatments.
In the immune system, B cells, dendritic cells, NK cells, and T lymphocytes all respond to signals received via ligand binding to receptors and coreceptors. While the specificity of T cell recognition is determined by interaction of T cell receptors with MHC/peptide complexes, the development of T cells in the thymus and their sensitivity to antigen are also dependent on coreceptor molecules CD8 (for MHCI) and CD4 (for MHCII). The CD8αβ heterodimer is a potent coreceptor for T cell activation, but efforts to understand its function fully have been hampered by ignorance of structural details of its interactions with MHCI. Here we describe the structure of CD8αβ in complex with the murine MHCI molecule H-2Dd at 2.6 Å resolution. The focus of the CD8αβ interaction is the acidic loop (residues 222-228) of the α3 domain of H-2Dd. The β subunit occupies a T cell membrane-proximal position, defining the relative positions of the CD8α and CD8β subunits. Unlike the CD8αα homodimer, CD8αβ does not contact MHCIα2 or β2-microglobulin domains. Movements of the CD8α complementarity determining region-(CDR) 2 and CD8β CDR1 and CDR2 loops as well as flexibility of the H-2Dd CD loop facilitate the monovalent interaction. The structure resolves inconclusive data on the topology of the CD8αβ/MHCI interaction, indicates that CD8β is crucial in orienting the CD8αβ heterodimer, provides a framework for understanding the mechanistic role of CD8αβ in lymphoid cell signaling, and offers a tangible context for design of structurally altered coreceptors for tumor and viral immunotherapy.
cell surface molecules; T cells; MHC; T cell receptors
Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8+ cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8+ T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2Dd better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8+ T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.
The purpose of this study was to examine rages and define their associated clinical and diagnostic conditions systematically. Children's severe anger outbursts, sometimes called “rages,” have been associated with many disorders, including mania, “severe mood dysregulation,” and oppositional defiant/conduct disorder. Although reactive aggression has been studied extensively, there are almost no data on this important and disabling clinical phenomenon.
A total of 130 different 5–12 year olds were hospitalized over 151 consecutive admissions were evaluated diagnostically with information from parents, children, doctors, nursing staff, and teachers. Rages were operationally defined as agitated/angry behaviors requiring seclusion or medication because the child could not be verbally redirected to “time out.” Rage behaviors were categorized as they occurred with the specially designed Children's Agitation Inventory. Hypotheses were that rages would be associated with prior treatment failure, and that children with rages would have the most co-morbidities, including learning/language disorders. We did not expect narrow-phenotype bipolar disorder to be specifically associated with rages.
Of 130 children, 71 (54.6%) were admitted for rages. Preadmission rages and admission taking an atypical antipsychotic significantly predicted the subsequent number of in-hospital rages. Attention-deficit/hyperactivity disorder with learning/language disorder significantly predicted the occurrence and number of rages. Bipolar disorder was the referring diagnosis in 17/49 (34.7%) admissions with rages and 15/102 (14.7%) of admissions without rages (odds ratio [OR] 3.05, confidence interval [CI] 1.36, 6.80). However, a consensus diagnosis of bipolar disorder occurred in 5 (9.1%) of the sample with rages and 5 (5.8%) in the rest of admissions.
Psychiatrically hospitalized children with multiple rages have complex, chronic neuropsychiatric disorders and have failed prior conventional treatment. One third of children with rages had been given a bipolar diagnosis prior to admission. However, only 9% of children with rages were given that diagnosis after careful observation.
Immuno-receptor tyrosine based inhibitory motif (ITIM)-containing receptors play an essential role in modulating immune responses. Leukocyte associated inhibitory receptor (LAIR)-1, also known as CD305, is an ITIM-containing inhibitory receptor, expressed by all leukocytes, that binds collagens. In this report, we investigate the effect of a conservative R65K mutation on LAIR-1 ligand binding and function. Compared to LAIR-1 wild-type (wt) expressing cells, LAIR-1 R65K cells show markedly reduced binding to collagen, which correlates with a reduced level of LAIR-1 polarization to the site of interaction with collagens. Both LAIR-1 wt and R65K cells can generate intracellular signals when ligated by anti-LAIR-1 mAb, but only LAIR-1 wt cells respond to collagens or matrigel. In agreement, surface plasmon resonance (SPR) analyses showed that LAIR-1 R65K protein has markedly reduced avidity for collagen type I compared to LAIR-1 wt. Likewise, LAIR-1 R65K protein has decreased avidity for cells expressing transmembrane collagen XVII. Thus, a single residue, Arg 65, is critical for the interaction of LAIR-1 with collagens.
Natural Killer Cells; Cell Surface Molecules; Signal Transduction; Cell Activation
drug design; protein–protein interactions; SCF–KIT; supramolecular chemistry; synthetic inhibitors
The mouse cytomegalovirus (CMV), a β-herpesvirus, exploits its large (~230 kb) double-stranded DNA genome for both essential and non-essential functions. Among the non-essential functions are those that offer the virus selective advantage in eluding both the innate and adaptive immune responses of the host. Several non-essential genes of MCMV are thought to encode MHC-I-like genes and to function as immunoevasins. To understand further the evolution and function of these viral MHC-I (MHC-Iv) molecules, X-ray structures of several of them have been determined, confirming the overall MHC-I-like structure, but also elucidating features unique to this family. Future efforts promise to clarify the nature of the molecular ligands of these molecules, their evolution in the context of the adapting immune response of the murine host, and by analogy the evolution of the host response to human CMV as well.
MHC recognition; NK cells; T cells; immunoevasins; X-ray crystallography; MHC-Iv molecules; cytomegalovirus
Angry outbursts, sometimes called “rages”, are a major impetus for children's psychiatric hospitalization. In hospital, such outbursts are a management problem and a diagnostic puzzle. Among 130 4-to-12 year olds successively admitted to a child psychiatry unit, those having in-hospital outbursts were likely to be younger, have been in special education, have a pre-admission history of outbursts, and a longer hospital stay. Three subsets of behaviors, coded as they occurred in 109 outbursts, expressed increasing levels of anger; two other subsets expressed increasing levels of distress. Factor structure, temporal organization and age trends indicated that outbursts are exacerbations of ordinary childhood tantrums. Diagnostically, children with outbursts were more likely to have language difficulty and a trend towards ADHD. Outbursts of children with anxiety diagnoses showed significantly more distress relative to anger. Outbursts were not especially associated with our small sample of bipolar diagnoses.
We examined TCR: MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IAd-restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6–8wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IAd/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IAd/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.
Antigen presentation/processing; Autoimmunity; TCR; Tg/knockout mice
Adequate presentation of self and foreign antigens is a key factor for efficient T-cell immunosurveillance against pathogens and tumors. Cells presenting foreign antigens usually comprise a rare population and are difficult to detect even at the peak of infection. Here we demonstrate a CD8+ T-cell-based approach that allows detection of specific antigen-presenting cells (APC) at a frequency of less than 0.0005%. When T cells are in excess, they form rosettes with rare APCs, which appear as single spots in an IFN-γ ELISPOT assay. Using this RELISPOT (Rosette ELISPOT) method we demonstrate the dynamic interplay between CD8 T cells and professional and non-professional APCs following virus challenge.
Mouse cytomegalovirus (MCMV), a β-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted MHC-I structure. Functions attributed to some family members include downregulation of host MHC-I (m152) and NKG2D ligands (m145, m152, m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require β2m or peptide, and is expressed at the surface of MCMV-infected cells. Its 2.4 Å crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended amino terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family.
Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/β2-microglobulin (β2m) complex at 1.9 Å resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable α1, α2, and α3 domains. A unique disulfide bond links the α1 helix to the b-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the α1 and α2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the α2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.
X-ray structure; MHC-Iv; immunoevasin; NK recognition; viral virulence
This paper describes the approach and progress Children's Hospital has made in the development and implementation of a generic medical document handling system as part of a Progressively Automated Medical Record. In addition, a description of our character cell based implementation of Patient Word Processing and Chart Browser applications are presented. Emphasis is given to the problems, objectives, and solutions associated with the generation of patient-oriented word processing documents.
Children's Hospital has chosen to develop an interface between its central, relational database and its vendor-supplied laboratory systems. We describe here the relational model for objective results which we are using in this implementation. The model presented addresses a variety of data types, permits hierarchical structuring of test results, and is flexible. The major parts of the entity-relationship model are covered, including a discussion of some of the major design decisions.