Repression of the Glucocorticoid Receptor Increases Hypoxic-Ischemic Brain Injury in the Male Neonatal Rat.

Hypoxic-ischemic encephalopathy (HIE) resulting from asphyxia is the most common cause of neonatal brain damage and results in significant neurological sequelae, including cerebral palsy. The current therapeutic interventions are extremely limited in improving neonatal outcomes. The present study tests the hypothesis that the suppression of endogenous glucocorticoid receptors (GRs) in the brain increases hypoxic-ischemic (HI) induced neonatal brain injury and worsens neurobehavioral outcomes through the promotion of increased inflammation. A mild HI treatment of P9 rat pups with ligation of the right common carotid artery followed by the treatment of 8% O2 for 60 min produced more significant brain injury with larger infarct size in female than male pups. Intracerebroventricular injection of GR siRNAs significantly reduced GR protein and mRNA abundance in the neonatal brain. Knockdown of endogenous brain GRs significantly increased brain infarct size after HI injury in male, but not female, rat pups. Moreover, GR repression resulted in a significant increase in inflammatory cytokines TNF-α and IL-10 at 6 h after HI injury in male pups. Male pups treated with GR siRNAs showed a significantly worsened reflex response and exhibited significant gait disturbances. The present study demonstrates that endogenous brain GRs play an important role in protecting the neonatal brain from HI induced injury in male pups, and suggests a potential role of glucocorticoids in sex differential treatment of HIE in the neonate.

Distinct roles for IκB kinases alpha and beta in regulating pulmonary endothelial angiogenic function during late lung development.

Pulmonary angiogenesis is essential for alveolarization, the final stage of lung development that markedly increases gas exchange surface area. We recently demonstrated that activation of the nuclear factor kappa-B (NFκB) pathway promotes pulmonary angiogenesis during alveolarization. However, the mechanisms activating NFκB in the pulmonary endothelium, and its downstream targets are not known. In this study, we sought to delineate the specific roles for the NFκB activating kinases, IKKα and IKKß, in promoting developmental pulmonary angiogenesis. Microarray analysis of primary pulmonary endothelial cells (PECs) after silencing IKKα or IKKß demonstrated that the 2 kinases regulate unique panels of genes, with few shared targets. Although silencing IKKα induced mild impairments in angiogenic function, silencing IKKß induced more severe angiogenic defects and decreased vascular cell adhesion molecule expression, an IKKß regulated target essential for both PEC adhesion and migration. Taken together, these data show that IKKα and IKKß regulate unique genes in PEC, resulting in differential effects on angiogenesis upon inhibition, and identify IKKß as the predominant regulator of pulmonary angiogenesis during alveolarization. These data suggest that therapeutic strategies to specifically enhance IKKß activity in the pulmonary endothelium may hold promise to enhance lung growth in diseases marked by altered alveolarization.

Repression of the Glucocorticoid Receptor Aggravates Acute Ischemic Brain Injuries in Adult Mice.

Strokes are one of the leading causes of mortality and chronic morbidity in the world, yet with only limited successful interventions available at present. Our previous studies revealed the potential role of the glucocorticoid receptor (GR) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). In the present study, we investigate the effect of GR knockdown on acute ischemic brain injuries in a model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in adult male CD1 mice. GR siRNAs and the negative control were administered via intracerebroventricular (i.c.v.) injection 48 h prior to MCAO. The cerebral infarction volume and neurobehavioral deficits were determined 48 h after MCAO. RT-qPCR was employed to assess the inflammation-related gene expression profiles in the brain before and after MCAO. Western Blotting was used to evaluate the expression levels of GR, the mineralocorticoid receptor (MR) and the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling. The siRNAs treatment decreased GR, but not MR, protein expression, and significantly enhanced expression levels of pro-inflammatory cytokines (IL-6, IL-1ß, and TNF-α) in the brain. Of interest, GR knockdown suppressed BDNF/TrkB signaling in adult mice brains. Importantly, GR siRNA pretreatment significantly increased the infarction size and exacerbated the neurobehavioral deficits induced by MCAO in comparison to the control group. Thus, the present study demonstrates the important role of GR in the regulation of the inflammatory responses and neurotrophic BDNF/TrkB signaling pathway in acute ischemic brain injuries in adult mice, revealing a new insight into the pathogenesis and therapeutic potential in acute ischemic strokes.

A novel xenograft model to study the role of TSLP-induced CRLF2 signals in normal and malignant human B lymphopoiesis.

Thymic stromal lymphopoietin (TSLP) stimulates in-vitro proliferation of human fetal B-cell precursors. However, its in-vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in-vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing.

BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

Corticosteroids and perinatal hypoxic-ischemic brain injury.

Perinatal hypoxic-ischemic (HI) brain injury is the major cause of neonatal mortality and severe long-term neurological morbidity. Yet, the effective therapeutic interventions currently available are extremely limited. Corticosteroids act on both mineralocorticoid (MR) and glucocorticoid (GR) receptors and modulate inflammation and apoptosis in the brain. Neuroinflammatory response to acute cerebral HI is a major contributor to the pathophysiology of perinatal brain injury. Here, we give an overview of current knowledge of corticosteroid-mediated modulations of inflammation and apoptosis in the neonatal brain, focusing on key regulatory cells of the innate and adaptive immune response. In addition, we provide new insights into targets of MR and GR in potential therapeutic strategies that could be beneficial for the treatment of infants with HI brain injury.