Practical Murine Hematopathology: A Comparative Review and Implications for Research

Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation.Abbreviations: GEM, genetically engineered mouse; NMB, new methylene blue; nRBC, nucleated RBC; RDW, RBC distribution width; TNCC, total nucleated cell countHematology is an important adjunct to both clinical medicine and biomedical research, with more than 1700 currently funded NIH projects¹⁰⁹ and more than 3400 research articles published over the past 5 years using mouse models.¹²⁰ There are now more than 6000 genetically engineered mouse (GEM) models of disease, with 500 new GEM created each year at the Jackson Laboratory alone, and several large projects are underway to thoroughly phenotype each new mutant mouse strain (https://www.komp.org/).^13,176 A mouse tumor database (http://tumor.informatics.jax.org/mtbwi/index.do) is available to provide information regarding mouse models of human cancer, and the Mouse Phenome Database at the Jackson Laboratory provides links to phenotypic data for many GEM models (http://phenome.jax.org/).⁸ The defined components to complete the phenotyping of GEM models have been recently reviewed.^13,157,176 In addition, 21 inbred strains of mice are commonly used for investigations into such topics as response to infectious and genetically induced disease and dietary and pharmacologic therapies. These commonly used laboratory mouse strains have, for example, inherent differences in immunology or iron trafficking, which can affect research outcomes.^16,47,137 These interstrain differences are important to recognize and understand as a component of effective study design and prior to strain selection for laboratory investigations, especially when hematologic responses to disease need to be considered.^13,16,137For any appropriately designed experiment, concurrent age-, sex-, and strain-matched control mice must be included to accurately compare the effects of a disease, genetic manipulation or therapeutic intervention;^13,155 alternatively, individual mice can be used as their own controls in some studies. Several important guidelines exist to ensure that appropriate numbers of experimental and control mice are incorporated into a study design to maximize statistical power yet minimize waste.^{13,40-42,71,72,176} During and between studies, consistent blood collection methods are essential for accurate comparative analyses. Species-appropriate hematologic instrumentation and timely analysis of fresh blood are necessary to minimize preanalytic hematologic errors.^3,37,71 Especially important for mice and their restricted available blood volume are the use of practical, accurate, species-specific, and up-to-date hematologic methods.Here we comprehensively review murine hematology and hematopathologic responses to disease in the context of biomedical research, discovery, and phenotyping studies. To maximize the opportunity for detecting phenotypes, disease, and responses to therapeutic interventions in mice, we focus on providing a practical summary of methods and analysis for accurate hematologic studies and on describing the morphologic assessment of mouse hematopathology in peripheral blood and bone marrow in ways that will be useful to those—veterinarians and researchers alike—who work with murine species.     

Comparative Genomic and Proteomic Anatomy of Mycobacterium Ubiquitous Esx Family Proteins: Implications in Pathogenicity and Virulence

Secreted proteins are among the most important molecules involved in host—pathogen interaction of Mycobacterium tuberculosis, the etiological agent of human tuberculosis (TB). M. tuberculosis encodes five types of VII secretion systems (ESX-1 to ESX-5) responsible for the exportation of many proteins. This system mediated substrates including members of the Esx family implicated in tuberculosis pathogenesis and survival within host cells. However, the distribution and evolution of this family remain elusive. To explore the evolution and distribution of Esx family proteins, we analyzed all available Mycobacteria genomes. Interestingly, amino mutations among M. tuberculosis esx family proteins may relate to their functions. We further analyzed the differences between pathogenic Mycobacteria, the attenuated Mycobacteria and non-pathogenic Mycobacteria. The stability, the globular domains and the phosphorylation of serine/threonine residues of M. tuberculosis esx proteins with their homologies among other Mycoabcteria were analyzed. Our comparative genomic and proteomic analysis found that the change of stability, gain or loss of globular domains and phosphorylation of serine/threonine might be responsible for the difference between the pathogenesis and virulence of the esx proteins and its homolog widespread among Mycobacteria and related species, which may provide clues for novel anti-tuberculosis drug targets.     

Th17 Pathway As a Target for Multipotent Stromal Cell Therapy in Dogs: Implications for Translational Research

Detrimental Th17 driven inflammatory and autoimmune disease such as Crohn’s disease, graft versus host disease and multiple sclerosis remain a significant cause of morbidity and mortality worldwide. Multipotent stromal/stem cell (MSC) inhibit Th17 polarization and activation in vitro and in rodent models. As such, MSC based therapeutic approaches are being investigated as novel therapeutic approaches to treat Th17 driven diseases in humans. The significance of naturally occurring diseases in dogs is increasingly recognized as a realistic platform to conduct pre-clinical testing of novel therapeutics. Full characterization of Th17 cells in dogs has not been completed. We have developed and validated a flow-cytometric method to detect Th17 cells in canine blood. We further demonstrate that Th17 and other IL17 producing cells are present in tissues of dogs with naturally occurring chronic inflammatory diseases. Finally, we have determined the kinetics of a canine specific Th17 polarization in vitro and demonstrate that canine MSC inhibit Th17 polarization in vitro, in a PGE₂ independent mechanism. Our findings provide fundamental research tools and suggest that naturally occurring diseases in dogs, such as inflammatory bowel disease, may be harnessed to translate novel MSC based therapeutic strategies that target the Th17 pathway.     

The Endocranial Anatomy of Therizinosauria and Its Implications for Sensory and Cognitive Function

Background

Therizinosauria is one of the most enigmatic and peculiar clades among theropod dinosaurs, exhibiting an unusual suite of characters, such as lanceolate teeth, a rostral rhamphotheca, long manual claws, and a wide, opisthopubic pelvis. This specialized anatomy has been associated with a shift in dietary preferences and an adaptation to herbivory. Despite a large number of discoveries in recent years, the fossil record for Therizinosauria is still relatively poor, and cranial remains are particularly rare.

Methodology/Principal Findings

Based on computed tomographic (CT) scanning of the nearly complete and articulated skull of Erlikosaurus andrewsi, as well as partial braincases of two other therizinosaurian taxa, the endocranial anatomy is reconstructed and described. The wider phylogenetic range of the described specimens permits the evaluation of sensory and cognitive capabilities of Therizinosauria in an evolutionary context. The endocranial anatomy reveals a mosaic of plesiomorphic and derived characters in therizinosaurians. The anatomy of the olfactory apparatus and the endosseous labyrinth suggests that olfaction, hearing, and equilibrium were well-developed in therizinosaurians and might have affected or benefited from an enlarged telencephalon.

Conclusion/Significance

This study presents the first appraisal of the evolution of endocranial anatomy and sensory adaptations in Therizinosauria. Despite their phylogenetically basal position among maniraptoran dinosaurs, therizinosaurians had developed the neural pathways for a well developed sensory repertoire. In particular olfaction and hearing may have played an important role in foraging, predator evasion, and/or social complexity.     

The Endoplasmic Reticulum Stress Response in Neuroprogressive Diseases: Emerging Pathophysiological Role and Translational Implications

The endoplasmic reticulum (ER) is the main cellular organelle involved in protein synthesis, assembly and secretion. Accumulating evidence shows that across several neurodegenerative and neuroprogressive diseases, ER stress ensues, which is accompanied by over-activation of the unfolded protein response (UPR). Although the UPR could initially serve adaptive purposes in conditions associated with higher cellular demands and after exposure to a range of pathophysiological insults, over time the UPR may become detrimental, thus contributing to neuroprogression. Herein, we propose that immune-inflammatory, neuro-oxidative, neuro-nitrosative, as well as mitochondrial pathways may reciprocally interact with aberrations in UPR pathways. Furthermore, ER stress may contribute to a deregulation in calcium homoeostasis. The common denominator of these pathways is a decrease in neuronal resilience, synaptic dysfunction and even cell death. This review also discusses how mechanisms related to ER stress could be explored as a source for novel therapeutic targets for neurodegenerative and neuroprogressive diseases. The design of randomised controlled trials testing compounds that target aberrant UPR-related pathways within the emerging framework of precision psychiatry is warranted.     

脑发育性静脉畸形9例临床影像特征分析及文献复习

目的:研究脑发育性静脉畸形(Cerebral Developmental Venous Anomalies,CDVA)临床及影像学特征及复习CDVA文献。方法:回顾性收集了自2011年11月至2014年3月我科确诊的9例CDVA的病人,对其临床特征、影像学检查方法包括电子计算机断层扫描(Computed Tomography,CT)、核磁共振成像(Magnetic Resonance Imaging,MRI)、数字减影血管造影(Digital Subtraction Angiography,DSA)及特征进行分析并对相关文献进行复习。结果:(1)临床症状:9例病人的临床症状包括头晕4例(4/9)、头痛4例(4/9)、恶心不适2例(2/9)、站立不稳1例((1/9)、小脑出血史1例(1/9)、眼部症状行眼科检查偶然发现小脑CDVA1例(1/9);(2)病变部位:病变位于幕上4例(4/9);幕下5例(5/9);(3)影像学检查:9例病人中,6例行CT平扫或增强扫描(3例平扫,3例平扫+增强);4例行MRI(1例平扫,3例平扫+增强);3例行DSA检查;(4)影像学特点:CT增强及重建、MRI的T1WI增强、SWI、MRA及DSA静脉期像均可显示出髓静脉及其形成的特征性"海蛇头"征象和其引流静脉。结论:CT、MRI、DSA影像学方法均可用于CDVA的诊断,在临床实践中需根据需要优化选择联合应用。     

Challenges in Translational Research: The Views of Addiction Scientists

Objectives

To explore scientists'' perspectives on the challenges and pressures of translating research findings into clinical practice and public health policy.

Methods

We conducted semi-structured interviews with a purposive sample of 20 leading scientists engaged in genetic research on addiction. We asked participants for their views on how their own research translates, how genetic research addresses addiction as a public health problem and how it may affect the public''s view of addiction.

Results

Most scientists described a direct translational route for their research, positing that their research will have significant societal benefits, leading to advances in treatment and novel prevention strategies. However, scientists also pointed to the inherent pressures they feel to quickly translate their research findings into actual clinical or public health use. They stressed the importance of allowing the scientific process to play out, voicing ambivalence about the recent push to speed translation.

Conclusions

High expectations have been raised that biomedical science will lead to new prevention and treatment modalities, exerting pressure on scientists. Our data suggest that scientists feel caught in the push for immediate applications. This overemphasis on rapid translation can lead to technologies and applications being rushed into use without critical evaluation of ethical, policy, and social implications, and without balancing their value compared to public health policies and interventions currently in place.     

Synthesis and Phosphorylation of Maize Acidic Ribosomal Proteins : Implications in Translational Regulation

The objective of this research was to determine the role of acidic ribosomal protein (ARP) phosphorylation in translation. Ribosomes (Rbs) from germinated maize (Zea mays L.) axes had four ARP bands within 4.2 to 4.5 isoelectric points when analyzed by isoelectric focusing. Two of these bands disappeared after alkaline phosphatase hydrolysis. During germination a progressive change from nonphosphorylated (0 h) to phosphorylated ARP (24 h) forms was observed in the Rbs; a free cytoplasmic pool of nonphosphorylated ARPs was also identified by immunoblot and isoelectric focusing experiments. De novo ARP synthesis initiated very slowly early in germination, whereas ARP phosphorylation occurred rapidly within this period. ARP-phosphorylated versus ARP-nonphosphorylated Rbs were tested in an in vitro reticulocyte lysate translation system. Greater in vitro mRNA translation rates were demonstrated for the ARP-phosphorylated Rbs than for the non-ARP-phosphorylated ones. Rapamycin application to maize axes strongly inhibited S6 ribosomal protein phosphorylation, but did not interfere with the ARP phosphorylation reaction. We conclude that ARP phosphorylation does not depend on ARP synthesis or on ARP assembly into Rbs. Rather, this process seems to be part of a translational regulation mechanism.     

Phenotype Ontologies and Cross-Species Analysis for Translational Research

The use of model organisms as tools for the investigation of human genetic variation has significantly and rapidly advanced our understanding of the aetiologies underlying hereditary traits. However, while equivalences in the DNA sequence of two species may be readily inferred through evolutionary models, the identification of equivalence in the phenotypic consequences resulting from comparable genetic variation is far from straightforward, limiting the value of the modelling paradigm. In this review, we provide an overview of the emerging statistical and computational approaches to objectively identify phenotypic equivalence between human and model organisms with examples from the vertebrate models, mouse and zebrafish. Firstly, we discuss enrichment approaches, which deem the most frequent phenotype among the orthologues of a set of genes associated with a common human phenotype as the orthologous phenotype, or phenolog, in the model species. Secondly, we introduce and discuss computational reasoning approaches to identify phenotypic equivalences made possible through the development of intra- and interspecies ontologies. Finally, we consider the particular challenges involved in modelling neuropsychiatric disorders, which illustrate many of the remaining difficulties in developing comprehensive and unequivocal interspecies phenotype mappings.     

The Hypocretin/Orexin System: Implications for Drug Reward and Relapse

Hypocretins (also known as orexins) are hypothalamic neuropeptides involved in the regulation of sleep/wake states and feeding behavior. Recent studies have also demonstrated an important role for the hypocretin/orexin system in the addictive properties of drugs of abuse, consistent with the reciprocal innervations between hypocretin neurons and brain areas involved in reward processing. This system participates in the primary reinforcing effects of opioids, nicotine, and alcohol. Hypocretins are also involved in the neurobiological mechanisms underlying relapse to drug-seeking behavior induced by drug-related environmental stimuli and stress, as mainly described in the case of psychostimulants. Based on these preclinical studies, the use of selective ligands targeting hypocretin receptors could represent a new therapeutical strategy for the treatment of substance abuse disorders. In this review, we discuss and update the current knowledge about the participation of the hypocretin system in drug addiction and the possible neurobiological mechanisms involved in these processes regulated by hypocretin transmission.     

PaVESy: Pathway Visualization and Editing System

A data managing system for editing and visualization of biological pathways is presented. The main component of PaVESy (Pathway Visualization and Editing System) is a relational SQL database system. The database design allows storage of biological objects, such as metabolites, proteins, genes and respective relations, which are required to assemble metabolic and regulatory biological interactions. The database model accommodates highly flexible annotation of biological objects by user-defined attributes. In addition, specific roles of objects are derived from these attributes in the context of user-defined interactions, e.g. in the course of pathway generation or during editing of the database content. Furthermore, the user may organize and arrange the database content within a folder structure and is free to group and annotate database objects of interest within customizable subsets. Thus, we allow an individualized view on the database content and facilitate user customization. A JAVA-based class library was developed, which serves as the database programming interface to PaVESy. This API provides classes, which implement the concepts of object persistence in SQL databases, such as entries, interactions, annotations, folders and subsets. We created editing and visualization tools for navigation in and visualization of the database content. User approved pathway assemblies are stored and may be retrieved for continued modification, annotation and export. Data export is interfaced with a range of network visualization programs, such as Pajek or other software allowing import of SBML or GML data format. AVAILABILITY: http://pavsey.mpimp-golm.mpg.de