The prototypical interferonopathy: Aicardi-Goutières syndrome from bedside to bench.

Aicardi-Goutières syndrome (AGS) is a progressive genetic encephalopathy caused by pathogenic mutations in genes controlling cellular anti-viral responses and nucleic acid metabolism. The mutations initiate autoinflammatory processes in the brain and systemically that are triggered by chronic overproduction of type I interferon (IFN), including IFN-alpha. Emerging disease-directed therapies aim to dampen autoinflammation and block cellular responses to IFN production, creating an urgent and unmet need to understand better which cells, compartments, and mechanisms underlying disease pathogenesis. In this review, we highlight existing pre-clinical models of AGS and our current understanding of how causative genetic mutations promote disease in AGS, to promote new model development and a continued focus on improving and directing future therapies.

Rocky Mountain Spotted Fever in Children along the US‒Mexico Border, 2017-2023.

Rocky mountain spotted fever (RMSF) causes significant illness and death in children. Although historically rare in California, USA, RMSF is endemic in areas of northern Mexico that border California. We describe 7 children with RMSF who were hospitalized at a tertiary pediatric referral center in California during 2017-2023. Five children had recent travel to Mexico with presumptive exposure, but 2 children did not report any travel outside of California. In all 7 patients, Rickettsia rickettsii DNA was detected by plasma microbial cell-free next-generation sequencing, which may be a useful diagnostic modality for RMSF, especially early in the course of illness, when standard diagnostic tests for RMSF are of limited sensitivity. A high index of suspicion and awareness of local epidemiologic trends remain most critical to recognizing the clinical syndrome of RMSF and initiating appropriate antimicrobial therapy in a timely fashion.

Proinflammatory immune cells disrupt angiogenesis and promote germinal matrix hemorrhage in prenatal human brain.

Germinal matrix hemorrhage (GMH) is a devastating neurodevelopmental condition affecting preterm infants, but why blood vessels in this brain region are vulnerable to rupture remains unknown. Here we show that microglia in prenatal mouse and human brain interact with nascent vasculature in an age-dependent manner and that ablation of these cells in mice reduces angiogenesis in the ganglionic eminences, which correspond to the human germinal matrix. Consistent with these findings, single-cell transcriptomics and flow cytometry show that distinct subsets of CD45+ cells from control preterm infants employ diverse signaling mechanisms to promote vascular network formation. In contrast, CD45+ cells from infants with GMH harbor activated neutrophils and monocytes that produce proinflammatory factors, including azurocidin 1, elastase and CXCL16, to disrupt vascular integrity and cause hemorrhage in ganglionic eminences. These results underscore the brain's innate immune cells in region-specific angiogenesis and how aberrant activation of these immune cells promotes GMH in preterm infants.

Pathogen kinetics and detection by next-generation sequencing in pediatric complicated pneumonia.

Pediatric pneumonia can be severe and result in empyema. Next-generation sequencing (NGS) may broadly detect pathogens though, optimal timing and impact of sample type on diagnostic yield is unknown. This is a prospective, single-center pilot study of children aged 3 months through 17 years admitted to the PICU with a primary diagnosis of complicated pneumonia. Plasma, endotracheal, nasopharyngeal, and pleural fluid samples were collected at three time points during hospitalization. After nucleic acid extraction, combined libraries were enriched with an NGS enrichment panel kit (RPIP, Illumina), sequenced and quantitative organism detections were analyzed. NGS identified the same bacterial pathogen as traditional testing in all samples, regardless of antibiotic pre-treatment or time collected. Conventional culture methods only identified the pathogen reliably in invasively obtained pleural fluid or endotracheal aspirates. Future application of NGS may allow for non-invasive pathogen detection at a broader range of time points and more targeted antibiotic coverage.

The inactive X chromosome drives sex differences in microglial inflammatory activity in human glioblastoma.

Biological sex is an important risk factor in cancer, but the underlying cell types and mechanisms remain obscure. Since tumor development is regulated by the immune system, we hypothesize that sex-biased immune interactions underpin sex differences in cancer. The male-biased glioblastoma multiforme (GBM) is an aggressive and treatment-refractory tumor in urgent need of more innovative approaches, such as considering sex differences, to improve outcomes. GBM arises in the specialized brain immune environment dominated by microglia, so we explored sex differences in this immune cell type. We isolated adult human TAM-MGs (tumor-associated macrophages enriched for microglia) and control microglia and found sex-biased inflammatory signatures in GBM and lower-grade tumors associated with pro-tumorigenic activity in males and anti-tumorigenic activity in females. We demonstrated that genes expressed or modulated by the inactive X chromosome facilitate this bias. Together, our results implicate TAM-MGs, specifically their sex chromosomes, as drivers of male bias in GBM.

Novel variants in TECRL leading to catecholaminergic polymorphic ventricular tachycardia.

Pathogenic and likely pathogenic variants in the TECRL gene are known to be associated with recessive catecholaminergic polymorphic ventricular tachycardia 3, which can include prolonged QT intervals (MIM#614021). We report a case of cardiac arrest in a previously healthy adolescent male in the community. The patient was found to have a novel maternally inherited likely pathogenic variant in TECRL (c.915T>G [p.Tyr305Ter]) and an additional 19-kb duplication encompassing multiple exons of TECRL (chr4:65165944-65185287, dup [4q13.1]) not identified in the mother. Genetic results were revealed via rapid whole-genome sequencing, which allowed appropriate treatment and prognostication.

Microglial lipid droplet accumulation in tauopathy brain is regulated by neuronal AMPK.

The accumulation of lipid droplets (LDs) in aging and Alzheimer's disease brains is considered a pathological phenomenon with unresolved cellular and molecular mechanisms. Utilizing stimulated Raman scattering (SRS) microscopy, we observed significant in situ LD accumulation in microglia of tauopathy mouse brains. SRS imaging, combined with deuterium oxide (D2O) labeling, revealed heightened lipogenesis and impaired lipid turnover within LDs in tauopathy fly brains and human neurons derived from induced pluripotent stem cells (iPSCs). Transfer of unsaturated lipids from tauopathy iPSC neurons to microglia induced LD accumulation, oxidative stress, inflammation, and impaired phagocytosis. Neuronal AMP-activated protein kinase (AMPK) inhibits lipogenesis and promotes lipophagy in neurons, thereby reducing lipid flux to microglia. AMPK depletion in prodromal tauopathy mice increased LD accumulation, exacerbated pro-inflammatory microgliosis, and promoted neuropathology. Our findings provide direct evidence of native, aberrant LD accumulation in tauopathy brains and underscore the critical role of AMPK in regulating brain lipid homeostasis.

Rapid Whole-Genome Sequencing and Clinical Management in the PICU: A Multicenter Cohort, 2016-2023.

OBJECTIVES: Analysis of the clinical utility of rapid whole-genome sequencing (rWGS) outside of the neonatal period is lacking. We describe the use of rWGS in PICU and cardiovascular ICU (CICU) patients across four institutions. DESIGN: Ambidirectional multisite cohort study. SETTING: Four tertiary children's hospitals. PATIENTS: Children 0-18 years old in the PICU or CICU who underwent rWGS analysis, from May 2016 to June 2023. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 133 patients underwent clinical, phenotype-driven rWGS analysis, 36 prospectively. A molecular diagnosis was identified in 79 patients (59%). Median (interquartile range [IQR]) age was 6 months (IQR 1.2 mo-4.6 yr). Median time for return of preliminary results was 3 days (IQR 2-4). In 79 patients with a molecular diagnosis, there was a change in ICU management in 19 patients (24%); and some change in clinical management in 63 patients (80%). Nondiagnosis changed management in 5 of 54 patients (9%). The clinical specialty ordering rWGS did not affect diagnostic rate. Factors associated with greater odds ratio (OR [95% CI]; OR [95% CI]) of diagnosis included dysmorphic features (OR 10.9 [95% CI, 1.8-105]) and congenital heart disease (OR 4.2 [95% CI, 1.3-16.8]). Variables associated with greater odds of changes in management included obtaining a genetic diagnosis (OR 16.6 [95% CI, 5.5-62]) and a shorter time to genetic result (OR 0.8 [95% CI, 0.76-0.9]). Surveys of pediatric intensivists indicated that rWGS-enhanced clinical prognostication ( p < 0.0001) and contributed to a decision to consult palliative care ( p < 0.02). CONCLUSIONS: In this 2016-2023 multiple-PICU/CICU cohort, we have shown that timely genetic diagnosis is feasible across institutions. Application of rWGS had a 59% (95% CI, 51-67%) rate of diagnostic yield and was associated with changes in critical care management and long-term patient management.

Emerging Role of Plasma Microbial Cell-free DNA in the Diagnosis of Pediatric Mucormycosis.

Mucormycosis is a rare and devastating angioinvasive infection that can be challenging to diagnose due to the low sensitivity of current noninvasive diagnostics and the lack of a "gold standard" reference test. We describe a retrospective case series of children with suspected mucormycosis where plasma microbial cell-free DNA testing was utilized in the diagnostic evaluation to illustrate the ways in which microbial cell-free DNA testing can noninvasively contribute to the evaluation and management of at-risk, immunosuppressed patients suspected of mucormycosis.

Application of cell-free plasma next-generation sequencing technology in the diagnosis and management of pediatric meningitis.

This retrospective study evaluates the clinical utility of CFPNGS in the diagnosis and management of pediatric meningitis. CFPNGS identified a causative pathogen in 36% of 28 subjects, compared to 50% for diverse conventional testing (57% combined). CFPNGS may be considered as an adjunct to standard testing.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.

Gaps in diagnosing suspected infection in immunocompromised children with cancer: A systematic review.

While the survival of children with cancer has improved over time, infection remains a major morbidity and mortality risk. We conducted a systematic literature review to determine the unmet needs in diagnosing infection in immunocompromised children with cancer. The comprehensive search strategy followed the guidelines established by the Cochrane Handbook for Systematic Reviews of Interventions and the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 statement, and spanned multiple bibliographic databases and other public sources from January 1, 2012 to June 23, 2022. From 5188 records, 34 unique pediatric-focused studies met inclusion criteria. This review highlights the lack of published data on infectious disease testing in pediatric oncology patients, and the need for well-designed clinical impact and cost-effectiveness studies of both existing and novel diagnostic platforms. Such studies are necessary to optimize diagnostic and antimicrobial stewardship, leading to improvement in patient outcomes.

A maelstrom of migrating monocytes drives neurodegeneration.

Neurodegeneration is a devastating complication of Langerhans cell histiocytosis (LCH), but it is not clear how it develops. In this issue of Immunity, Wilk et al. demonstrate that circulating BRAFV600E+ myeloid cells damage the blood-brain barrier and infiltrate the brain. Dual inhibition of the MAPK and senescence pathways can block parenchymal injury, providing a potential therapeutic avenue for histiocytic neurodegeneration.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

Nicole G. Coufal.

Physician-scientist Nicole Coufal cares for critically ill children in the pediatric ICU and studies microglial contributions to neurodevelopmental and neurodegenerative disease. She discusses with Neuron balancing basic and clinical research and how chance moments-from reading Oliver Sacks to a rotation in the burn unit-influenced her career and joy for mentorship.

Human microglia maturation is underpinned by specific gene regulatory networks.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes.

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity.

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFß and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFß-SMAD signaling axis.

3D genome mapping identifies subgroup-specific chromosome conformations and tumor-dependency genes in ependymoma.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

Human microglial models to study host-virus interactions.

Microglia, the resident macrophage of the central nervous system, are increasingly recognized as contributing to diverse aspects of human development, health, and disease. In recent years, numerous studies in both mouse and human models have identified microglia as a "double edged sword" in the progression of neurotropic viral infections: protecting against viral replication and cell death in some contexts, while acting as viral reservoirs and promoting excess cellular stress and cytotoxicity in others. It is imperative to understand the diversity of human microglial responses in order to therapeutically modulate them; however, modeling human microglia has been historically challenging due to significant interspecies differences in innate immunity and rapid transformation upon in vitro culture. In this review, we discuss the contribution of microglia to the neuropathogenesis of key neurotropic viral infections: human immunodeficiency virus 1 (HIV-1), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Herpes simplex virus (HSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We pay special attention to recent work with human stem cell-derived microglia and propose strategies to leverage these powerful models to further uncover species- and disease-specific microglial responses and novel therapeutic interventions for neurotropic viral infections.

Genomic sequencing has a high diagnostic yield in children with congenital anomalies of the heart and urinary system.

Background: Congenital heart defects (CHD) and congenital anomalies of the kidney and urinary tract (CAKUT) account for significant morbidity and mortality in childhood. Dozens of monogenic causes of anomalies in each organ system have been identified. However, even though 30% of CHD patients also have a CAKUT and both organs arise from the lateral mesoderm, there is sparse overlap of the genes implicated in the congenital anomalies for these organ systems. We sought to determine whether patients with both CAKUT and CHD have a monogenic etiology, with the long-term goal of guiding future diagnostic work up and improving outcomes. Methods: Retrospective review of electronic medical records (EMR), identifying patients admitted to Rady Children's Hospital between January 2015 and July 2020 with both CAKUT and CHD who underwent either whole exome sequencing (WES) or whole genome sequencing (WGS). Data collected included demographics, presenting phenotype, genetic results, and mother's pregnancy history. WGS data was reanalyzed with a specific focus on the CAKUT and CHD phenotype. Genetic results were reviewed to identify causative, candidate, and novel genes for the CAKUT and CHD phenotype. Associated additional structural malformations were identified and categorized. Results: Thirty-two patients were identified. Eight patients had causative variants for the CAKUT/CHD phenotype, three patients had candidate variants, and three patients had potential novel variants. Five patients had variants in genes not associated with the CAKUT/CHD phenotype, and 13 patients had no variant identified. Of these, eight patients were identified as having possible alternative causes for their CHD/CAKUT phenotype. Eighty-eight percent of all CAKUT/CHD patients had at least one additional organ system with a structural malformation. Conclusions: Overall, our study demonstrated a high rate of monogenic etiologies in hospitalized patients with both CHD and CAKUT, with a diagnostic rate of 44%. Thus, physicians should have a high suspicion for genetic disease in this population. Together, these data provide valuable information on how to approach acutely ill patients with CAKUT and CHD, including guiding diagnostic work up for associated phenotypes, as well as novel insights into the genetics of CAKUT and CHD overlap syndromes in hospitalized children.