Contribution of circulating Mfge8 to human T2DM and cardiovascular disease.

MFGE8 is a major exosome (EV) protein known to mediate inflammation and atherosclerosis in type 2 diabetes mellitus (T2DM) in animal studies. The pathophysiological role of this protein in obesity, T2DM, and cardiovascular disease is less investigated in humans. Earlier we reported a rare Asian Indian population-specific missense variant (rs371227978; Arg148His) in the MFGE8 gene associated with increased circulating Mfge8 and T2DM. We have further investigated the role of Mfge8 with T2DM risk in additional Asian Indians (n = 4897) and Europeans and other multiethnic cohorts from UK Biobank (UKBB) (n = 455,808) and the US (n = 1150). We also evaluated the exposure of Mfge8-enriched human EVs in zebrafish (ZF) for their impact on cardiometabolic organ system. Most individual carriers of Arg148His variant not only had high circulating Mfge8 but also revealed a positive significant correlation with glucose (r = 0.42; p = 4.9 × 10-04), while the non-carriers showed a negative correlation of Mfge8 with glucose (r = -0.38; p = 0.001) in Asian Indians. The same variant was monomorphic in non-South Asian ethnicities. Even without the variant, serum Mfge8 correlated significantly with blood glucose in other non-South Asian ethnicities (r = 0.47; p = 2.2 × 10-13). Since Mfge8 is an EV marker, we tested the exposure of Mfge8-enriched human EVs to ZF larvae as an exploratory study. The ZF larvae showed rapid effects on insulin-sensitive organs, developing fatty liver disease, heart hypertrophy and exhibiting redundant growth with poor muscular architecture with and without the high-fat diet (HFD). In contrast, the control group fishes developed fatty liver disease and heart hypertrophy only after the HFD feeding. Backed with strong support from animal studies on the role of Mfge8 in obesity, insulin resistance, and atherosclerosis, the current research suggests that circulating Mfge8 may become a potential marker for predicting the risk of T2DM and cardiovascular disease in humans.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, Ags. B cells also reside and develop in the thymus, although their functions are less clear. During "thymic involution," a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ∼1% of human neonatal thymocytes but up to ∼10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. In this article, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- versus postinvolution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells postinvolution. We also performed RNA sequencing on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is, to our knowledge, the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution and establishing the highly genetically manipulatable zebrafish model as a template for involution studies.

The hepatitis E virus ORF1 hypervariable region confers partial cyclophilin dependency.

Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, but mortality rates in some populations such as pregnant women can reach 30 %. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilins A and B (CypA/B) are the two most abundant Cyps in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using sub-genomic replicons. Using a combination of pharmacological inhibition by cyclosporine A (CsA), and silencing by small hairpin RNA we find that CypA and CypB are not essential for HEV replication. However, we find that silencing of CypB reduces replication of some HEV isolates in some cells. Furthermore, sensitivity to Cyp silencing appears to be partly conferred by the sequence within the hypervariable region of the viral polyprotein. These data suggest HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype- and sequence-specific.

Dynamic Changes in Lymphocyte Populations Establish Zebrafish as a Thymic Involution Model.

The thymus is the site of T lymphocyte development and T cell education to recognize foreign, but not self, antigens. B cells also reside and develop in the thymus, although their functions are less clear. During 'thymic involution,' a process of lymphoid atrophy and adipose replacement linked to sexual maturation, thymocytes decline. However, thymic B cells decrease far less than T cells, such that B cells comprise ~1% of human neonatal thymocytes, but up to ~10% in adults. All jawed vertebrates possess a thymus, and we and others have shown zebrafish (Danio rerio) also have thymic B cells. Here, we investigated the precise identities of zebrafish thymic T and B cells and how they change with involution. We assessed the timing and specific details of zebrafish thymic involution using multiple lymphocyte-specific, fluorophore-labeled transgenic lines, quantifying the changes in thymic T- and B-lymphocytes pre- vs. post-involution. Our results prove that, as in humans, zebrafish thymic B cells increase relative to T cells post-involution. We also performed RNA sequencing (RNA-seq) on D. rerio thymic and marrow lymphocytes of four novel double-transgenic lines, identifying distinct populations of immature T and B cells. Collectively, this is the first comprehensive analysis of zebrafish thymic involution, demonstrating its similarity to human involution, and establishing the highly genetically-manipulatable zebrafish model as a template for involution studies.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Veterinary Clinical Education Delivery Models: A Conceptual Framework.

The sustainability of the traditional university-owned and -operated veterinary teaching hospital has been discussed for many years. Concerns around the shortage and lack of diversity of clinical faculty, financial sustainability, and suitability of secondary and tertiary case load for the development of Doctor of Veterinary Medicine students' Day One Competences are perennial issues. Consequently, many schools have been looking at alternative ways of delivering veterinary clinical education. This article provides a conceptual framework for evaluating the delivery of veterinary clinical education, providing putative advantages and disadvantages of each model for further empirical investigation. Four different models are proposed-owner, third party, embedded distributive, and fully distributive-that can broadly be defined along two dimensions: the degree of integration of the clinical enterprise with the academic enterprise and the degree of authority of the dean/head of school with respect to clinical enterprise governance and their role in budgetary, investment, and hiring decisions. The author offers a typology that may assist deans/heads of schools make strategic decisions about the mode of delivery of veterinary clinical education for their school.

Association of Combined Focal 22q11.22 Deletion and IKZF1 Alterations With Outcomes in Childhood Acute Lymphoblastic Leukemia.

IMPORTANCE: Alterations in the IKZF1 gene drive B-cell acute lymphoblastic leukemia (B-ALL) but are not routinely used to stratify patients by risk because of inconsistent associations with outcomes. We describe a novel deletion in 22q11.22 that was consistently associated with very poor outcomes in patients with B-ALL with IKZF1 alterations. OBJECTIVE: To determine whether focal deletions within the λ variable chain region in chromosome 22q11.22 were associated with patients with B-ALL with IKZF1 alterations with the highest risk of relapse and/or death. DESIGN, SETTING, AND PARTICIPANTS: This cohort study included 1310 primarily high-risk pediatric patients with B-ALL who were taken from 6 independent clinical cohorts, consisting of 3 multicenter cohorts (AALL0232 [2004-2011], P9906 [2000-2003], and patients with Down syndrome who were pooled from national and international studies) and 3 single-institution cohorts (University of Utah [Salt Lake City], Children's Hospital of Philadelphia [Philadelphia, Pennsylvania], and St. Jude Children's Hospital [Memphis, Tennessee]). Data analysis began in 2011 using patients from the older studies first, and data analysis concluded in 2021. EXPOSURES: Focal 22q11.22 deletions. MAIN OUTCOMES AND MEASURES: Event-free and overall survival was investigated. The hypothesis that 22q11.22 deletions stratified the prognostic effect of IKZF1 alterations was formulated while investigating nearby deletions in VPREB1 in 2 initial cohorts (n = 270). Four additional cohorts were then obtained to further study this association (n = 1040). RESULTS: This study of 1310 patients with B-ALL (717 male [56.1%] and 562 female patients [43.9%]) found that focal 22q11.22 deletions are frequent (518 of 1310 [39.5%]) in B-ALL and inconsistent with physiologic V(D)J recombination. A total of 299 of 1310 patients with B-ALL had IKZF1 alterations. Among patients with IKZF1 alterations, more than half shared concomitant focal 22q11.22 deletions (159 of 299 [53.0%]). Patients with combined IKZF1 alterations and 22q11.22 deletions had worse outcomes compared with patients with IKZF1 alterations and wild-type 22q11.22 alleles in every cohort examined (combined cohorts: 5-year event-free survival rates, 43.3% vs 68.5%; hazard ratio [HR], 2.18; 95% CI, 1.54-3.07; P < .001; 5-year overall survival rates, 66.9% vs 83.9%; HR, 2.05; 95% CI, 1.32-3.21; P = .001). While 22q11.22 deletions were not prognostic in patients with wild-type IKZF1 , concomitant 22q11.22 deletions in patients with IKZF1 alterations stratified outcomes across additional risk groups, including patients who met the IKZF1plus criteria, and maintained independent significance in multivariate analysis for event-free survival (HR, 2.05; 95% CI, 1.27-3.29; P = .003) and overall survival (HR, 1.83; 95% CI, 1.01-3.34; P = .05). CONCLUSIONS AND RELEVANCE: This cohort study suggests that 22q11.22 deletions identify patients with B-ALL and IKZF1 alterations who have very poor outcomes and may offer a new genetic biomarker to further refine B-ALL risk stratification and treatment strategies.

An Adverse Outcome Pathway for Decreased Lung Function Focusing on Mechanisms of Impaired Mucociliary Clearance Following Inhalation Exposure.

Adverse outcome pathways (AOPs) help to organize available mechanistic information related to an adverse outcome into key events (KEs) spanning all organizational levels of a biological system(s). AOPs, therefore, aid in the biological understanding of a particular pathogenesis and also help with linking exposures to eventual toxic effects. In the regulatory context, knowledge of disease mechanisms can help design testing strategies using in vitro methods that can measure or predict KEs relevant to the biological effect of interest. The AOP described here evaluates the major processes known to be involved in regulating efficient mucociliary clearance (MCC) following exposures causing oxidative stress. MCC is a key aspect of the innate immune defense against airborne pathogens and inhaled chemicals and is governed by the concerted action of its functional components, the cilia and airway surface liquid (ASL). The AOP network described here consists of sequences of KEs that culminate in the modulation of ciliary beat frequency and ASL height as well as mucus viscosity and hence, impairment of MCC, which in turn leads to decreased lung function.

Zebrafish B cell acute lymphoblastic leukemia: new findings in an old model.

Acute lymphoblastic leukemia (ALL) is the most common pediatric, and ninth most common adult, cancer. ALL can develop in either B or T lymphocytes, but B-lineage ALL (B-ALL) exceeds T-ALL clinically. As for other cancers, animal models allow study of the molecular mechanisms driving ALL. Several zebrafish (Danio rerio) T-ALL models have been reported, but until recently, robust D. rerio B-ALL models were not described. Then, D. rerio B-ALL was discovered in two related zebrafish transgenic lines; both were already known to develop T-ALL. Here, we report new B-ALL findings in one of these models, fish expressing transgenic human MYC (hMYC). We describe B-ALL incidence in a large cohort of hMYC fish, and show B-ALL in two new lines where T-ALL does not interfere with B-ALL detection. We also demonstrate B-ALL responses to steroid and radiation treatments, which effect ALL remissions, but are usually followed by prompt relapses. Finally, we report gene expression in zebrafish B lymphocytes and B-ALL, in both bulk samples and single B- and T-ALL cells. Using these gene expression profiles, we compare differences between the two new D. rerio B-ALL models, which are both driven by transgenic mammalian MYC oncoproteins. Collectively, these new data expand the utility of this new vertebrate B-ALL model.

Tackling Acute Lymphoblastic Leukemia-One Fish at a Time.

Despite advancements in the diagnosis and treatment of acute lymphoblastic leukemia (ALL), a need for improved strategies to decrease morbidity and improve cure rates in relapsed/refractory ALL still exists. Such approaches include the identification and implementation of novel targeted combination regimens, and more precise upfront patient risk stratification to guide therapy. New curative strategies rely on an understanding of the pathobiology that derives from systematically dissecting each cancer's genetic and molecular landscape. Zebrafish models provide a powerful system to simulate human diseases, including leukemias and ALL specifically. They are also an invaluable tool for genetic manipulation, in vivo studies, and drug discovery. Here, we highlight and summarize contributions made by several zebrafish T-ALL models and newer zebrafish B-ALL models in translating the underlying genetic and molecular mechanisms operative in ALL, and also highlight their potential utility for drug discovery. These models have laid the groundwork for increasing our understanding of the molecular basis of ALL to further translational and clinical research endeavors that seek to improve outcomes in this important cancer.

Variability in body temperature in healthy adults and in patients receiving chemotherapy: prospective observational cohort study.

Between-individual variability of body temperature has been little investigated, but is of clinical importance: for example, in detection of neutropenic sepsis during chemotherapy. We studied within-person and between-person variability in temperature in healthy adults and those receiving chemotherapy using a prospective observational design involving 29 healthy participants and 23 patients undergoing chemotherapy. Primary outcome was oral temperature. We calculated each patient's mean temperature, standard deviation within each patient (within-person variability), and between patients (between-person variability). Secondary analysis explored temperature changes in the three days before admission for neutropenic sepsis. 1,755 temperature readings were returned by healthy participants and 1,765 by chemotherapy patients. Mean participant temperature was 36.16 C (95% CI 36.07-36.26) in healthy participants and 36.32 C (95% CI 36.18-36.46) in chemotherapy patients. Healthy participant within-person variability was 0.40 C (95% CI 0.36-0.44) and between-person variability was 0.26 C (95% CI 0.16-0.35). Chemotherapy patient within-person variability was 0.39 C (95% CI 0.34-0.44) and between-person variability was 0.34 C (95% CI 0.26-0.48). Thus, use of a population mean rather than personalised baselines is probably sufficient for most clinical purposes as between-person variability is not large compared to within-person variability. Standardised guidance and provision of thermometers to patients might help to improve recording and guide management.

Targeted sequencing of candidate genes of dyslipidemia in Punjabi Sikhs: Population-specific rare variants in GCKR promote ectopic fat deposition.

Dyslipidemia is a well-established risk factor for cardiovascular diseases. Although, advances in genome-wide technologies have enabled the discovery of hundreds of genes associated with blood lipid phenotypes, most of the heritability remains unexplained. Here we performed targeted resequencing of 13 bona fide candidate genes of dyslipidemia to identify the underlying biological functions. We sequenced 940 Sikh subjects with extreme serum levels of hypertriglyceridemia (HTG) and 2,355 subjects were used for replication studies; all 3,295 participants were part of the Asian Indians Diabetic Heart Study. Gene-centric analysis revealed burden of variants for increasing HTG risk in GCKR (p = 2.1x10-5), LPL (p = 1.6x10-3) and MLXIPL (p = 1.6x10-2) genes. Of these, three missense and damaging variants within GCKR were further examined for functional consequences in vivo using a transgenic zebrafish model. All three mutations were South Asian population-specific and were largely absent in other multiethnic populations of Exome Aggregation Consortium. We built different transgenic models of human GCKR with and without mutations and analyzed the effects of dietary changes in vivo. Despite the short-term of feeding, profound phenotypic changes were apparent in hepatocyte histology and fat deposition associated with increased expression of GCKR in response to a high fat diet (HFD). Liver histology of the GCKRmut showed severe fatty metamorphosis which correlated with ~7 fold increase in the mRNA expression in the GCKRmut fish even in the absence of a high fat diet. These findings suggest that functionally disruptive GCKR variants not only increase the risk of HTG but may enhance ectopic lipid/fat storage defects in absence of obesity and HFD. To our knowledge, this is the first transgenic zebrafish model of a putative human disease gene built to accurately assess the influence of genetic changes and their phenotypic consequences in vivo.

Isolating Malignant and Non-Malignant B Cells from lck:eGFP Zebrafish.

Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.

Simultaneous B and T cell acute lymphoblastic leukemias in zebrafish driven by transgenic MYC: implications for oncogenesis and lymphopoiesis.

Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly- penetrant zebrafish pre-B ALL, driven by human MYC. Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)-which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC-driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies.

Characteristics predicting outcomes of allogeneic stem-cell transplantation in relapsed acute myelogenous leukemia.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation (ahsct) is associated with significant morbidity and mortality, but it can cure carefully selected patients with acute myeloid leukemia (aml) in second remission (cr2). In a cohort of patients with aml who underwent ahsct in cr2, we determined the pre-transplant factors that predicted for overall survival (os), relapse, and non-relapse mortality. We also sought to validate the prognostic risk groups derived by Michelis and colleagues in this independent population. METHODS: In a retrospective chart review, we obtained data for 55 consecutive patients who underwent ahsct for aml in cr2. Hazard ratios were used to describe the independent effects of pre-transplant variables on outcome, and Kaplan-Meier curves were used to assess outcomes in the three prognostic groups identified by Michelis and colleagues. RESULTS: At 1, 3, and 5 years post-transplant, os was 60%, 45.5%, and 37.5% respectively. Statistically significant differences in os, relapse mortality, and non-relapse mortality were not identified between the prognostic risk groups identified by Michelis and colleagues. Women were less likely than men to relapse, and a modified European Society for Blood and Marrow Transplantation (mebmt) score of 3 or less was associated with a lower non-relapse mortality. CONCLUSIONS: The 37.5% 5-year os in this cohort suggests that, compared with other options, ahsct offers patients with aml in cr2 a better chance of cure. Our study supports the use of the mebmt score to predict non-relapse mortality in this population.

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 µM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.