Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2014-06

PROJECT SUMMARY/ABSTRACT Hypoxic ischemic encephalopathy (HIE), a major cause of perinatal mortality and long-term morbidity, affects 1- 5/1000 live births. Hypoxic ischemic insults (HII) lead to HIE through a cascade of neuronal injury that continues for hours to days. Therapeutic hypothermia (TH) proved that brain injury from HII can be avoided, revolutionizing treatment of HIE. Despite TH success, additional improvements are urgently needed as up to 63% of infants still die or have long term cognitive deficits. Also, guidelines for treatment are highly subjective, with no consensus on when to treat mild HIE. Our first premise is that adverse outcomes are due to ongoing neuronal injury after HII. As neurons are the primary consumer of oxygen, measures of cerebral oxygen metabolism (CMRO2) would provide a potential means to monitor neuronal health and the evolution of injury. Our second premise is that hemodynamic instability contributes to adverse outcomes through secondary neuronal injury. Secondary injury may be preventable with bedside measures of cerebral blood flow (CBF) to ensure the brain’s metabolic needs are met. In fact, direct CBF measures enable assessment of cerebral autoregulation (CA) and neurovascular coupling (NVC), which reflect hemodynamic stability. Bedside measures of CMRO2 and CBF could enable treatment optimization to prevent secondary injury and inform decisions on who to treat. In R01HD076258, our major achievement was performing >500 Frequency-Domain NIRS and Diffuse Correlation Spectroscopy (FDNIRS- DCS) measurements in >100 neonates with HIE, demonstrating that routine direct bedside measurement of CBF and CMRO2 are possible. Our major finding is that CMRO2 in the days after TH was the only early predictor of outcome, with higher CMRO2 strongly associated (r=0.62, P=0.002) with better 18-month cognitive scores of Bayley Scales of Infant Development 3rd edition (BSID-3). Our renewal goal is to perform early, continuous bedside monitoring of CMRO2 and CBF, demonstrating their potential as vital signs with three aims: Aim 1: Extend current infrastructure to enable continuous, real-time bedside monitoring of neonatal CBF and CMRO2 at 100Hz, integrated with data from other clinical monitors. Aim 2: In neonates with HIE, determine evolution of CMRO2, CA and NVC during TH and their association with post-TH CMRO2; validate that post-TH CMRO2 predicts 2-year outcome. Aim 3: In controls and neonates at risk for HIE but not meeting criteria for TH, determine CMRO2, CA and NVC during the first 24 hours; determine if day 1 CMRO2 predicts 2-year outcome. In the first grant, we demonstrated that bedside measures of CBF and CMRO2 are feasible and discovered that CMRO2 after TH predicts of outcome. In this renewal, we propose to turn CMRO2 and CBF into early, continuous, real-time, bedside vital signs. Our goal is to determine if there is evidence of neuronal injury (decreased CMRO2) mediated by cerebral hemodynamic instability (impaired CA and NVC). If successful, CMRO2 and CBF monitoring may improve neonatal outcomes by enabling medical optimization of hemodynamic stability during TH as well as to screen for neonates at risk for HIE who may benefit from TH.

NIH Research Projects · FY 2025 · 2013-07

PROJECT SUMMARY Mutations in transmembrane channel-like gene 1 (TMC1) underlie dominant, progressive hearing loss (DFNA36) and recessive nonsyndromic hearing loss (DFNB7/B11) in humans (Kurima et al., 2002). Similarly, semidominant and recessive alleles of Tmc1 cause hearing loss in Beethoven (Bth) and deafness (dn) mutant mice (Vreugde et al.,2002; Kurima et al., 2002). Tmc1 is a member of the Tmc gene family that includes seven other paralogs in mammals (Keresztes et al., 2003). Tmc1 and closely related Tmc2 are expressed in auditory and vestibular hair cells of the mouse inner ear and are necessary for mechanosensory transduction. In prior work, funded by this RO1, we demonstrated that TMC1 is a pore-forming subunit of the hair cell transduction channel and contains four transmembrane domains (S4-S7) that line the ion channel pore (Pan et al., 2018). With compelling evidence in hand demonstrating that TMC1 is a major component of the channel, we can now use this information to tackle both basic science and translational research questions that were previously intractable. This project is organized around five independent, but related specific aims focused on understanding the structure and function of TMC1 in isolated systems and in native hair cells. In addition, we will explore novel techniques for restoration of auditory function in mouse models of the human genetic inner ear disorders DFNA36 and DFNB7/11. We have optimized protocols for expression and purification of high-quality mammalian TMC1 protein. For aims 1 and 2, we will use purified protein to generate the cryo-EM structures of mammalian TMC1 and will examine the function of isolated TMC1 channels in heterologous systems. For aim 3, we will characterize the N- and C- termini of TMC1 and TMC2 and identify motifs of these regions that confer unique properties to these proteins. We will express TMC1 and TMC2 proteins bearing N-terminal mutations in hair cells of Tmc1/Tmc2 double mutant mice and assay for changes in the properties of hair cell sensory transduction. Likewise, for aim 4, we will examine the interaction between TMC1 and its binding partner TMIE using Tmie-null mice. We will express WT or mutant TMIE constructs in hair cells of Tmie-null mice and assay for functional differences in hair cell transduction. For aim 5 we will explore a novel therapeutic approach for targeting all dominant and recessive Tmc1 mutations in auditory and vestibular hair cells using a single therapeutic strategy. Based on new information about the structure and function of TMC1, projects included in this proposal will allow us to expand our understanding of sensory transduction in auditory hair cells and develop a cutting-edge translational approach suitable for treating both dominant and recessive TMC1 mutations that cause genetic hearing loss in humans.

NIH Research Projects · FY 2025 · 2013-03

Project Summary Botulinum neurotoxins (BoNTs) are a family of bacterial protein toxins. They are one of the six most dangerous potential bioterrorism agents. Members of BoNTs, BoNT/A (Botox) and BoNT/B, are also FDA-approved for treating a growing list of disorders, as well as for reducing wrinkles. Understanding the molecular mechanisms for this family of toxins, engineering toxins to improve their therapeutic uses, and developing effective countermeasures have been the focus of our lab. Our prior funding cycle has led to major discoveries in understanding toxin receptors, identification of novel BoNT-like toxins, and development of new treatment that can neutralize BoNTs inside motor neurons. In this renewal proposal, we will address both side of BoNTs through protein engineering approaches: Aim 1 seeks to develop a novel chimeric toxin that can preferentially bind to the toxin receptor isoform dominantly expressed in autonomic and sensory neurons and evaluate its improvement over the standard Botox for treating overactive bladder conditions in rodent models. Aim 2 seeks to develop and evaluate novel nanobody-based triple-epitopic antibodies (NTAb) for neutralizing and clearing BoNTs in the circulatory system. NTAb is constructed by fusion of three nanobodies targeting distinct regions on BoNTs and then to a Fc, thus generating an artificial antibody that can achieve binding of three NTAb to a single toxin molecule, which promotes toxin clearance. In this proposal, we will develop two sets of NTAb, targeting two most common toxins, BoNT/A and BoNT/B, respectively, and thoroughly evaluate their efficacy, pharmacokinetics, and toxin clearance using rodent and guinea pig models.

NIH Research Projects · FY 2024 · 2011-09

The leading cause of death in diabetic patients is cardiovascular disease (CVD). Our long-term goal is to identify new therapeutic targets for the prevention of CVD in diabetic patients. In the first grant cycle, we identified the enzyme flavin-containing monooxygenase 3 (FMO3) as a potential mediator of diabetes-associated cardiovascular disease via a non-biased transcriptomics approach. In the second grant cycle, we found that FMO3 exerted many of its effects via the key metabolite, trimethylamine N-oxide (TMAO); we further found the endoplasmic reticulum stress kinase, PERK, to be a receptor for TMAO. Multiple clinical studies have now shown that TMAO is increased with insulin resistance, as well as atherosclerosis, confirming that this pathway is dysregulated in humans, and suggesting that inhibition of the FMO3/TMAO pathway may have beneficial effects. TMAO is synthesized from the metabolite trimethylamine (TMA), which is in turn produced by the gut microbes. Therefore, an attractive strategy would be to inhibit the production of TMA by the gut microbes. In our unpublished, preliminary data, we screened a drug repurposing library. The advantage of using repurposed drugs is that they are already known to be safe in humans, reducing the time and expense needed to bring them into the clinic. We identified a compound that inhibits the microbial enzyme that generates TMA and can lower TMAO levels in mice. The goals of the current cycle are to fill the key remaining gaps in our mechanistic understanding of the TMAO pathway, and to test the therapeutic potential of lowering TMAO. We hypothesize that TMAO, which is increased with diabetes, induces PERK to promote dyslipidemia, inflammation and diabetes-associated atherosclerosis. Our aims are to elucidate the mechanisms by which PERK promotes metabolic dysfunction; to determine the extent to which hepatic deletion of PERK can prevent TMAO-induced dyslipidemia, inflammation and atherosclerosis; and to test whether the novel compound identified in our drug repurposing screen can prevent diabetes-induced atherosclerosis in mice. We expect that these studies will lead to a novel, orthogonal approach to reducing CVD risk in patients with diabetes.

NIH Research Projects · FY 2026 · 2011-08

Abstract - Overall Influenza virus evolves in response to pressure from herd immunity, resulting in progressive variation of viral antigenicity and limiting vaccine efficacy. The goal of this Program project is to establish a secure mechanistic foundation for novel vaccination strategies that might confer long-lasting immunity in face of rapid antigenic variation of currently circulating subtypes and in anticipation of potential introduction of zoonotic subtypes into the human population. The breadth of expertise in our four collaborative Projects and three scientific Cores and Results from the current and previous grant cycles allow us to pursue the following Specific Aims. (1) Can a suitable vaccination strategy modify or redirect immune "imprinting" -- the observed influence of an individual's history of infection of vaccination on their response to an antigenically drifted strain or novel subtype? We will determine the full scope of the human humoral response to vaccination in individuals of all ages (infants to seniors) and hence of varying exposures, and we will carry out, in non-human primates (NHPs), experiments to compare imprinting by infection and vaccination. (2) We have found (in mice) a strong effect of the anatomical location of immunization with protein antigen on the response to a subsequent immunization. We will determine whether this effect extends to infection and whether it is also true in NHPs. Experiments in mice will determine whether components of TFH cell memory are similarly localized and test the contribution of conserved T-cell epitopes to humoral imprinting. (3) We will design novel vaccine antigens to extend previous studies of human immune responses (focused on hemagglutinin) to influenza neuraminidase (NA) and to determine the importance of T-cell epitope diversity for design of optimized immunogens. We will also create a model for co-evolution of virus (immune escape) and humoral immunity (antibody affinity maturation) using directed molecular evolution in the laboratory.

NIH Research Projects · FY 2024 · 2010-07

Project Summary This competing continuation proposal will provide an opportunity for selected residents in the neurology residency training programs of the Beth Israel Deaconess Medical Center (BIDMC) and Boston Children's Hospital (BCH) to participate for 6 to 36 months in an intensive, mentored, research educational experience during the final year of residency and subsequent fellowship years. This training will be designed to prepare participating residents for successful competition for NIH funded independent mentored research awards, and will facilitate the transition from resident/fellow to clinician-scientist. Each participant will work with one of 57 mentors, who have been recruited from the faculties of BCH, BIDMC, and Harvard Medical School (HMS). All have active NIH funding and a history of training clinician-scientists. The proposed mentors cover all major areas of the clinical and basic neurosciences, and include 30 investigators from BCH, 15 investigators from BIDMC, seven investigators from HMS and five investigators from other Harvard and Harvard Hospital affiliates. Thirty of these investigators are engaged in clinical/translational neuroscience research and forty-two are engaged in basic neuroscience research. Mentors have been drawn not only from the Departments of Neurology/Neurobiology, but also from Divisions/Departments of Anaesthesia, Cell Biology, Genetics, Neurosurgery, Ophthalmology, Pathology, Radiology, Developmental Medicine, and Psychiatry. Selected resident participants will learn state-of-the-art laboratory skills and will acquire the critical expertise necessary for the conduct of responsible research. Data collected and analyzed will serve for publications as well as for future NIH proposals. The program will be governed by a Steering Committee consisting of the PD/PIs, the Department Chairs, and the Residency Directors of the participating residency programs. This Committee will work together to recruit and select trainees, to monitor their progress, and to evaluate the effectiveness of the training experience.

NIH Research Projects · FY 2025 · 2010-02

Novel Biologic Therapies for GVHD Abstract: For patients with high-risk malignant and non-malignant hematologic diseases, hematopoietic stem cell transplant (HCT) often represents the only option for cure. However, HCT is fraught with complications, leading to high rates of toxicity and patient death. The principal cause of early non-relapse mortality is acute graft-versus-host disease (AGVHD), with death from infection a close second. These two are interrelated, as intensifying global immune suppression to control AGVHD increases infection risk. Moreover, augmented immunosuppression can also increase the risk of malignant relapse. To move the field forward, we must develop targeted, evidence-based prevention and treatment strategies, designed to specifically control alloreactivity while preserving anti-tumor and anti-viral protective immunity. Perhaps the greatest need for these targeted therapeutics is in GI AGVHD, the leading cause of AGVHD- related death. Two issues predominate, which significantly impede progress: (1) We do not adequately understand the distinct mechanisms controlling GI T cell infiltration versus tissue residency or damage, which inhibits development of specific therapies. (2) We do not understand the mechanisms driving steroid-refractory GI AGVHD, significantly slowing treatment development for this most deadly type of AGVHD. The goal of this proposal is to address these unmet needs, and thereby discover the next generation of therapeutics for GI AGVHD. We will do so by completing the following three Specific Aims: (1): Prioritize and validate next-generation GI-targeted therapeutics with a novel translational pipeline. We will test the hypothesis that a pipeline from patient/NHP-derived therapeutic candidates → mouse → NHP AGVHD models can prioritize new AGVHD therapeutics. (2) Determine the impact of Notch ligand blockade on the blood and GI immune landscape in NHP and transplant patients. We will test the hypothesis that Notch ligand blockade with the anti-DLL4 mAb REGN421 protects against GI AGVHD, and that it does so by inhibiting effector T cell infiltration into the GI tract. (3) Identify predictors of steroid responsive and resistant GI AGVHD in patients. We will test the hypothesis that unifying mechanisms of steroid response and resistance can be identified in patients by comprehensive linked immune studies of the blood and GI tract at AGVHD diagnosis. By completing these aims, we will discern the mechanisms driving GI AGVHD, and thus fundamentally advance our ability to care for patients undergoing HCT.

NIH Research Projects · FY 2024 · 2009-09

ABSTRACT Dramatic advances in surgical repair and cardiac intervention have improved survival in even the most complex forms of congenital heart disease (CHD). With this notable success, there has been a shift from perioperative to chronic cardiac morbidity and accelerated mortality. Right ventricular (RV) dysfunction is an important determinant of long-term outcomes in children and adults with many forms of CHD. Outcomes such as RV dysfunction and associated comorbidities are currently thought of primarily in terms of hemodynamic or physiological factors. However, routine clinical and imaging variables have explained only a small percentage of the variability in RV function and clinical outcomes in CHD patients, suggesting an important role for as-yet- unrecognized contributors. We hypothesize that multiple genetic factors contribute to the unexplained variation in RV performance and patient outcomes. To investigate the relationship of genomic factors and clinical outcomes, we will study two exemplars of CHD for which right ventricular (RV) dysfunction especially impacts outcomes: tetralogy of Fallot (TOF) and hypoplastic left heart syndrome (HLHS). Our proposed study population will leverage a unique clinical and genetic database developed by the PCGC, as well cohorts within individual PCGC centers and other consortia. In Aim 1, we will study the effects of rare damaging variants identified in patients with TOF and HLHS on RV function, clinical outcomes, and anatomical subtypes influencing outcomes. Our primary outcome will be RV ejection fraction by cardiac MRI (CMR). Secondary outcomes will include other CMR measures of systolic and diastolic function, as well as clinical outcomes such as transplant-free survival, sustained ventricular and atrial tachycardias, and heart failure defined as New York Heart Association Class III or IV. In Aim 2, we will study the effects of common variants identified in patients with TOF and HLHS on RV function, clinical outcomes, and anatomical subtypes influencing outcomes. Primary and secondary outcomes will be identical to those in Aim 1. Aim 3 will assess the effects of rare and common variants associated with outcomes on cardiomyocyte function, metabolism, gene expression, and chromatin accessibility in isogenic induced pluripotent stem cells differentiated into cardiomyocytes (iPSC-CMs) and CHD tissues. We will define outcome-associated common variants using bioinformatic and functional assays, and derive iPSC-CMs with CHD variants, alone or in addition to rare and common outcome-associated variants. We will assess contraction relaxation, energetic parameters, and transcriptional activities in iPSC-CMs. We will complement these studies with single cell nuclear sequencing (NucSeq) and ATACseq analyses of CHD tissues to explore how outcome-associated variants influence in vivo cardiomyocyte biology. By identifying genes affecting outcomes, our proposal will advance mechanistic insights, improve risk-stratification and provide resources for more precise personalized therapies for CHD patients across the lifespan.

NIH Research Projects · FY 2026 · 2009-04

Abstract Infantile hemangioma (IH) is an extraordinary example of vascular overgrowth wherein vessels form rapidly over a year, then undergo a slow spontaneous involution that leaves behind a fibrofatty residuum. IH is common: it occurs in 5% of infants, equating to ~183,200 infants/year in the U.S. alone. 10-15% of IH will cause complications – e.g., destroy facial structures and impair vision, breathing and feeding depending on the location. Propranolol was discovered serendipitously to be effective therapy for IH, yet some do not respond, regrowth occurs in ~20% of cases, and surgery is needed in 37% of patients to correct IH residua. In the current funding cycle, we showed the non-beta blocker R+ enantiomers of propranolol and atenolol prevent hemangioma endothelial differentiation by directly interfering with the activity of the transcription factor SOX18 in hemangioma stem cells (HemSC). Further we showed that R+ propranolol and R+ atenolol, along with the small molecule SOX18 inhibitor Sm4, block hemangioma vessel formation in vivo in a pre-clinical model that uses IH patient derived HemSC. Our findings elucidate a novel etiological component of IH and also validate a molecular target, which opens new research directions for discovery and drug repurposing. Using R-propranolol as a molecular probe, we uncovered by transcriptional profiling of HemSC that the most decreased biological process during HemSC endothelial differentiation is the mevalonate pathway. From this data, we propose an entirely novel SOX18-mevalonate pathway axis as a central regulatory process that underpins IH-vascular overgrowth. We suggest a new conceptual framework and clinical research directions in which R+ propranolol, R+ atenolol, statins, or tipifarnib (all mevalonate pathway blockers) have the potential to be repurposed to prevent vascular overgrowth. This molecular strategy should reduce side effects seen with racemic (traditional) propranolol therapy due to beta-adrenergic receptor blockade and provide rapid clinical benefit, especially for non-responder patients. Our goals in this renewal are to decipher how the SOX18-mevalonate pathway contributes to IH formation and investigate the gene regulatory networks that govern the vasculogenic and adipogenic transitions. In addition, we aim to determine whether the SOX18-mevalonate axis is an etiological component in other vascular anomalies (VA), which could lead to repurposing these drugs to a wide range of VA.

NIH Research Projects · FY 2026 · 2008-12

Project Summary/Abstract of Research Plans for the Extension Period The first two specific aims of this R37 grant were to test our hypothesis that cohesin-mediated loop extrusion plays a key role in lgH class switch recombination (CSR) and lg variable region exon somatic hypermutation (SHM). A third aim was to test our hypothesis that Peyer's Patch (PP) germinal centers(GCs) expand rare V(D)J clonotypes with high intrinsic SHM levels. For these studies, we developed powerful assays for CSR, SHM and chromatin interactions. We also employed our RAG2-defcient blastocyst complementation approach (RDBC) to generate ES cell-based V(D)J passenger allele and V(D)J-rearranging mouse models for in vivo studies. Despite pandemic challenges, we made substantial progress in the first 3.5 years.

NIH Research Projects · FY 2025 · 2007-07

Project summary Hair cells of the inner ear are the primary receptors of the auditory system. They transduce mechanical information, associated with sound waves, into electro-mechanical (outer hair cells) and electro-chemical (Inner hair cells) signals, which lead to amplification of the initial signal and activation of afferent neuronal fibers, respectively. While hair cells and neuronal fibers appear before birth in mice, development and maturation of the hair cells, neurons and synapses proceeds until hearing onset, ~postnatal day 12. This process is believed to be dynamic and modulated by hair cell activity. In particular, recent work has shown that lack of hair cell transmission, due to absence of functional synapses or defective mechanosensation, leads to altered neuronal maturation and specification. Successful outcomes for new therapies, including gene therapy, aimed at restoring hair cell function after birth, may depend on restoration of auditory circuits, including mature and functional hair cell synapses and neuronal fibers. Here we propose to assess how disruption or loss of sensory transduction in several mouse models affects hair cell function, synaptic maturation and spiral ganglion specification. Furthermore, we will determine if inner ear gene therapy is capable of reversing any of these observed changes and identify the conditions for optimal recovery of auditory function. We will combine state-of-the-art technologies to address these important questions, including high-resolution imaging, electrophysiology, single cell RNA sequencing and localization of RNA transcripts.

NIH Research Projects · FY 2026 · 2007-06

Human genetics reveals underlying biological mechanisms for human disease susceptibility and quantitative trait variability. Random inheritance of parental alleles provides a natural experiment to test genes for a causal role in human phenotypes. Understanding the variants, genes, and mechanisms that underlie human phenotypic variability can have important therapeutic implications – drugs whose targets are supported by human genetic evidence are more likely to be efficacious. Despite improvements in treatments for obesity (particularly newly-approved terzepatide, whose targets both have support from our prior genetic studies), obesity remains a pressing public health need, and the most effective therapies carry significant risks and cost. Our prior and ongoing genome-wide association studies (GWAS) have implicated both known and novel genes for anthropometric traits, including measures of obesity and height (the classical model polygenic trait). However, translating genetic discovery into new biology and actionable mechanistic hypotheses remains challenging. Fortunately, improved tools and new resources now enable more rapid progress on the journey from genetic discovery to biological knowledge. We can benchmark and therefore rationally select powerful computational tools and data sets that, especially in combination, more precisely implicate causal variants, genes, and pathways. Newly available and much larger studies of rare variation, especially coding variation, also help pinpoint relevant genes. Whole genome sequence data sets now allow queries of structural variants whose often large effect sizes can reveal regulatory effects on causal genes. Expanded sample sizes for non- European ancestries will increase power and permit a broader delineation of the phenotypic consequences of causal variants. Functional studies can test hypotheses emerging from genetic and computational analyses, especially if the functional assays are shown to be relevant by benchmarking them against genetic data. This proposal builds on the ongoing, successful and global collaborations we established within the GIANT consortium, which led to discovery of most of the common variants known to be associated with anthropometric traits. We will leverage new collaborations and newly available genetic resources, with larger and more diverse sample sizes. By covering the range of variants - common and rare, coding, noncoding, and structural - we will more completely define the genetic basis of measures of obesity (BMI and WHR) and height. We will use data-driven approaches to benchmark and select computational methods (to turn genetic data into hypotheses about causal variants, genes, and mechanisms) and functional assays (to test these hypotheses). Finally, we will examine, in diverse populations, the phenotypic and metabolic implications of polygenic risk scores composed of associated variants (including meaningfully selected subsets). Our proposal will also address as yet unanswered questions about the genetic architecture of polygenic traits, using novel approaches to study sex differences and genetic interactions.

NIH Research Projects · FY 2026 · 2007-03

PROJECT SUMMARY / ABSTRACT The goal of the Boston Children's Hospital K12 CHRCDA Program is to develop independent pediatrician physician-scientists who will decipher the pathobiology of childhood disease and develop transformative new therapies. Our Scholars will perform laboratory-based basic and translational research under the mentorship of outstanding scientists at Boston Children's Hospital and other affiliated institutions. Our program has been established to ensure the continued development of outstanding independent pediatrician-scientists in the field. In this proposal, we describe a program that includes intensive mentoring, a comprehensive didactic program, and appropriate supervision and support to ensure that our Scholars reach their maximum scientific potential. Programmatic oversight will be provided by an External Advisory Committee and an Internal Steering Committee. Plans are in place for programmatic self-evaluation and for review of Scholar Progress. We propose to continue the funding of three Scholar positions. Scholars will be funded for a minimum of two years, contingent on satisfactory progress on research and career development activities, and the overall pool of Scholar candidates. Scholars will be faculty members most often at the rank of Instructor, but Assistant Professors who are early in the development of their independent careers will also be eligible to apply. Scholars will be considered at any point from the beginning of their Instructorship until they are considered to be two years away from submitting their first R-level NIH grant or equivalent, though no more than four years beyond the end of fellowship training. Scholars will work in broad areas of scientific investigation relevant to child health. Past and present Scholars have studied the genomics of rare childhood disease, developmental biology, mucosal immunology, host- microbe interactions, monogenic kidney diseases, genetics of epilepsy, and environmental effects on early brain development. Our Scholars are expected to elucidate molecular mechanisms of disease, leading to the development of transformative new therapies for a broad range of diseases of childhood.

NIH Research Projects · FY 2025 · 2006-07

Retinopathy of prematurity (ROP) affects ~16,000 premature infants per year in the US. At preterm birth, there is loss of both ω3 and ω6 long-chain polyunsaturated fatty acid (LCPUFA), normally provided by the maternal/placental interface in utero. prominently contributes to initiation and progression of ROP. Docosahexaenoic acid (DHA, ω3) alone prevents ROP in some but not all studies. If DHA is given alone serum arachidonic acid (AA, ω6) decreases further, and lack of AA also contributes to ROP. We must understand how DHA and AA together prevent ROP to develop potent safe interventions based on physiology. In premature infants developing severe ROP, decreased mitochondrial number and increased peroxisomal activity (cleaving lipids ≥22 carbons) was found. However, knowledge of mitochondrial and peroxisomal lipid oxidation in retinal diseases is limited. Pilot work suggests DHA and AA control peroxisomal activity in a phase 1 ROP model. We will determine DHA/AA effects on retinal metabolism, particularly mitochondrial and peroxisomal fatty acid oxidation in early vessel loss in ROP. DHA/AA controls neurovascular development in phase 1 ROP by improving metabolism. In hyperglycemic mice in phase I ROP (vessel growth suppression and retinal neuronal dysfunction) we will: i) investigate if AA adds to DHA protection against retinal neurovascular abnormalities; ii) determine if DHA/AA alters retinal mitochondrial metabolism, and iii) determine if DHA/AA controls peroxisomal β-fatty acid oxidation. SUMMARY: These studies will determine if AA adds to DHA protection in phase I ROP (improving retinal metabolism) and uncover novel lipid metabolic associations. Supplementing DHA and AA orally will likely help prevent ROP and other retinopathies

NIH Research Projects · FY 2026 · 2003-09

ABSTRACT There is growing evidence that subcortical regions of the early visual pathway are not simple relays of primary visual information but play a greater role in visual system processing and plasticity than previously acknowledged. One such example is the primary visual thalamus, the dorsal lateral geniculate nucleus (dLGN). Studies across different species have demonstrated that circuits in this nucleus can change over development and even in the adult. The Chen Lab uses the mouse visual system as an experimental model to study synaptic plasticity of the connection between retinal ganglion cells (RGCs) and thalamocortical (TC) neurons in the dLGN. Our studies uncovered a late developmental period when visual deprivation can alter the strength and number of convergent retinal inputs onto TC neurons. We refer to this window of experience-dependent plasticity as the thalamic sensitive period. Visual deprivation before or after this period does not elicit similar changes in retinogeniculate connectivity. In the prior funding period, we showed that enhanced visual experience during the thalamic sensitive period can also trigger plasticity. Select exposure to a feature, referred to as select experience rearing (SER), can shift the population representation for that feature in dLGN. Moreover, these circuit changes are long-lasting, even if mice subjected to SER are subsequently re-exposed to normal vision. Our results support the idea that changes in retinogeniculate connectivity underlie SER plasticity and demonstrate that visual experience is not just permissive for plasticity but can instruct long-term changes in the thalamic circuits. In this current proposal, we turn our attention to the mature thalamus, asking whether plasticity also exists in the adult. Our preliminary studies suggest that there is plasticity in adult mice, however, unlike the developing brain, these changes do not persist. Here we propose to identify mechanisms that distinguish the transient nature of adult plasticity when compared to long-lasting changes that can occur during development. We will use a combination of tools including longitudinal in vivo two photon imaging of response preferences of thalamocortical neurons, in vivo single unit dLGN recordings, in vitro dLGN slice recordings and chemogenetics to characterize adult SER plasticity. We will also take advantage of mouse genetics, immunohistochemistry and 3-dimensional EM reconstruction to test the hypothesis that with age, glial ensheathment of retinal boutons in dLGN restricts rewiring of retinogeniculate connections, thus contributing to the difference between developmental and adult plasticity.

NIH Research Projects · FY 2026 · 2001-09

PROJECT SUMMARY For the past century, a major focus for Transfusion Medicine has been the immune compatibility of blood components with added attention to the safe and appropriate use of blood products. The success of these investigations has fueled expansive growth in the field with efforts focused on developing novel blood components for transfusion and to generate cellular therapies. These activities require a concerted effort to link advances in basic science with therapeutic interventions in the clinic. Transfusion Medicine Scientists and Specialists lead the effort to train individuals to advance blood and hematopoietic cellular therapies. The overall goal of the Program in Transfusion Biology and Cellular Therapies continues to be the preparation of M.D., M.D./Ph.D. or Ph.D. postdoctoral fellows for biomedical research careers. This long-standing program entering its third decade has supported 80 postdoctoral fellows supporting 8 postdoctoral fellows each for 2-3 years enabling them to move on to leading positions in academia or industry. As the field of Transfusion Medicine continues to grow, as does the program to include the scientific areas around Immunobiology, Hematopoiesis, and Cellular Therapy. Major strengths of our multi-disciplinary program include utilizing the strong clinical and basic research environments at Harvard Medical School involving Boston Children's Hospital, Dana-Farber Cancer Institute, Brigham & Women's Hospital, and Massachusetts General Hospital. In addition to scientific investigation, we have developed programs to a) educate trainees in responsible conduct of research, grant writing, laboratory management, and ensuring rigor and reproducibility of data. These opportunities allow trainees to develop skills in public speaking and to maintain academic oversight for scholarship to advance their career development. Dr. John Manis will continue his role as overall PI and has expanded the number of Program co-Directors to effectively represent both basic science and clinical Transfusion Medicine. These leaders will be responsible for the scientific direction of the training program, the mentoring of trainees as well as junior faculty, and monitoring milestones to ensure continued success. We have assembled our faculty roster this year to increase contemporary scientific representation from disciplines related to Transfusion and to better reflect the needs of our applicant pool. We believe our approach of including broad representation of clinical, basic and translational research talent is a fundamental strength of our program and fits well with the evolving field of Transfusion Biology and Cellular Therapies. This distinctive training approach has generated a unique environment for trainees in the Harvard Medical Area and ours remains a highly sought-after program. The outstanding pool of applicants and recruitment of faculty dedicated to the program has facilitated this goal. Our evolving Training Program strives to provide a unique and rich training environment for academic transfusion biology and cellular therapies.

NIH Research Projects · FY 2026 · 2001-05

Project Summary/Abstract The Pediatric Emergency Medicine Research Training Program (PEMRTP), established in 2001, offers a unique and comprehensive training experience for MD and PhD biomedical researchers focused on investigating acute illnesses and injuries in pediatric emergency medicine (PEM). With millions of children visiting emergency departments (EDs) annually, the need for evidence-based care and effective diagnostics and treatments is paramount. As pediatric research enters a new paradigm driven by data-driven care, artificial intelligence, genomics, and optimization of electronic health record systems, the PEMRTP aims to advance the care of acutely ill and injured children by training independent researchers, with a strong focus on informatics, genomics, and artificial intelligence. The COVID-19 pandemic also highlighted the central role of pediatric EDs in children's healthcare, and as a critical link in national public health surveillance programs. This competing renewal builds upon the assets of Boston Children's Hospital, housing the largest PEM faculty worldwide, to create a culture of rigorous research and training. The program's goals remain to prepare trainees for independent research in PEM and address the critical need for research mentors in the field. The next funding cycle plans to include 12 participating trainees. Trainees in the PEMRTP will progress through four interrelated phases, including mentored research projects, ongoing didactic instruction, formal coursework (optional), and preparation of a research grant. Additionally, trainees have the opportunity to pursue formal coursework for advanced degrees such as Master of Medical Science or Master of Public Health. Benefiting from the extensive research laboratories, clinical systems, academic programs, experienced faculty, and connections to multisite research networks at Boston Children's Hospital, the Computational Health Informatics Program, and the Department of Biomedical Informatics at Harvard Medical School, the PEMRTP provides an unparalleled environment for mentoring trainees to become future leaders in pediatric emergency medicine research.

NIH Research Projects · FY 2025 · 1999-07

Project Summary/Abstract The Developmental Neurology Training Program at Boston Children's Hospital is designed to produce trainees who are equipped for and deeply engaged in state-of-the-art research in developmental neuroscience from a detailed and mechanistic perspective, while also cognizant of the clinical importance of their field and the clinical challenges and opportunities today. A key aspect of the program is the attention given to experimental design, statistics, and quantitative skills that contribute to rigorous science. We take advantage of the presence of an extraordinary community of basic neuroscientists at Children's Hospital and its affiliated institutions. We select outstanding candidates who will work in one of 30 mentor laboratories and engage in fundamental mechanistic research. These highly interactive laboratories employ genetics, molecular biology, biochemistry, imaging, electrophysiology, anatomy and behavior and use diverse experimental systems. Consequently, our trainees are exposed to the full breadth of the field and are prepared to make informed strategic decisions. The goal of the program is not to distract from an intensive research experience in fundamental molecular mechanisms of neurodevelopment, but rather to ensure the highest standards of rigor in experimental design and analysis and to inform the trainees of the context of the research in human health. The training program is also designed to have a spill-over effect of enhancing the attention to rigor and quantitative skills in the broader community of neuro-research trainees and their mentors. Each trainee is also paired with a clinician or clinician-scientist as a co-mentor with the goal of helping trainees to understand the relationship of basic research to health and disease. This co-mentoring relationship will take advantage of the hospital setting and augment the rich opportunities for learning about translational research. The research experience of the trainee also enhanced by an extensive mentoring relationship with the Directors of the Training Program, Prof. Thomas Schwarz and Prof. Elizabeth Engle, who meet with and advise the trainees to offer feedback and career guidance, and by monthly trainee group meetings and a trainee-organized seminar series. The training experience is further supplemented by a wealth of career-guidance instruction, including presentation skills, job-seeking skills, information about diverse career options, and of course the responsible conduct of research. All of this is situated within an environment of extraordinary resources and intellectual life. We have recruited a racially and ethnically diverse faculty of mentors and are committed to enhancing the diversity of the community of neuroscientists through our Training Program. There are few challenges in neuroscience as great as understanding the processes that result in neurodevelopmental disorders and intellectual disabilities and there is a growing awareness that many affective disorders arise from errors in development. To face this challenge, the next generation will require rigorous training and strong quantitative skills. Without deeper mechanistic understanding of these processes, the clinical challenges will remain.

NIH Research Projects · FY 2026 · 1998-08

It is appreciated that the high prevalence of asthma over the last decades reflects the interaction of susceptibility genes in affected individuals with environmental and social changes ushered by modernity [1]. A key feature of asthma is chronic airway inflammation driven and/or exacerbated by ongoing exposure to allergens and pollutants, most notably ambient particulate matter (PMs). The persistence of such inflammation suggests the derailment of normative countervailing immune regulatory mechanisms that would otherwise limit its scope. In that regard, we have identified Notch4 expression in lung regulatory T (TR) cells as a key mechanism that disrupts lung tissue homeostatic immune regulation to promote allergic airway inflammation. Notch4 is upregulated by IL-6 signaling in TR cells to activate downstream intermediates including the Wnt and Hippo pathways to promote airway Th2 and Th17 immune responses, respectively. Notch4 expression is augmented by PM, which act to induce the expression of the Notch receptor ligand Jag1 in alveolar macrophages as well as the production of IL-6. Finally, Notch4 is also upregulated on lung TR cells in the context of viral infections, thus providing a potential link between viral infections and exacerbation of allergic airway inflammation. Accordingly, our central hypothesis is that Notch4 signaling in TR cells integrates cues by allergens, pollutants and viruses to license tissue allergic inflammation. To address this hypothesis, we propose under Aim 1 to investigate the mechanisms by which Notch4 subverts TR cell function to promote allergic airway inflammation. We will investigate the role of the Notch4-activated Hippo pathway in TR cell destabilization, leading to the generation of tissue resident ex-TR cells that promote airway inflammation. We will also investigate the role of the Wnt pathway downstream of Notch4 in driving Th2 pathology, as well as the interaction of Notch4 with environmental inputs such as PMs of different sources and physiochemical properties to promote airway inflammation. We further propose under Aim 2 to investigate the role of Growth and Differentiation Factor 15 (GDF15) as a cytokine produced by lung TR cells in a Notch4-Wnt pathway-dependent manner that may contribute to tissue inflammation and pathology by acting via its receptor GFRAL on ILC2 to drive their activation and expansion. The relationship between TR cell Notch4, GDF15, IL-6 and BMI will be explored in a cohort of asthmatic subjects. Finally, under Aim 3 we will examine the role of TR cell-intrinsic viral sensing pathways alone and in synergy with Notch4 in promoting virus and allergen-induced inflammation. Our studies will help elucidate novel mechanisms fundamental to the biology of allergic airway inflammation and its augmentation by pollutants, obesity and viral infections.

NIH Research Projects · FY 2026 · 1997-12

Project Summary: Epilepsy is a debilitating disease affecting 1 in 26 people (3-4% lifetime risk), making it the third most common neurological disorder in the United States. Antiepileptic drugs (AEDs) remain the mainstay of epilepsy therapy, but the long-term sequelae of uncontrolled epilepsy, including underachievement, social isolation, and heightened rates of depression and sudden death, bring into consideration more aggressive strategies for patients whose disease is recalcitrant to pharmacotherapy. The most prevalent form of intractable, non-lesional focal epilepsy in adults is mesial temporal lobe epilepsy (mTLE), and the causal mechanisms of mTLE remain poorly understood. mTLE often starts in adolescence or early adulthood, often following a history of earlier febrile seizures or head trauma, and often becoming more intractable with time. Sclerotic changes of the hippocampus (HS) often appear radiographically. With proper localization, mTLE is amenable to surgical intervention, resulting in seizure freedom or improvement in up to two thirds of cases. Our preliminary data, based on the genomic study of these resected tissues samples, implicate somatic activating variants in multiple RAS pathway genes in mTLE. The major goals of this study are to: sequence larger numbers of mTLE samples resected for epilepsy control, in order to confirm and extend our preliminary findings on the role of RAS activation in mTLE, and to identify other potential genetic regulators of mTLE; delineate the transcriptional effects of somatic mutation on specific cell types within the epileptic tissue ; and to identify the exact cell type(s) that carry mosaic variants, and the transcriptional effects on those variant- positive cells, compared to surrounding variant-negative cells in mTLE. Our prior experience in the development, use, and analysis of single cell genomic data, together with our promising preliminary data on mTLE and the availability of high-quality samples in-hand, indicate a high likelihood that this study will be successful. Our proposed work promises to provide valuable insight into the genetic causes of mTLE. It will further use that information to investigate the molecular mechanisms underlying epilepsy.

NIH Research Projects · FY 2025 · 1997-09

Project Summary The Harvard Digestive Disease Center (HDDC) is a community of 66 Principal (Full) Members with over $39M annual funding for research directly related to the digestive diseases (this is our Research Base, 31% NIDDK). Center members focus on understanding the cell, tissue, and developmental biology of the mucosal surfaces lining the alimentary tract: this is the Center’s Theme. We address the fundamental mechanisms that underlie normal digestive tract function and the pathogenesis of digestive diseases. Center members work in 4 major Research Areas that address the basis for most diseases of the alimentary tract: n Cell, Developmental, and Stem Cell Biology of the Alimentary Tract; n Innate and Adaptive Mucosal Immunology and Microbial Pathogenesis; n Gut Microbiology and Metabolism; and n Clinical and Translational Human Studies. The Center also includes 55 Affiliate Members (not included in Research Base), who participate in Center activities, use our Cores, but who conduct research that falls outside the theme of the HDDC. Our Members’ resources are amplified through services, equipment and training in 3 Biomedical Cores that provide: (Core B) high-resolution microscopy & histopathology, (Core C) diverse technologies to study epithelial cell function & mucosal immunology, and (Core D) technologies in gnotobiotic mice, microbiological and metabolic analyses. Our Cores helped produce 424 original papers. The HDDC Clinical Component supports clinical and translational GI research through subsidized biostatistical and bio-repository services. The Center fosters scientific collaborations through an Enrichment Program reflecting our theme, including an annual symposium, a biennial regional conference "Frontiers in Mucosal Immunology", and monthly seminars and workshops focused on young investigators. The HDDC also promotes careers of young scientists through a competitive Pilot-Feasibility Grant Program that has supported 62 investigators since 2006: 92% were awarded major independent funding within 5 years of their award, and all, but one, remain active in digestive diseases-related research. Center Director Wayne Lencer (PI) and Associate Director Richard Blumberg (Co-PI) are highly accomplished physician-scientists, currently Division Chiefs of Pediatric and Adult GI at two major Harvard teaching hospitals, and both are Directors of NIH-funded T32 training programs in Gastroenterology. They are assisted in HDDC leadership by an Executive Committee that includes all Core Directors and Research Area (Affinity-group) Directors, and guided by an External Advisory Board who are all highly accomplished scientists and leaders in GI-related research. The HDDC’s overarching mission is to foster and expand basic and translational science in digestive diseases by n connecting people, n creating opportunity, and n extending resources.

NIH Research Projects · FY 2026 · 1995-05

PROJECT SUMMARY/ABSTRACT Anxiety disorders are the most common psychiatric illnesses and are often resistant to treatment. Adolescence is a core risk period for the development and exacerbation of anxiety, which often has a chronic course, negatively affecting academic, social, and adaptive functioning, and increasing the risk for mental illness through adulthood. Research has highlighted a number of risk factors that likely contribute to the development and maintenance of anxiety. However, there is limited understanding of the earliest precursors of anxiety or how multiple risk factors interact within and across development to influence anxiety risk. Prospective studies beginning in infancy are needed to explicate the origins of anxiety so that (a) biomarkers can be discovered that identify at-risk youth prior to the emergence of symptoms and (b) preventive strategies can be developed and implemented with those at risk. The overall goal of the current project is to test the combined effects of neural, physiological, behavioral, and environmental risk factors on anxiety from infancy through adolescence. The study aims will be accomplished by following our established longitudinal cohort (R01 MH078829; N=807), who have provided a rich dataset, including repeated assessments of neurophysiology (EEG, ERP), physiological stress reactivity, behavioral indicators of threat reactivity, and environmental risk (e.g., maternal psychopathology, negative life events, COVID-19 related stressors) between infancy and age 7 years. In the current proposal, we seek funds to support a follow-up study to age 13 years. We will phenotype our cohort for anxiety symptomatology and diagnoses, across multiple phenotypes, and implement a battery of brain-based measures, physiological and behavioral protocols, and assessments of environmental exposures, including exposures of particular relevance in adolescence and exposures related to the COVID-19 pandemic. We will apply a combination of established and novel analysis approaches to develop diagnostic neural biomarkers of anxiety in adolescence; identify positive and negative environmental characteristics that influence anxiety- relevant neural signatures in adolescence, that affect anxiety-related neural trajectories from infancy to adolescence, and that moderate the effects of neural reactivity on anxiety risk; determine how COVID-19 related stressors interact with childhood pre-pandemic characteristics (neural and behavioral threat reactivity, physiological stress reactivity) to influence adolescent anxiety risk; and to develop assay profiles comprising neural, physiological, behavioral, and/or environmental characteristics from infancy through adolescence that robustly predict anxiety trajectories across development. We expect that the findings will (a) improve our understanding of the neural circuitry underlying anxiety risk in youth, (b) contribute to the discovery of robust developmentally-informed multi-modal profiles that can identify at-risk children, and (c) inform the design of innovative strategies to prevent the emergence of anxiety and to treat more precisely symptomatic youth by addressing and correcting atypical neural processes and their downstream behavioral manifestations.

NIH Research Projects · FY 2025 · 1994-12

Project Summary Deciphering the cell lineage of the human brain is vital for understanding the expansion of brain size and cognitive capabilities in humans and for elucidating the causes of, and finding treatments for, a growing number of human neurological disorders. Somatic mutations are the drivers of brain cancer and are the most common cause of intractable pediatric epilepsy resulting in brain surgery. They have also been implicated in adult temporal lobe epilepsy, autism spectrum disorders, and schizophrenia, and may contribute to neurodegeneration. Work from our lab and others have shown that somatic mutations occurring during fetal development represent clonal marks that can be used to trace the lineage of the cells. Each cell division is associated with ≈2-4 somatic single nucleotide variants, so that every brain cell carries a unique DNA “barcode” that can be exploited to reconstruct lineage relationships between cell types. Recent technical developments in single-cell RNA sequencing allow analysis of cell types in postmortem human brain at a level of detail previously thought impossible, allowing description of the many transcriptional patterns that characterize distinct neuronal and glial cell types in human brain. The proposed experiments will integrate DNA lineage marks with RNA analysis of cell types, and describe additional new methods that can outline the major features of clonal dynamics that generate neuronal and glial types in human brain and clonal dispersion that underlies the architecture of the functional subdivision of the human cerebral cortex. Our research will advance three specific aims: Lineage Analysis of Neurons: We will investigate the late divergence of excitatory and inhibitory neuron lineages in the human frontal lobe, using deep whole genome sequencing (WGS) of sorted neuronal populations. By understanding these lineages, we hope to clarify how distinct neuronal types emerge and interconnect, which is critical for grasping the functional organization of the brain. Oligodendrocyte Lineage Dynamics: Oligodendrocytes exhibit unique mutational patterns. We will analyze their lineage and proliferation through WGS and single-cell RNA sequencing. This aim seeks to reveal how oligodendrocyte precursor cells (OPCs) contribute to brain health and disease. High-Throughput cell type-specific lineage and aging: Utilizing the innovative Duplex Multiome technology, we will explore the accumulation of age-related somatic mutations across different cell types. This approach will enable us to identify patterns of mutation and lineage in a single experiment. These data provide three major discoveries not obtainable by any other means: 1] direct cell lineage data from the adult human brain, which is essential for understanding how our brain develops and how developmental somatic mutations cause disease, 2] preliminary lineage maps connecting neuronal and glial cell classes, and 3] cell type-informed understanding of the age-related process of mutation accumulation.

NIH Research Projects · FY 2024 · 1994-09

ABSTRACT The Harvard-wide pediatric HSR Fellowship is a 30 year collaboration of Boston Children’s Hospital, Massachusetts General Hospital for Children, and the Harvard Department of Population Medicine/Harvard Pilgrim Healthcare Institute. This program has an amazing training record- the 154 alumni include 18 graduates from race/ethnicity groups underrepresented in medicine, 61 in leadership positions including 16 CMOs or Senior VPs, and four elected to the National Academy of Medicine. The curriculum is grounded in rigorous child health services research training and in-depth implementation science training, aligned with AHRQ learning health system (LHS) competencies. While our multisite collaborative leadership structure has remained constant for two decades, our list of multidisciplinary, innovative faculty is updated regularly. Pediatric HSR training programs are needed because critical epidemiologic, fiscal, and workforce differences, such as a mental health crisis or a relative dearth of quality measures and interventions, set pediatrics apart from adult medicine. Our overarching aim remains: to provide outstanding, comprehensive training across the spectrum of pediatric health services research methods that both advances understanding of equitable health and healthcare systems, and tests and evaluates implementation of novel approaches that demonstrably improve child health. Toward this aim, the objectives for this fellowship are to: 1) recruit and retain diverse candidates; 2) provide exemplary training in health services research and implementation science within LHS; 3) support our dedicated faculty; 4) continuously improve the training program and update the curriculum; and 5) continue to develop diverse graduates to lead the nation in innovative pediatric health services research, including research that accelerates the development of LHS. To meet this goal, training focus areas include: cutting edge and emerging methods in health systems research; application of implementation science and related methods; LHS competencies including embedded research and health equity research within health systems; and patient and family-centered research. The program will support 7 postdoctoral trainees per year for the 2-year postdoctoral fellowship to create a critical mass of trainees across subspecialties, areas of research, and methodological domains. In addition to conducting closely mentored research, each trainee will complete coursework from the Harvard T.H. Chan School of Public Health for core skills of biostatistics and epidemiology and participate in advanced health services research methods training. Over the past 30 years, our program has evolved in response to feedback from trainees, faculty, and our advisory board. In this cycle, we will expand LHS training consistent with AHRQ’s new health equity and embedded research competencies and add an alumni mentoring program. Renewal of our T32 Program will address a critical deficiency in the pediatric subspecialist and generalist healthcare delivery system scientists that will foster the development and success of learning healthcare systems to improve child health.

NIH Research Projects · FY 2026 · 1993-09

ABSTRACT/SUMMARY: Our goal is to elucidate how apical membrane glycosphingolipids of barrier epithelial cells sense enterotoxin and virus binding to induce a form of cell-autonomous host defense – mediated by blocking toxin/virus entry. Several years ago, we discovered that loss of the cell polarity gene PARD6B selectively diminished apical endosome function in polarized epithelia (1). We then discovered that in response to certain viruses and bacterial toxins, PARD6B together with aPKC (a second component of the apical PARD6B-polarity complex) underwent rapid proteasome-dependent degradation by the intestinal cell, and this down regulated the apical endosome preventing further toxin/virus entry. The basolateral endosome functioned normally. The signal for degradation was initiated by perturbation of apical membrane glycosphingolipids (GSLs) caused by toxin or virus binding (1, 2). New studies show that signaling in this pathway requires perturbation of specific GSL species with affinity for membrane nanodomains; and that binding the apical membrane GSLs leads also to unique cytokine responses amplifying host defense – including the rapid and apically-polarized secretion of the anti-viral type III interferon IFN and evidence for cytokine-induced degradation of PARD6B - thus phenocopying toxin/virus binding. These discoveries frame our central hypothesis: that polarized epithelial cells of mucosal surfaces harness GSL membrane nanodomains to sense and respond to environmental signals in the gut lumen by effecting a form of innate cell autonomous host defense mediated by down-regulation of the apical recycling endosome and lumenal secretion of anti-viral cytokines - which we term the GSL-PARD6B-pathway - or the GSL-pathway for short. The topic addresses a new concept in innate host defense operating at the immediate host-microbial interface; and it addresses large gaps in our knowledge for how barrier epithelial cells can rapidly sense and effect resistance to toxin and viral invasion at mucosa surfaces. Aim 1 will elucidate how intestinal epithelial cells use the GSLs to sense enterotoxin/virus binding and induce PARD6B/aPKC degradation. Hypothesis-driven studies using cell biological, lipid structural, and genetic approaches - employing cell line and organoid models - will test how an outer leaflet membrane lipid can effect transmembrane signal transduction, and what exactly the GSLs sense as a “danger” signal. Aim 2 will test how toxin/virus-affected barrier epithelial cells may propagate a danger signal to neighboring unaffected cells for broader protection of the mucosal tissue. Two mechanisms of signal transduction will be studied using cell biological and biochemical approaches – signaling by soluble paracrine factors, including the polarized lumenal secretion of IFN, and signaling through cell-cell contact sites via alteration of intercellular adherens junctions effected by dynamics of the apical cortical cytoskeleton. Aim 3 will test the GSL- pathway in vivo and mechanism of GSL-induced signal transduction using WT mice and mice lacking essential components of the pathway as defined in vitro (conditional PARD6B KO, Flotillin 1/2 KO).