Children’s Discovery Institute

As SARS-CoV-2 continues to spread globally, questions have emerged regarding the strength and durability of immune responses in specific populations. In this study, we evaluated humoral immune responses in 69 children and adolescents with asymptomatic or mild symptomatic SARS-CoV-2 infection. We detected robust IgM, IgG, and IgA antibody responses to a broad array of SARS-CoV-2 antigens at the time of acute infection and 2 and 4 months after acute infection in all participants. Notably, these antibody responses were associated with virus-neutralizing activity that was still detectable 4 months after acute infection in 94% of children. Moreover, antibody responses and neutralizing activity in sera from children and adolescents were comparable or superior to those observed in sera from 24 adults with mild symptomatic infection. Taken together, these findings indicate that children and adolescents with mild or asymptomatic SARS-CoV-2 infection generate robust and durable humoral immune responses that can likely contribute to protection from reinfection.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Ventricular chamber growth and development during perinatal circulatory transition is critical for functional adaptation of the heart. However, the chamber-specific programs of neonatal heart growth are poorly understood. We used integrated systems genomic and functional biology analyses of the perinatal chamber specific transcriptome and we identified Wnt11 as a prominent regulator of chamber-specific proliferation. Importantly, downregulation of Wnt11 expression was associated with cyanotic congenital heart defect (CHD) phenotypes and correlated with O2 saturation levels in hypoxemic infants with Tetralogy of Fallot (TOF). Perinatal hypoxia treatment in mice suppressed Wnt11 expression and induced myocyte proliferation more robustly in the right ventricle, modulating Rb1 protein activity. Wnt11 inactivation was sufficient to induce myocyte proliferation in perinatal mouse hearts and reduced Rb1 protein and phosphorylation in neonatal cardiomyocytes. Finally, downregulated Wnt11 in hypoxemic TOF infantile hearts was associated with Rb1 suppression and induction of proliferation markers. This study revealed a previously uncharacterized function of Wnt11-mediated signaling as an important player in programming the chamber-specific growth of the neonatal heart. This function influences the chamber-specific development and pathogenesis in response to hypoxia and cyanotic CHDs. Defining the underlying regulatory mechanism may yield chamber-specific therapies for infants born with CHDs.

Physician-scientists represent a critical component of the biomedical and health research workforce. However, the proportion of physicians who spend a significant amount of effort on scientific research has declined over the past 40 years. This trend has been particularly noticeable in pediatrics despite recent scientific work revealing that early life influences, exposures, and health status play a significant role in lifelong health and disease. To address this problem, the Duke University Department of Pediatrics developed the Duke Pediatric Research Scholars Program for Physician-Scientist Development (DPRS). The DPRS is focused on research training during pediatric residency and fellowship. We aim to provide sufficient research exposure and support to help scholars develop a research niche and scholarly products as well as identify the career pathways that will enable them to achieve their research goals. Herein, we describe the DPRS's organizational structure, core components, recruitment strategies, and initial results, and we discuss implementation challenges and solutions. Additionally, we detail the program's integration with the department's residency and fellowship training programs (with particular reference to the challenges of integrating research into small- to medium-sized residency programs) and describe the development and integration of related initiatives across Duke University School of Medicine. The program served as the basis for 2 successful National Institutes of Health Stimulating Access to Research in Residency (R38) applications, and we hope it will serve as a model to integrate formalized research training for residents and fellows who wish to pursue research careers in academic medicine.

Human cytomegalovirus (HCMV) profoundly impacts host T and NK cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III-expressing (FcγRIII-expressing) CD8+ T cells following HCMV exposure in utero. Most FcγRIII+CD8+ T cells express the canonical αβ T cell receptor (TCR), but a proportion express noncanonical γδ TCR. FcγRIII+CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+CD8+ T cells mediate antibody-dependent IFN-γ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+CD8+ T cell Fc effector functions were further enhanced by IL-15, as has been observed in neonatal NK cells. Our study reveals that FcγRIII+CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

Background: Fetal to neonatal transition of heart is an elaborate process, during which, neonatal cardiomyocytes undergo functional maturation and terminal exit from the cell cycle. However, transcriptome programming in neonatal cardiac chambers during perinatal stages is understudied. In particular, the changes in long non-coding RNAs (lncRNAs) in neonatal heart have not been explored. Objective: To achieve transcriptome-wide analysis of lncRNAs in neonatal left ventricle (LV) and right ventricle (RV) during maturation stages using deep RNA-Sequencing Methods: Deep RNA-sequencing was performed on male newborn mouse (C57 BL) LV and RV at 3 time points of perinatal circulatory transition: P0, P3 and P7. Reads were mapped to mouse genome (mm10). The lncRNAs annotated in NONCODE database were identified. Differentially expressed lncRNAs were defined as those with coefficient of variation ≥0.2, at a false discovery rate ≤0.05, and expressed at ≥3 RPKM in at least one sample. Correlated lncRNAs/ gene pairs were identified using Pearson’s (r2≥0.8, P≤0.05). A subset of LncRNAs/gene expression was validated using qRT-PCR. Results: Out of the 70, 983 observed unique lncRNAs, approximately 7000 were identified exhibiting significant variation during maturation windows with highly spatial-temporal dependent expression patterns, including approximately 5000 known and 2000 novel lncRNAs. Notably, 20% of these lncRNAs were located within 50 KB of a protein coding gene. Out of a total of 2400 lncRNAs/gene pairs, 10 % exhibited significantly concordant (lncRNA/gene) expression patterns. These correlated genes were significantly enriched in metabolism, cell cycle and contractility functional ontology. Interestingly, some of these lncRNAs exhibited concordance with their neighboring gene in human tissues with congenital heart defects, suggesting conserved, potentially significant, regulatory function. Conclusions: Transcriptome programming during neonatal heart maturation involves global changes in lncRNAs. Their expression concordance with neighboring protein coding genes implicates potential important regulatory role of lncRNAs in neonatal heart chamber specification and congenital diseases.

Background: Chamber-specific and temporally orchestrated perinatal cardiac growth and maturation is critical to functional adaptation of heart to the dramatic changes in hemodynamic load and nutrient environment, and perturbation of this process may have major implications in congenital heart defects (CHDs). However, the responsible molecular regulatory mechanisms are still poorly understood. Objective: To determine the factors that control chamber specificity of neonatal heart growth during perinatal maturation Methods: Total transcriptome was analyzed from deep RNA-seq in male newborn mouse (C57BL) left ventricle (LV) and right ventricle (RV) at postnatal day 0, 3 and 7. Functional validation was performed in Neonatal Rat Ventricular Myocytes (NRVMs) and intact neonatal mice. Results: Chamber specific- regulation of cell cycle genes was correlated with chamber specific- expression of the non-canonical Wnt11. Exogenous expression of Wnt11 induced maturation markers and suppressed cell cycle genes in myocytes. In contrast, targeted Wnt11 inhibition induced myocyte proliferation associated with upregulated cell cycle genes both in cultured NRVMs and in the intact heart. Notably, Wnt inhibition also significantly enhanced β-catenin phosphorylation without impacting JNK, PKCα, or p38- activities. Remarkably, both Wnt mediated signals and cell cycle markers were altered in heart samples from infants cyanotic CHDs. Furthermore, hypoxia exposure suppressed Wnt11 and induced cell cycle genes in cultured cardiomyocytes. Conclusions: Differential expression of Wnt11 and downstream cell cycle genes are novel molecular basis for chamber specific maturation. Wnt11 mediated-signaling has potential impact on cyanotic CHDs. Differential role of Wnt signaling in chamber specific post-natal heart maturation can lead to potential chamber-specific diagnoses and therapies for congenital heart diseases.