Seattle Children's Research Institute

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

at the L2 larval stage, which provided >50-fold "shotgun" cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type-specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.

Mental health symptoms and disorders are common among elite athletes, may have sport related manifestations within this population and impair performance. Mental health cannot be separated from physical health, as evidenced by mental health symptoms and disorders increasing the risk of physical injury and delaying subsequent recovery. There are no evidence or consensus based guidelines for diagnosis and management of mental health symptoms and disorders in elite athletes. Diagnosis must differentiate character traits particular to elite athletes from psychosocial maladaptations. Management strategies should address all contributors to mental health symptoms and consider biopsychosocial factors relevant to athletes to maximise benefit and minimise harm. Management must involve both treatment of affected individual athletes and optimising environments in which all elite athletes train and compete. To advance a more standardised, evidence based approach to mental health symptoms and disorders in elite athletes, an International Olympic Committee Consensus Work Group critically evaluated the current state of science and provided recommendations.

We report the updated classification of Inborn Errors of Immunity/Primary Immunodeficiencies, compiled by the International Union of Immunological Societies Expert Committee. This report documents the key clinical and laboratory features of 430 inborn errors of immunity, including 64 gene defects that have either been discovered in the past 2 years since the previous update (published January 2018) or were characterized earlier but have since been confirmed or expanded upon in subsequent studies. The application of next-generation sequencing continues to expedite the rapid identification of novel gene defects, rare or common; broaden the immunological and clinical phenotypes of conditions arising from known gene defects and even known variants; and implement gene-specific therapies. These advances are contributing to greater understanding of the molecular, cellular, and immunological mechanisms of disease, thereby enhancing immunological knowledge while improving the management of patients and their families. This report serves as a valuable resource for the molecular diagnosis of individuals with heritable immunological disorders and also for the scientific dissection of cellular and molecular mechanisms underlying inborn errors of immunity and related human diseases.

We report the updated classification of primary immunodeficiencies compiled by the Primary Immunodeficiency Expert Committee (PID EC) of the International Union of Immunological Societies (IUIS). In the two years since the previous version, 34 new gene defects are reported in this updated version. For each disorder, the key clinical and laboratory features are provided. In this new version we continue to see the increasing overlap between immunodeficiency, as manifested by infection and/or malignancy, and immune dysregulation, as manifested by auto-inflammation, auto-immunity, and/or allergy. There is also an increased number of genetic defects that lead to susceptibility to specific organisms which reflects the finely tuned nature of immune defense systems. This classification is the most up to date catalogue of all known and published primary immunodeficiencies and acts as a current reference of the knowledge of these conditions and is an important aid for the genetic and molecular diagnosis of patients with these rare diseases.

remission rate was 93% in patients who received a CAR T-cell product and 100% in the subset of patients who received fludarabine and cyclophosphamide lymphodepletion. Twenty-three percent of patients developed reversible severe cytokine release syndrome and/or reversible severe neurotoxicity. These data demonstrate that manufacturing a defined-composition CD19 CAR T cell identifies an optimal cell dose with highly potent antitumor activity and a tolerable adverse effect profile in a cohort of patients with an otherwise poor prognosis. This trial was registered at www.clinicaltrials.gov as #NCT02028455.

Malformations of cerebral cortical development include a wide range of developmental disorders that are common causes of neurodevelopmental delay and epilepsy. In addition, study of these disorders contributes greatly to the understanding of normal brain development and its perturbations. The rapid recent evolution of molecular biology, genetics and imaging has resulted in an explosive increase in our knowledge of cerebral cortex development and in the number and types of malformations of cortical development that have been reported. These advances continue to modify our perception of these malformations. This review addresses recent changes in our perception of these disorders and proposes a modified classification based upon updates in our knowledge of cerebral cortical development.

Importance: Zika virus infection can be prenatally passed from a pregnant woman to her fetus. There is sufficient evidence to conclude that intrauterine Zika virus infection is a cause of microcephaly and serious brain anomalies, but the full spectrum of anomalies has not been delineated. To inform pediatric clinicians who may be called on to evaluate and treat affected infants and children, we review the most recent evidence to better characterize congenital Zika syndrome. Observations: We reviewed published reports of congenital anomalies occurring in fetuses or infants with presumed or laboratory-confirmed intrauterine Zika virus infection. We conducted a comprehensive search of the English literature using Medline and EMBASE for Zika from inception through September 30, 2016. Congenital anomalies were considered in the context of the presumed pathogenetic mechanism related to the neurotropic properties of the virus. We conclude that congenital Zika syndrome is a recognizable pattern of structural anomalies and functional disabilities secondary to central and, perhaps, peripheral nervous system damage. Although many of the components of this syndrome, such as cognitive, sensory, and motor disabilities, are shared by other congenital infections, there are 5 features that are rarely seen with other congenital infections or are unique to congenital Zika virus infection: (1) severe microcephaly with partially collapsed skull; (2) thin cerebral cortices with subcortical calcifications; (3) macular scarring and focal pigmentary retinal mottling; (4) congenital contractures; and (5) marked early hypertonia and symptoms of extrapyramidal involvement. Conclusions and Relevance: Although the full spectrum of adverse reproductive outcomes caused by Zika virus infection is not yet determined, a distinctive phenotype-the congenital Zika syndrome-has emerged. Recognition of this phenotype by clinicians for infants and children can help ensure appropriate etiologic evaluation and comprehensive clinical investigation to define the range of anomalies in an affected infant as well as determine essential follow-up and ongoing care.

OBJECTIVE: Cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, continues to present diagnostic challenges. Newborn screening and an evolving understanding of CF genetics have prompted a reconsideration of the diagnosis criteria. STUDY DESIGN: To improve diagnosis and achieve standardized definitions worldwide, the CF Foundation convened a committee of 32 experts in CF diagnosis from 9 countries to develop clear and actionable consensus guidelines on the diagnosis of CF and to clarify diagnostic criteria and terminology for other disorders associated with CFTR mutations. An a priori threshold of ≥80% affirmative votes was required for acceptance of each recommendation statement. RESULTS: After reviewing relevant literature, the committee convened to review evidence and cases. Following the conference, consensus statements were developed by an executive subcommittee. The entire consensus committee voted and approved 27 of 28 statements, 7 of which needed revisions and a second round of voting. CONCLUSIONS: It is recommended that diagnoses associated with CFTR mutations in all individuals, from newborn to adult, be established by evaluation of CFTR function with a sweat chloride test. The latest mutation classifications annotated in the Clinical and Functional Translation of CFTR project (http://www.cftr2.org/index.php) should be used to aid in diagnosis. Newborns with a high immunoreactive trypsinogen level and inconclusive CFTR functional and genetic testing may be designated CFTR-related metabolic syndrome or CF screen positive, inconclusive diagnosis; these terms are now merged and equivalent, and CFTR-related metabolic syndrome/CF screen positive, inconclusive diagnosis may be used. International Statistical Classification of Diseases and Related Health Problems, 10th Revision codes for use in diagnoses associated with CFTR mutations are included.

BACKGROUND: We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS: We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS: All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS: Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

The gene expression program underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of gene expression and chromatin accessibility in fetal tissues. For gene expression, we applied three-level combinatorial indexing to >110 samples representing 15 organs, ultimately profiling ~4 million single cells. We leveraged the literature and other atlases to identify and annotate hundreds of cell types and subtypes, both within and across tissues. Our analyses focused on organ-specific specializations of broadly distributed cell types (such as blood, endothelial, and epithelial), sites of fetal erythropoiesis (which notably included the adrenal gland), and integration with mouse developmental atlases (such as conserved specification of blood cells). These data represent a rich resource for the exploration of in vivo human gene expression in diverse tissues and cell types.

Recent studies have identified broadband phenomena in the electric potentials produced by the brain. We report the finding of power-law scaling in these signals using subdural electrocorticographic recordings from the surface of human cortex. The power spectral density (PSD) of the electric potential has the power-law form P(f ) approximately Af(-chi) from 80 to 500 Hz. This scaling index, chi = 4.0+/-0.1, is conserved across subjects, area in the cortex, and local neural activity levels. The shape of the PSD does not change with increases in local cortical activity, but the amplitude, A, increases. We observe a "knee" in the spectra at f(0) approximately 75 Hz, implying the existence of a characteristic time scale tau = (2pif(0))(-1) approximately 2 - 4ms. Below f(0), we explore two-power-law forms of the PSD, and demonstrate that there are activity-related fluctuations in the amplitude of a power-law process lying beneath the alpha/beta rhythms. Finally, we illustrate through simulation how, small-scale, simplified neuronal models could lead to these power-law observations. This suggests a new paradigm of non-oscillatory "asynchronous," scale-free, changes in cortical potentials, corresponding to changes in mean population-averaged firing rate, to complement the prevalent "synchronous" rhythm-based paradigm.

OBJECTIVE: The purpose of this study was to examine the performance characteristics and validity of the Patient Health Questionnaire-9 Item (PHQ-9) as a screening tool for depression among adolescents. METHODS: The PHQ-9 was completed by 442 youth (aged 13-17 years) who were enrolled in a large health care-delivery system and participated in a study on depression outcomes. Criterion validity and performance characteristics were assessed against an independent structured mental health interview (the Child Diagnostic Interview Schedule [DISC-IV]). Construct validity was tested by examining associations between the PHQ-9 and a self-report measure of functional impairment, as well as parental reports of child psychosocial impairment and internalizing symptoms. RESULTS: A PHQ-9 score of 11 or more had a sensitivity of 89.5% and a specificity of 77.5% for detecting youth who met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for major depression on the DISC-IV. Receiver-operator-curve analysis revealed that the PHQ-9 had an area under the curve of 0.88 (95% confidence interval: 0.82-0.94), and the cut point of 11 was optimal for maximizing sensitivity without loss of specificity. Increasing PHQ-9 scores were significantly correlated with increasing levels of functional impairment, as well as parental report of internalizing symptoms and psychosocial problems. CONCLUSIONS: Although the optimal cut point is higher among adolescents, the sensitivity and specificity of the PHQ-9 are similar to those of adult populations. The brief nature and ease of scoring of this instrument make this tool an excellent choice for providers and researchers seeking to implement depression screening in primary care settings.

Beginning in 1970, a committee was constituted under the auspices of the World Health Organization (WHO) to catalog primary immunodeficiencies. Twenty years later, the International Union of Immunological Societies (IUIS) took the remit of this committee. The current report details the categorization and listing of 354 (as of February 2017) inborn errors of immunity. The growth and increasing complexity of the field have been impressive, encompassing an increasing variety of conditions, and the classification described here will serve as a critical reference for immunologists and researchers worldwide.

Administration of lymphodepletion chemotherapy followed by CD19-specific chimeric antigen receptor (CAR)-modified T cells is a remarkably effective approach to treating patients with relapsed and refractory CD19(+) B-cell malignancies. We treated 7 patients with B-cell acute lymphoblastic leukemia (B-ALL) harboring rearrangement of the mixed lineage leukemia (MLL) gene with CD19 CAR-T cells. All patients achieved complete remission (CR) in the bone marrow by flow cytometry after CD19 CAR-T-cell therapy; however, within 1 month of CAR-T-cell infusion, 2 of the patients developed acute myeloid leukemia (AML) that was clonally related to their B-ALL, a novel mechanism of CD19-negative immune escape. These reports have implications for the management of patients with relapsed and refractory MLL-B-ALL who receive CD19 CAR-T-cell therapy.

We report the updated classification of primary immunodeficiencies (PIDs) compiled by the Expert Committee of the International Union of Immunological Societies. In comparison to the previous version, more than 30 new gene defects are reported in this updated version. In addition, we have added a table of acquired defects that are phenocopies of PIDs. For each disorder, the key clinical and laboratory features are provided. This classification is the most up-to-date catalog of all known PIDs and acts as a current reference of the knowledge of these conditions and is an important aid for the molecular diagnosis of patients with these rare diseases.

BACKGROUND: This is an update of the original Cochrane review first published in Issue 1, 2003, and previously updated in 2009 and 2012. Chronic pain affects many children, who report severe pain, disability, and distressed mood. Psychological therapies are emerging as effective interventions to treat children with chronic or recurrent pain. This update focuses specifically on psychological therapies delivered face-to-face, adds new randomised controlled trials (RCTs), and additional data from previously included trials. OBJECTIVES: There were three objectives to this review. First, to determine the effectiveness on clinical outcomes of pain severity, disability, depression, and anxiety of psychological therapy delivered face-to-face for chronic and recurrent pain in children and adolescents compared with active treatment, waiting-list, or standard medical care. Second, to evaluate the impact of psychological therapies on depression and anxiety, which were previously combined as 'mood'. Third, we assessed the risk of bias of the included studies and the quality of outcomes using the GRADE criteria. SEARCH METHODS: Searches were undertaken of CENTRAL, MEDLINE, EMBASE, and PsycINFO. We searched for further RCTs in the references of all identified studies, meta-analyses, and reviews. Trial registry databases were also searched. The date of most recent search was January 2014. SELECTION CRITERIA: RCTs with at least 10 participants in each arm post-treatment comparing psychological therapies with active treatment, standard medical care, or waiting-list control for children or adolescents with episodic, recurrent or persistent pain were eligible for inclusion. Only trials conducted in person (face-to-face) were considered. Studies that delivered treatment remotely were excluded from this update. DATA COLLECTION AND ANALYSIS: All included studies were analysed and the quality of outcomes were assessed. All treatments were combined into one class, psychological treatments. Pain conditions were split into headache and non-headache. Both conditions were assessed on four outcomes: pain, disability, depression, and anxiety. Data were extracted at two time points; post-treatment (immediately or the earliest data available following end of treatment) and at follow-up (between three and 12 months post-treatment). MAIN RESULTS: Seven papers were identified in the updated search. Of these papers, five presented new trials and two presented follow-up data for previously included trials. Five studies that were previously included in this review were excluded as therapy was delivered remotely. The review thus included a total of 37 studies. The total number of participants completing treatments was 2111. Twenty studies addressed treatments for headache (including migraine); nine for abdominal pain; two for mixed pain conditions including headache pain, two for fibromyalgia, two for recurrent abdominal pain or irritable bowel syndrome, and two for pain associated with sickle cell disease.Analyses revealed psychological therapies to be beneficial for children with chronic pain on seven outcomes. For headache pain, psychological therapies reduced pain post-treatment and at follow-up respectively (risk ratio (RR) 2.47, 95% confidence interval (CI) 1.97 to 3.09, z = 7.87, p < 0.01, number needed to treat to benefit (NNTB) = 2.94; RR 2.89, 95% CI 1.03 to 8.07, z = 2.02, p < 0.05, NNTB = 3.67). Psychological therapies also had a small beneficial effect at reducing disability in headache conditions post-treatment and at follow-up respectively (standardised mean difference (SMD) -0.49, 95% CI -0.74 to -0.24, z = 3.90, p < 0.01; SMD -0.46, 95% CI -0.78 to -0.13, z = 2.72, p < 0.01). No beneficial effect was found on depression post-treatment (SMD -0.18, 95% CI -0.49 to 0.14, z = 1.11, p > 0.05). At follow-up, only one study was eligible, therefore no analysis was possible and no conclusions can be drawn. Analyses revealed a small beneficial effect for anxiety post-treatment (SMD -0.33, 95% CI -0.61 to -0.04, z = 2.25, p < 0.05). However, this was not maintained at follow-up (SMD -0.28, 95% CI -1.00 to 0.45; z = 0.75, p > 0.05).Analyses revealed two beneficial effects of psychological treatment for children with non-headache pain. Pain was found to improve post-treatment (SMD -0.57, 95% CI -0.86 to -0.27, z = 3.74, p < 0.01), but not at follow-up (SMD -0.11, 95% CI -0.41 to 0.19, z = 0.73, p > 0.05). Psychological therapies also had a beneficial effect for disability post-treatment (SMD -0.45, 95% CI -0.71 to -0.19, z = 3.40, p < 0.01), but this was not maintained at follow-up (SMD -0.35, 95% CI -0.71 to 0.02, z = 1.87, p > 0.05). No effect was found for depression or anxiety post-treatment (SMD -0.07, 95% CI -0.30 to 0.17, z = 0.54, p > 0.05; SMD -0.15, 95% CI -0.36 to 0.07, z = 1.33, p > 0.05) or at follow-up (SMD 0.06, 95% CI -0.16 to 0.28, z = 0.53, p > 0.05; SMD 0.05, 95% CI -0.24 to 0.33, z = 0.32, p > 0.05). AUTHORS' CONCLUSIONS: Psychological treatments delivered face-to-face are effective in reducing pain intensity and disability for children and adolescents (<18 years) with headache, and therapeutic gains appear to be maintained, although this should be treated with caution for the disability outcome as only two studies could be included in the follow-up analysis. Psychological therapies are also beneficial at reducing anxiety post-treatment for headache. For non-headache conditions, psychological treatments were found to be beneficial for pain and disability post-treatment but these effects were not maintained at follow-up. There is limited evidence available to estimate the effects of psychological therapies on depression and anxiety for children and adolescents with headache and non-headache pain. The conclusions of this update replicate and add to those of the previous review which found that psychological therapies were effective in reducing pain intensity for children with headache and non-headache pain conditions, and these effects were maintained at follow-up for children with headache conditions.

Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.

Since 2013, the International Union of Immunological Societies (IUIS) expert committee (EC) on Inborn Errors of Immunity (IEI) has published an updated phenotypic classification of IEI, which accompanies and complements their genotypic classification into ten tables. This phenotypic classification is user-friendly and serves as a resource for clinicians at the bedside. There are now 430 single-gene IEI underlying phenotypes as diverse as infection, malignancy, allergy, autoimmunity, and autoinflammation. We herein report the 2019 phenotypic classification, including the 65 new conditions. The diagnostic algorithms are based on clinical and laboratory phenotypes for each of the ten broad categories of IEI.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.