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Frontline Science: Splenic progenitors aid in maintaining high neutrophil numbers at sites of sterile chronic inflammation

Wed, 11/16/2016 - 8:56am

Neutrophils are constantly generated from hematopoietic stem and progenitor cells in the bone marrow to maintain high numbers in circulation. A considerable number of neutrophils and their progenitors have been shown to be present in the spleen too; however, their exact role in this organ remains unclear. Herein, we sought to study the function of splenic neutrophils and their progenitors using a mouse model for sterile, peritoneal inflammation. In this microcapsule device implantation model, we show chronic neutrophil presence at implant sites, with recruitment from circulation as the primary mechanism for their prevalence in the peritoneal exudate. Furthermore, we demonstrate that progenitor populations in the spleen play a key role in maintaining elevated neutrophil numbers. Our results provide new insight into the role for splenic neutrophils and their progenitors and establish a model to study neutrophil function during sterile inflammation.

Needles in Hay II: Detecting Cardiac Pathology by the Pediatric Chest Pain Standardized Clinical Assessment and Management Plan

Wed, 11/16/2016 - 8:56am

OBJECTIVES: To determine if patients evaluated using the pediatric chest pain standardized clinical assessment and management plan (SCAMP) in cardiology clinic were later diagnosed with unrecognized cardiac pathology, and to determine if other patients with cardiac pathology not enrolled in the SCAMP would have been identified using the algorithm.

STUDY DESIGN: Patients 7-21 years of age, newly diagnosed with hypertrophic or dilated cardiomyopathy, coronary anomalies, pulmonary embolus, pulmonary hypertension, pericarditis, or myocarditis were identified from the Boston Children's Hospital (BCH) cardiac database between July 1, 2010 and December 31, 2012. Patients were cross-referenced to the SCAMP database or retrospectively assessed with the SCAMP algorithm.

RESULTS: Among 98 patients with cardiac pathology, 34 (35%) reported chest pain, of whom 10 were diagnosed as outpatients. None of these patients were enrolled in the SCAMP because of alternate chief complaints (n = 4) or referral to BCH for management of the new diagnosis (n = 6). Each of these patients would have had an echocardiogram recommended by retrospective application of the SCAMP algorithm. Two other patients with cardiac pathology were among the 1124 patients assessed by the SCAMP. One patient initially diagnosed with noncardiac chest pain presented 18 months later and was diagnosed with myocarditis as an inpatient. One patient seen initially in the emergency department was subsequently diagnosed with pericarditis as an outpatient.

CONCLUSIONS: Patients assessed by the chest pain SCAMP at BCH were not later diagnosed with cardiac pathology that was missed at the initial encounter. Nonenrolled outpatients with cardiac pathology and chest pain would have been successfully identified with the SCAMP algorithm.

Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans

Wed, 11/16/2016 - 8:56am

The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

Identification and Characterization of MicroRNA Modulators in Caenorhabditis Elegans: A Dissertation

Tue, 11/15/2016 - 8:45pm

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that posttranscriptionally regulate gene expression primarily through binding to the 3’ untranslated region (3’UTR) of target mRNAs, and are known to play important roles in various developmental and physiological processes. The work presented in this thesis was centered on understanding how Caenorhabditis elegans miRNAs are modulated by genetic, environmental, or physiological factors and how these small RNAs function to maintain the robustness of developmental processes under stressful conditions.

To identify modulators of the miRNA pathway, I developed sensitized genetic backgrounds that consist of a panel of miRNA gene mutants and miRNA biogenesis factor mutants with partially penetrant phenotypes. First, I found that upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an opportunistic pathogen of diverse plants and animals, let-7 family miRNAs are engaged in reciprocal regulatory interactions with the p38 MAPK innate immune pathway to maintain robust developmental timing despite the stress of pathogen infection. These let-7 family miRNAs, along with other developmental timing regulators, are also integrated into innate immune regulatory networks to modulate immune responses. Next, I demonstrated that loss-of-function mutations of Staufen (stau-1), a double-stranded RNA-binding protein, increase miRNA activity for several miRNA families, and this negative modulation of Staufen on miRNA activity acts downstream of miRNA biogenesis, possibly by competing with miRNAs for binding to target mRNA 3’UTRs.

In summary, these studies provide a better understanding on how miRNAs are modulated by various environmental and cellular components, and further support the role of the miRNA pathway in conferring robustness to developmental processes under these perturbations.

Small RNA Regulation of the Innate Immune Response: A Role for Dicer in the Control of Viral Production and Sensing of Nucleic Acids: A Dissertation

Tue, 11/15/2016 - 8:45pm

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

Computational Approaches for the Analysis of Chromosome Conformation Capture Data and Their Application to Study Long-Range Gene Regulation: A Dissertation

Tue, 11/15/2016 - 8:45pm

Over the last decade, development and application of a set of molecular genomic approaches based on the chromosome conformation capture method (3C), combined with increasingly powerful imaging approaches have enabled high resolution and genome-wide analysis of the spatial organization of chromosomes. The aim of this thesis is two-fold; 1), to provide guidelines for analyzing and interpreting data obtained from genome-wide 3C methods such as Hi-C and 3C-seq and 2), to leverage the 3C technology to solve genome function, structure, assembly, development and dosage problems across a broad range of organisms and disease models.

First, through the introduction of cWorld, a toolkit for manipulating genome structure data, I accelerate the pace at which *C experiments can be performed, analyzed and biological insights inferred. Next I discuss a set of practical guidelines one should consider while planning an experiment to study the structure of the genome, a simple workflow for data processing unique to *C data and a set of considerations one should be aware of while attempting to gain insights from the data.

Next, I apply these guidelines and leverage the cWorld toolkit in the context of two dosage compensation systems. The first is a worm condensin mutant which shows a reduction in dosage compensation in the hermaphrodite X chromosomes. The second is an allele-specific study consisting of genome wide Hi-C, RNA-Seq and ATAC-Seq which can measure the state of the active (Xa) and inactive (Xi) X chromosome. Finally I turn to studying specific gene – enhancer looping interactions across a panel of ENCODE cell-lines.

These studies, when taken together, further our understanding of how genome structure relates to genome function.

Investigating the Architecture and Vesicle Tethering Function of the Yeast Exocyst Complex: A Dissertation

Tue, 11/15/2016 - 8:45pm

The exocyst is an evolutionarily conserved, hetero-octameric protein complex proposed to serve as a multi-subunit tethering complex for exocytosis, although it remains poorly understood at the molecular level. The classification of the exocyst as a multisubunit tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the questions: why are so many subunits required for vesicle tethering and what are the contributions of each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex dynamics a required feature of exocyst function? We purified endogenous exocyst complexes from Saccharomyces cerevisiae, and showed that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. This conclusion contrasts starkly with current models suggesting that the yeast exocyst tethers vesicles by transient assembly of subcomplexes at sites of exocytosis. Using a combination of biochemical and auxininduced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as an octameric complex in yeast with a stably assembled, elongated structure. This is the first complete structure of a CATCHR family MTC and it differs greatly from the EM structures available for the partial COG and Dsl1 complexes. Future work will be necessary to determine whether exocyst conformational changes are a required feature of vesicle tethering and how such changes are regulated.

These architectural insights are now informing the design of the first in vitro functional assay for the exocyst complex. We developed methodology for attaching fluorescently-labeled exocyst complexes to glass slides and monitoring the capture of purified, endogenous secretory vesicles by single molecule TIRF microscopy. By this approach, we can monitor tethering events in real time and determine the required factors and kinetics of exocytic vesicle tethering.

Eosinophils as Drivers of the IL-23/IL-17 Axis: Implications for Acute Aspergillosis and Allergic Asthma: A Dissertation

Tue, 11/15/2016 - 8:44pm

Aspergillus fumigatus is an opportunistic fungal pathogen that causes lethal invasive pulmonary disease in immunocompromised hosts and allergic asthma in sensitized individuals. This dissertation explores how eosinophils may protect hosts from acute infection while driving asthma pathogenesis by co-producing IL-23 and IL-17 in both contexts. In an acute model of pulmonary aspergillosis, eosinophils were observed to associate with and kill A. fumigatus spores in vivo. In addition, eosinopenia was correlated with higher mortality rates, decreased recruitment of inflammatory monocytes to the lungs, and decreased expansion of lung macrophages. As IL-17 signaling must occur on a local level to elicit its stereotypical response, such as the up-regulation of antimicrobial peptides and specific chemokines from stromal cells, eosinophils were discovered to be a significant source of pulmonary IL-17 as well as one of its upstream inducers, IL-23. In the context of asthma, this discovery opens a new paradigm whereby eosinophils might be driving asthma pathogenesis.

A Role for the Lipid Droplet Protein HIG2 in Promoting Lipid Deposition in Liver and Adipose Tissue: A Dissertation

Tue, 11/15/2016 - 8:44pm

Chronic exposure of humans or rodents to high calorie diets leads to hypertriglyceridemia and ectopic lipid deposition throughout the body, resulting in metabolic disease. Cellular lipids are stored in organelles termed lipid droplets (LDs) that are regulated by tissue-specific LD proteins. These proteins are critical for lipid homeostasis, as humans with LD protein mutations manifest metabolic dysfunction. Identification of novel components of the LD machinery could shed light on human disease mechanisms and suggest potential therapeutics for Type 2 Diabetes.

Microarray analyses pinpointed the largely unstudied Hypoxia-Inducible Gene 2 (Hig2) as a gene that was highly expressed in obese human adipocytes. Imaging studies demonstrated that Hig2 localized to LDs in mouse hepatocytes and the human SGBS adipocyte cell line. Thus, this work examined the role of Hig2 as a LD protein in liver and adipose tissue.

Hig2 deficiency reduced triglyceride deposition in hepatocytes; conversely, ectopic Hig2 expression promoted lipid deposition. Furthermore, liver-specific Hig2-deficient mice displayed improved glucose tolerance and reduced liver triglyceride content. Hig2 deficiency increased lipolysis and -oxidation, accounting for the reduced triglyceride accumulation.

Similarly, adipocyte-specific Hig2-deficient mice displayed improved glucose tolerance, reduced adipose tissue weight and brown adipose tissue that was largely cleared of lipids. These improvements were abrogated when the animals were placed in thermoneutral housing and brown adipocyte-specific Hig2-deficient mice also displayed improved glucose tolerance, suggesting that active brown fat largely mediates the metabolic phenotype of Hig2 deletion. Thus, this work demonstrates that Hig2 localizes to LDs in liver and adipose tissue and promotes glucose intolerance.

Molecular Mechanisms of Assembly and Long-term Maintenance of Neuronal Architecture: A Dissertation

Tue, 11/15/2016 - 8:44pm

Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post-embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term.

Cytosolic and Endosomal DNA-Sensing Pathways Differentially Regulate Inflammatory Arthritis, Autoantibody Production, and Bone Remodeling: A Dissertation

Tue, 11/15/2016 - 8:44pm

Autoimmune diseases such as rheumatoid arthritis (RA) are associated with debilitating chronic inflammation, autoantibody production, articular bone erosions and systemic bone loss. The underlying mechanisms and cell types that initiate these diseases are not fully understood, and current therapies mainly address downstream mechanisms and do not fully halt disease progression in all patients. Moreover, previous studies have largely focused on the role of adaptive immunity in driving these diseases, and less attention has been given to the contribution of innate immune pathways such as DNA sensor signaling pathways in initiating and/or perpetuating autoimmunity and erosive inflammatory arthritis.

Detection of microbial nucleic acids by DNA sensors such as endosomal toll-like receptors (TLRs) and cytosolic sensors is an early form of antiviral defense. Upon detection of nucleic acid, TLRs dependent on Unc93B and cytosolic sensors dependent on the adaptor stimulator of interferon genes (STING) orchestrate production of type 1 interferons and pro-inflammatory cytokines to resolve infection. Additionally, the cytosolic DNA sensor absent in melanoma 2 (AIM2), which is not dependent on STING, also recognizes microbial DNA and coordinates the cleavage of pro-IL-1β. Previous studies have largely focused on the role of these DNA sensors in macrophages and dendritic cells in the context of antiviral immunity. In recent years, however, the inappropriate recognition of host nucleic acids by these sensors has been associated with several autoimmune diseases including RA.

This dissertation aims to delineate the mechanisms by which DNA sensors contribute to inflammatory arthritis and bone remodeling in the context of a murine model of autoimmunity. In DNase II deficient mice, excessive accrual of undegraded, endogenous DNA leads to robust production of type 1 interferons (IFNs) and proinflammatory cytokines. The high levels of type 1 IFNs result in anemia and embryonic lethality; therefore, the gene for the type 1 IFN receptor (IFNaR) has also been deleted so that the mice survive. DNase II-/- IFNaR-/- double knockout (DKO) mice develop erosive inflammatory arthritis, anti-nuclear antibodies, and splenomegaly not seen in the DNase II+/- IFNaR-/- (Het) control group. To evaluate whether cytosolic or endosomal DNA sensors contribute to the clinical manifestations of DKO mice, genes involved in TLR or cytosolic sensor signaling were deleted on the DKO background. Genetically altered mice include STING/DNaseII/IFNaR TKO (STING TKO), AIM2/DNase II/IFNaR TKO (AIM2 TKO), and Unc93b/DNase II/IFNaR TKO (Unc93 TKO) mice.

Our hypothesis was that the STING, AIM2, and/or Unc93 pathways would contribute to the autoimmune manifestations in DNase II deficient mice. Rigorous examination of inflammation in these lines revealed important roles for both the STING and AIM2 pathways in arthritis. Despite the substantial effects of the STING and AIM2 pathways on arthritis, STING TKO and AIM2 TKO mice still exhibited prominent autoantibody production. Interestingly, inflammation persisted in Unc93 TKO mice while autoantibody production to nucleic acids was abrogated. Collectively, these data indicate that innate immune pathways contribute to the initiation/perpetuation of inflammatory arthritis and demonstrate that cytosolic and endosomal pathways play distinct roles in the manifestations of autoimmunity. Moreover, they reveal a previously undescribed role for AIM2 as a sensor of endogenous nucleic acids in inflammatory arthritis. Thus, therapeutics that target the STING and AIM2 pathways may be beneficial for the treatment of inflammatory joint diseases.

While the role of hematopoietic cells in driving autoimmunity has been well established, the contribution of stromal elements to disease pathogenesis is less well understood. Therefore, we generated bone marrow chimeras to delineate the contribution of hematopoietic and non-hematopoietic cells to the various autoimmune manifestations in DKO mice. These studies revealed that both donor hematopoietic and host radioresistant cells are required for inflammation in the joint as well as for other features of autoimmunity in DKO mice, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. This data demonstrates that stromal host cells play a major role in DNA-driven autoimmunity. Moreover, these results suggest that targeting not only hematopoietic but also stromal elements may be advantageous in the setting of inflammatory arthritis.

In the final chapter of this thesis, a role for innate immune sensor pathways in bone is described. The majority of inflammatory arthritides have been shown to lead to systemic loss of bone. Surprisingly, however, we found that DKO mice accumulate trabecular bone in the long bones over time as well as ectopic bone in the spleens, both sites of robust DNA accrual. Moreover, deficiency of the STING pathway abrogated this bone accumulation. Collectively, these data demonstrate that DNA accrual promotes dysregulated bone remodeling through innate immune sensing pathways. These findings are the first to reveal a role for the STING pathway in bone and may unveil novel targets for the treatment of diseases associated with bone disorders.

Characterization of Higher-order Chromatin Structure in Bone Differentiation and Breast Cancer: A Dissertation

Tue, 11/15/2016 - 8:44pm

Higher-order genome organization is important for the regulation of gene expression by bringing different cis-regulatory elements and promoters in proximity. The establishment and maintenance of long-range chromatin interactions occur in response to cellular and environmental cues with the binding of transcription factors and chromatin modifiers. Understanding the organization of the nucleus in differentiation and cancer has been a long standing challenge and is still not well-understood. In this thesis, I explore the dynamic changes in the higher-order chromatin structure in bone differentiation and breast cancer. First, we show dynamic chromatin contact between a distal regulatory element and the promoter of Runx2 gene, which encodes the Runtrelated transcription factor 2 (RUNX2) that is essential for bone development. Next, via using a genome-wide approach, we show that breast cancer cells have altered long-range chromatin contacts among small, gene-rich chromosomes and at telomeres when compared with mammary epithelial cells. Furthermore, we assess the changes in nuclear structure and gene expression of breast cancer cells following Runt-related transcription factor 1 (RUNX1) deficiency, an event frequently observed in breast cancer. Finally, I present the role of the central ATPase subunit of the SWI/SNF complex, SMARCA4 (BRG1), in mediating nuclear structure and gene expression. Taken together, the research presented in this thesis reveals novel insight and paradigm for the dynamic changes in disease and differentiation, as well as uncovers previously unidentified roles for two chromatin regulatory proteins, RUNX1 and SMARCA4.

Adeno-associated virus-delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model

Mon, 11/14/2016 - 10:02pm

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, paralysis, and death within 5 years of diagnosis. About 10% of cases are inherited, of which 20% are due to mutations in the superoxide dismutase 1 (SOD1) gene. Riluzole, the only US Food and Drug Administration-approved ALS drug, prolongs survival by only a few months. Experiments in transgenic ALS mouse models have shown decreasing levels of mutant SOD1 protein as a potential therapeutic approach. We sought to develop an efficient adeno-associated virus (AAV)-mediated RNAi gene therapy for ALS.

METHODS: A single-stranded AAV9 vector encoding an artificial microRNA against human SOD1 was injected into the cerebral lateral ventricles of neonatal SOD1(G93A) mice, and impact on disease progression and survival was assessed.

RESULTS: This therapy extended median survival by 50% and delayed hindlimb paralysis, with animals remaining ambulatory until the humane endpoint, which was due to rapid body weight loss. AAV9-treated SOD1(G93A) mice showed reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons and significant improvements in multiple parameters including the numbers of spinal motor neurons, diameter of ventral root axons, and extent of neuroinflammation in the SOD1(G93A) spinal cord. Mice also showed previously unexplored changes in pulmonary function, with AAV9-treated SOD1(G93A) mice displaying a phenotype reminiscent of patient pathophysiology.

INTERPRETATION: These studies clearly demonstrate that an AAV9-delivered SOD1-specific artificial microRNA is an effective and translatable therapeutic approach for ALS.

Evaluating Harms in the Assessment of Net Benefit: A Framework for Newborn Screening Condition Review

Mon, 11/14/2016 - 10:02pm

BACKGROUND: The Department of Health and Human Services (HHS) Advisory Committee on Heritable Disorders in Newborns and Children ("Advisory Committee") makes recommendations to the HHS Secretary regarding addition of new conditions to the national Recommended Uniform Screening Panel for newborns. The Advisory Committee's decision-making process includes assessing the net benefit of screening for nominated conditions, informed by systematic evidence reviews generated by an independent Condition Review Workgroup. The evidence base regarding harms associated with screening for specific conditions is often more limited than that for benefits.

PROCEDURES: The process for defining potential harms from newborn screening reviewed the frameworks from other public health evidence-based review processes, adapted to newborn screening by experts in systematic review, newborn screening programs and bioethics, with input from and approval by the Advisory Committee.

MAIN FINDINGS: To support the Advisory Committee's review of nominated conditions, the Workgroup has developed a standardized approach to evaluation of harms and relevant gaps in the evidence. Types of harms include the physical burden to infants; psychosocial and logistic burdens to families from screening or diagnostic evaluation; increased risk of medical treatment for infants diagnosed earlier than children with clinical presentation; delayed diagnosis from false negative results; psychosocial harm from false positive results; uncertainty of clinical diagnosis, age of onset or clinical spectrum; and disparities in access to diagnosis or therapy.

CONCLUSIONS: Estimating the numbers of children at risk, the magnitude, timing and likelihood of harms will be integrated into Workgroup reports to the Advisory Committee.

Sleep Disorders Associated With Mild Traumatic Brain Injury Using Sport Concussion Assessment Tool 3

Mon, 11/14/2016 - 10:02pm

BACKGROUND: Sleep problems affect 30% to 80% of patients with mild traumatic brain injury. We assessed the prevalence of sleep disorders after mild traumatic brain injury and its correlation with other symptoms.

METHODS AND MATERIALS: Individuals with mild traumatic brain injury were assessed at the New York University Concussion Center during 2013-2014 with the Sports Concussion Assessment Tool, third edition, data following mild traumatic brain injury. The relationship between sleep problems (drowsiness, difficulty falling asleep, fatigue or low energy), psychiatric symptoms (sadness, nervousness or anxiousness), headache, and dizziness were analyzed by Spearman correlation and logistic regression using moderate to severe versus none to mild categorization.

RESULTS: Ninety-three patients were retrospectively considered. The most common injury causes were falls (34.4%) and motor vehicle accidents (21.5%). There was a positive correlation between dizziness, headache, psychiatric problems (sadness, anxiety, irritability), and sleep problems (fatigue, drowsiness, and difficulty falling asleep) (P < 0.001). Logistic regression showed a significant association between moderate to severe psychiatric symptoms and moderate to severe sleep symptoms (P < 0.05). Sleep symptoms became more severe with increased time interval from mild traumatic brain injury to Sport Concussion Assessment Tool 3 administration (odds ratio = 1.005, 1.006, and 1.008, P < 0.05). There was significant correlation between motor vehicle accident and drowsiness and difficulty falling asleep (P < 0.05). Medications given in the emergency department had a positive correlation with drowsiness (P < 0.05).

CONCLUSIONS: Individuals who report moderate to severe headache, dizziness, and psychiatric symptoms have a higher likelihood of reporting moderate to severe sleep disorders following mild traumatic brain injury and should be counseled and initiated with early interventions.

Use of a FITLINE to Support Families of Overweight and Obese Children in Pediatric Practices

Mon, 11/14/2016 - 10:02pm

BACKGROUND: The American Academy of Pediatrics (AAP) recommends a staged approach to pediatric weight management, starting with helping families to make targeted dietary and activity changes. This pilot study evaluated the preliminary efficacy of a pediatric practice-based referral program to support parents in helping their overweight/obese children improve their weight-related behaviors and BMI.

METHODS: A nonrandomized intervention study with contemporaneous control was used. Parents and their children ages 8-12 with BMI > /=85th percentile (N = 37) were recruited from a pediatric practice serving a low-income, multiethnic population. Providers delivered brief intervention and referred families to six weekly FITLINE telephone counseling sessions with a nutritionist who guided parents in helping their child make AAP-recommended changes. Child BMI and parent survey of child diet and physical activity were completed at baseline and 3 months. Medical record data from 44 children matched for age and BMI were collected.

RESULTS: Mean change in BMI from baseline to 3-month follow-up was -0.49 BMI units (standard deviation [SD], 0.95; p = 0.007) for the FITLINE group and 0.35 BMI units (SD, 0.96; p = 0.02) for the control group. Adjusting for baseline BMI, age, and sex, children in the FITLINE condition reduced BMI significantly more than children in the control condition (mean difference = -0.89; p = 0.0003). Significant improvements in many dietary and sedentary behaviors also were noted.

CONCLUSIONS: The FITLINE program reduced short-term BMI and improved dietary and sedentary behaviors. A randomized, controlled trial is warranted to assess the program's efficacy and potential to serve as a model for reducing obesity in pediatric practice.

Adeno-Associated Virus Type 2 and Hepatocellular Carcinoma

Mon, 11/14/2016 - 10:02pm

In a recent Nature Genetics letter, entitled “Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas,” Nault and colleaguesdocument that of 193 patients with hepatocellular carcinoma (HCC), 11 contained an integrated genome sequence of the wild-type adeno-associated virus 2 (AAV2), and suggest that AAV2 is associated with oncogenic insertional mutagenesis in human HCC.

Because AAV2 has long been known to be a nonpathogenic human parvovirus and, in fact, has been shown to possess antitumor activity, it is critical that the scientific and clinical implications of these studies be rigorously assessed to justify their conclusions. We have carefully analyzed the data presented by Nault and colleaguesand reached a conclusion that is at variance with that of the authors.

Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice

Mon, 11/14/2016 - 10:02pm

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

Management of Upper Gastrointestinal Bleeding in Children: Variceal and Nonvariceal

Mon, 11/14/2016 - 10:02pm

Upper gastrointestinal (UGI) bleeding is generally defined as bleeding proximal to the ligament of Treitz, which leads to hematemesis. There are several causes of UGI bleeding necessitating a detailed history to rule out comorbid conditions, medications, and possible exposures. In addition, the severity, timing, duration, and volume of the bleeding are important details to note for management purposes. Despite the source of the bleeding, acid suppression with a proton-pump inhibitor has been shown to be effective in minimizing rebleeding. Endoscopy remains the interventional modality of choice for both nonvariceal and variceal bleeds because it can be diagnostic and therapeutic.

New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors

Mon, 11/14/2016 - 10:02pm

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.