Publications by authors named "Gauthaman Sukumar"

22 Publications

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Response of human macrophages to gamma radiation is mediated via expression of endogenous retroviruses.

PLoS Pathog 2021 Feb 8;17(2):e1009305. Epub 2021 Feb 8.

Department of Pharmacology & Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.

Ionizing radiation-induced tissue damage recruits monocytes into the exposed area where they are differentiated to macrophages. These implement phagocytic removal of dying cells and elicit an acute inflammatory response, but can also facilitate tumorigenesis due to production of anti-inflammatory cytokines. Using primary human monocyte-derived macrophages (MDMs) and the THP1 monocytic cell line, we demonstrate that gamma radiation triggers monocyte differentiation toward the macrophage phenotype with increased expression of type I interferons (IFN-I) and both pro- and anti-inflammatory macrophage activation markers. We found that these changes correlate with significantly upregulated expression of 622 retroelements from various groups, particularly of several clades of human endogenous retroviruses (HERVs). Elevated transcription was detected in both sense and antisense directions in the HERV subgroups tested, including the most genetically homogeneous clade HML-2. The level of antisense transcription was three- to five-fold higher than of the sense strand levels. Using a proximity ligation assay and immunoprecipitation followed by RNA quantification, we identified an increased amount of the dsRNA receptors MDA-5 and TLR3 bound to an equivalent number of copies of sense and antisense chains of HERVK HML-2 RNA. This binding triggered MAVS-associated signaling pathways resulting in increased expression of IFN-I and inflammation related genes that enhanced the cumulative inflammatory effect of radiation-induced senescence. HML-2 knockdown was accompanied with reduced expression and secretion of IFNα, pro-inflammatory (IL-1β, IL-6, CCL2, CCL3, CCL8, and CCL20) and anti-inflammatory (IL10) modulators in irradiated monocytes and MDMs. Taken together, our data indicate that radiation stress-induced HERV expression enhances the IFN-I and cytokine response and results in increased levels of pro-inflammatory modulators along with expression of anti-inflammatory factors associated with the macrophage tumorigenic phenotype.
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http://dx.doi.org/10.1371/journal.ppat.1009305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895352PMC
February 2021

Distinguishing Smoking-Related Lung Disease Phenotypes Via Imaging and Molecular Features.

Chest 2021 Feb 16;159(2):549-563. Epub 2020 Sep 16.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, MA; Applied Chest Imaging Laboratory, Brigham and Women's Hospital, Boston, MA.

Background: Chronic tobacco smoke exposure results in a broad range of lung pathologies including emphysema, airway disease and parenchymal fibrosis as well as a multitude of extra-pulmonary comorbidities. Prior work using CT imaging has identified several clinically relevant subgroups of smoking related lung disease, but these investigations have generally lacked organ specific molecular correlates.

Research Question: Can CT imaging be used to identify clinical phenotypes of smoking related lung disease that have specific bronchial epithelial gene expression patterns to better understand disease pathogenesis?

Study Design And Methods: Using K-means clustering, we clustered participants from the COPDGene study (n = 5,273) based on CT imaging characteristics and then evaluated their clinical phenotypes. These clusters were replicated in the Detection of Early Lung Cancer Among Military Personnel (DECAMP) cohort (n = 360), and were further characterized using bronchial epithelial gene expression.

Results: Three clusters (preserved, interstitial predominant and emphysema predominant) were identified. Compared to the preserved cluster, the interstitial and emphysema clusters had worse lung function, exercise capacity and quality of life. In longitudinal follow-up, individuals from the emphysema group had greater declines in exercise capacity and lung function, more emphysema, more exacerbations, and higher mortality. Similarly, genes involved in inflammatory pathways (tumor necrosis factor-α, interferon-β) are more highly expressed in bronchial epithelial cells from individuals in the emphysema cluster, while genes associated with T-cell related biology are decreased in these samples. Samples from individuals in the interstitial cluster generally had intermediate levels of expression of these genes.

Interpretation: Using quantitative CT imaging, we identified three groups of individuals in older ever-smokers that replicate in two cohorts. Airway gene expression differences between the three groups suggests increased levels of inflammation in the most severe clinical phenotype, possibly mediated by the tumor necrosis factor-α and interferon-β pathways.

Clinical Trial Registration: COPDGene (NCT00608764), DECAMP-1 (NCT01785342), DECAMP-2 (NCT02504697).
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http://dx.doi.org/10.1016/j.chest.2020.08.2115DOI Listing
February 2021

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include Up-Regulated Innate Immune Modulators.

Front Immunol 2020 12;11:1219. Epub 2020 Jun 12.

Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

Formation of pathological anti-FVIII antibodies, or "inhibitors," is the most serious complication of therapeutic FVIII infusions, affecting up to 1/3 of severe Hemophilia A (HA) patients. Inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development vs. immune tolerance, achieved with or without Immune Tolerance Induction (ITI) therapy, are not well-understood. To address these questions, temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) was carried out for HA subjects with and without a current or historic inhibitor using RNA-Seq. PBMCs were isolated from 40 subjects in the following groups: HA with an inhibitor that resolved either following ITI or spontaneously; HA with a current inhibitor; HA with no inhibitor history and non-HA controls. PBMCs were stimulated with 5 nM FVIII and RNA was isolated 4, 16, 24, and 48 h following stimulation. Time-series differential expression analysis was performed and distinct transcriptional signatures were identified for each group, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. Subjects with a current inhibitor showed differential expression of 56 genes and a clustering analysis identified three major temporal profiles. Interestingly, gene ontology enrichments featured innate immune modulators, including , and and are associated with enhanced secretion of the pro-inflammatory cytokines IL-1β and TNFα, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. RNA-Seq results were validated by RT-qPCR, ELISAs, multiplex cytokine analysis, and flow cytometry. The inflammatory status of HA patients suffering from an ongoing inhibitor includes up-regulated innate immune modulators, which may act as ongoing danger signals that influence the responses to, and eventual outcomes of, ITI therapy.
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http://dx.doi.org/10.3389/fimmu.2020.01219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303277PMC
June 2020

Genetic and Virulence Profiles of Enteroaggregative (EAEC) Isolated From Deployed Military Personnel (DMP) With Travelers' Diarrhea.

Front Cell Infect Microbiol 2020 20;10:200. Epub 2020 May 20.

Department of Microbiology and Immunolgy, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

To discern if there was a particular genotype associated with clinical enteroaggregative (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of (SPATEs). A separate subset of the EAEC had and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic . None of these CS22-encoding EAEC formed biofilms or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an + - isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype + - and/or capacity to form biofilm may correlate to disease severity.
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http://dx.doi.org/10.3389/fcimb.2020.00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251025PMC
May 2020

Highly multiplexed proteomic assessment of human bone marrow in acute myeloid leukemia.

Blood Adv 2020 Jan;4(2):367-379

Laboratory of Myeloid Malignancies, Hematology Branch, National Heart, Lung, and Blood Institute, and.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by abnormal clonal proliferation of myeloid progenitor cells found predominantly within the bone marrow (BM) and blood. Recent studies suggest that genetic and phenotypic alterations in the BM microenvironment support leukemogenesis and allow leukemic cells to survive and evade chemotherapy-induced death. However, despite substantial evidence indicating the role of tumor-host interactions in AML pathogenesis, little is known about the complex microenvironment of the BM. To address this, we performed novel proteomic profiling of the noncellular compartment of the BM microenvironment in patients with AML (n = 10) and age- and sex-matched healthy control subjects (n = 10) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). We show that proteomic assessment of blood or RNA-sequencing of BM are suboptimal alternate screening strategies to determine the true proteomic composition of the extracellular soluble compartment of AML patient BM. Proteomic analysis revealed that 168 proteins significantly differed in abundance, with 91 upregulated and 77 downregulated in leukemic BM. A highly connected signaling network of cytokines and chemokines, including IL-8, was found to be the most prominent proteomic signature associated with AML in the BM microenvironment. We report the first description of significantly elevated levels of the myelosuppressive chemokine CCL23 (myeloid progenitor inhibitory factor-1) in both AML and myelodysplastic syndrome patients and perform functional experiments supportive of a role in the suppression of normal hematopoiesis. This unique paired RNA-sequencing and proteomics data set provides innovative mechanistic insights into AML and healthy aging and should serve as a useful public resource.
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http://dx.doi.org/10.1182/bloodadvances.2019001124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988405PMC
January 2020

FOXP3 protects conventional human T cells from premature restimulation-induced cell death.

Cell Mol Immunol 2021 Jan 28;18(1):194-205. Epub 2019 Oct 28.

Department of Pharmacology & Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD, 20817, USA.

The adaptive immune response relies on specific apoptotic programs to maintain homeostasis. Conventional effector T cell (Tcon) expansion is constrained by both forkhead box P3 (FOXP3)-regulatory T cells (Tregs) and restimulation-induced cell death (RICD), a propriocidal apoptosis pathway triggered by repeated stimulation through the T-cell receptor (TCR). Constitutive FOXP3 expression protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signaling strength. The role of transient FOXP3 induction in activated human CD4 and CD8 Tcons remains unresolved, but its expression is inversely correlated with acquired RICD sensitivity. Here, we describe a novel role for FOXP3 in protecting human Tcons from premature RICD during expansion. Unlike FOXP3-mediated protection from RICD in Tregs, FOXP3 protects Tcons through a distinct mechanism requiring de novo transcription that does not require SAP suppression. Transcriptome profiling and functional analyses of expanding Tcons revealed that FOXP3 enhances expression of the SLAM family receptor CD48, which in turn sustains basal autophagy and suppresses pro-apoptotic p53 signaling. Both CD48 and FOXP3 expression reduced p53 accumulation upon TCR restimulation. Furthermore, silencing FOXP3 expression or blocking CD48 decreased the mitochondrial membrane potential in expanding Tcons with a concomitant reduction in basal autophagy. Our findings suggest that FOXP3 governs a distinct transcriptional program in early-stage effector Tcons that maintains RICD resistance via CD48-dependent protective autophagy and p53 suppression.
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http://dx.doi.org/10.1038/s41423-019-0316-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852538PMC
January 2021

Sleep Deprivation Alters the Pituitary Stress Transcriptome in Male and Female Mice.

Front Endocrinol (Lausanne) 2019 9;10:676. Epub 2019 Oct 9.

Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

Poor sleep hygiene is a growing problem, with detrimental effects on many biological systems. The pituitary gland plays a crucial role in the regulation of sleep and the stress response, and its dysfunction leads to sleep-related disorders. However, the interaction between these critical functions remains unclear. Thus, we performed a comparative, whole-transcriptome, analysis to identify stress-induced genes and relevant pathways that may be affected by sleep deprivation. One day following 12 h of Paradoxical Sleep Deprivation (PSD), mice were restrained for 20 min. Gene expression changes in the pituitary were assessed via RNA-Seq and Gene Ontology in PSD and/or restrained groups compared to controls. We show that restraint triggers transcriptional responses involved in hormone secretion, the glucocorticoid response, and apoptosis in both sexes, with 285 differentially expressed genes in females and 93 in males. When PSD preceded restraint stress, the numbers of differentially expressed genes increased to 613 in females and 580 in males. The pituitary transcriptome of restraint+PSD animals was enriched for microglia and macrophage proliferation, cellular response to corticosteroids, and apoptosis, among others. Finally, we identify sex-specific differences in restraint-induced genes following PSD. These findings provide genetic targets to consider when studying sleep and the response to stress.
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http://dx.doi.org/10.3389/fendo.2019.00676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794367PMC
October 2019

Neuroinflammatory astrocytes generated from cord blood-derived human induced pluripotent stem cells.

J Neuroinflammation 2019 Aug 9;16(1):164. Epub 2019 Aug 9.

Center for Neuroscience and Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.

Background: Astrocytes respond to central nervous system (CNS) injury and disease by transforming to a reactive astrogliosis cell state that can contribute to either CNS dysfunction or repair. Neuroinflammation is a powerful driver of a harmful A1 astrogliosis phenotype associated with in vitro neurotoxicity and histopathology in human neurodegenerative diseases. Here we report a protocol for the rapid development of a human cell culture model of neuroinflammatory astrogliosis using induced pluripotent stem cells (iPSCs).

Methods: Using RNA sequencing and in vitro cell assays, we measured transcriptional and cellular effects of chronic exposure of human iPSC-derived astrocytes to the cytokines TNFα (tumor necrosis factor alpha) or IL-1β (interleukin-1 beta).

Results: We show TNFα and IL-1β induce pro-inflammatory gene signatures but by widely different magnitudes. TNFα treatment results in 606 differential expressed genes, the suppression of glutamate-uptake, and increased phagocytic activity in astrocyte cultures. In contrast, IL-1β effects are attenuated to 33 differential expressed genes and no significant effects on glutamate-uptake or increased phagocytic activity.

Conclusion: Our approach demonstrates a rapid tool for modeling neuroinflammatory human astrocytic responses in nervous system trauma and disease. In particular, we reveal a model for robust TNFα-induced human astrogliosis suitable for the study of neurotoxic A1 astrocytes.
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http://dx.doi.org/10.1186/s12974-019-1553-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688278PMC
August 2019

Comparative RNA-seq analysis reveals dys-regulation of major canonical pathways in ERG-inducible LNCaP cell progression model of prostate cancer.

Oncotarget 2019 Jul 2;10(42):4290-4306. Epub 2019 Jul 2.

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA.

Prostate Cancer (CaP) is the second leading cause of cancer related death in USA. In human CaP, gene fusion between androgen responsive regulatory elements at the 5'-untranslated region of TMPRSS2 and ETS-related genes (ERG) is present in at least 50% of prostate tumors. Here we have investigated the unique cellular transcriptome associated with over-expression of ERG in ERG-inducible LNCaP cell model system of human CaP. Comprehensive transcriptome analyses reveal a distinct signature that distinguishes ERG dependent and independent CaP in LNCaP cells. Our data highlight a significant heterogeneity among the transcripts. Out of the 526 statistically significant differentially expressed genes, 232 genes are up-regulated and 294 genes are down-regulated in response to ERG. These ERG-associated genes are linked to several major cellular pathways, cell cycle regulation being the most significant. Consistently our data indicate that ERG plays a key role in modulating the expression of genes required for G1 to S phase transition, particularly those that affect cell cycle arrest at G1 phase. Moreover, cell cycle arrest in response to ERG appears to be promoted by induction of p21 in a p53 independent manner. These findings may provide new insights into mechanisms that promote growth and progression of CaP.
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http://dx.doi.org/10.18632/oncotarget.27019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611515PMC
July 2019

Identification of a Small Molecule That Selectively Inhibits ERG-Positive Cancer Cell Growth.

Cancer Res 2018 07 30;78(13):3659-3671. Epub 2018 Apr 30.

Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences and the Walter Reed National Military Medical Center, Bethesda, Maryland.

Oncogenic activation of the ETS-related gene () by recurrent gene fusions (predominantly TMPRSS2-ERG) is one of the most validated and prevalent genomic alterations present in early stages of prostate cancer. In this study, we screened small-molecule libraries for inhibition of ERG protein in harboring VCaP prostate cancer cells using an In-Cell Western Assay with the highly specific ERG-MAb (9FY). Among a subset of promising candidates, 1-[2-Thiazolylazo]-2-naphthol (NSC139021, hereafter ERGi-USU) was identified and further characterized. ERGi-USU selectively inhibited growth of ERG-positive cancer cell lines with minimal effect on normal prostate or endothelial cells or ERG-negative tumor cell lines. Combination of ERGi-USU with enzalutamide showed additive effects in inhibiting growth of VCaP cells. A screen of kinases revealed that ERGi-USU directly bound the ribosomal biogenesis regulator atypical kinase RIOK2 and induced ribosomal stress signature. , ERGi-USU treatment inhibited growth of ERG-positive VCaP tumor xenografts with no apparent toxicity. Structure-activity-based derivatives of ERGi-USU recapitulated the ERG-selective activity of the parental compound. Taken together, ERGi-USU acts as a highly selective inhibitor for the growth of ERG-positive cancer cells and has potential for further development of ERG-targeted therapy of prostate cancer and other malignancies. A highly selective small-molecule inhibitor of ERG, a critical driver of early stages of prostate cancer, will be imperative for prostate cancer therapy. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2949DOI Listing
July 2018

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell-derived neurons in culture.

Glia 2018 04 12;66(4):725-748. Epub 2017 Dec 12.

Center for Neuroscience and Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland.

Human induced pluripotent stem (iPS) cell-derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell-derived neural stem cells (NSCs). We first demonstrate human iPS cell-derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co-culture on feeder layers of isogenic human iPS cell-derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor-channel function and occurred without changes in I , I , V , and AP properties of iPS cell-derived neurons. These data demonstrate co-culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell-derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell-derived neurons in culture. We present RNA-sequencing data for an immature, fetal-like status of our human iPS cell-derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co-culture system.
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http://dx.doi.org/10.1002/glia.23278DOI Listing
April 2018

The role of the immunoproteasome in interferon-γ-mediated microglial activation.

Sci Rep 2017 08 24;7(1):9365. Epub 2017 Aug 24.

Neuroscience Program, Uniformed Services University of the Health Sciences, F. Edward Hébert School of Medicine, Bethesda, MD, USA.

Microglia regulate the brain microenvironment by sensing damage and neutralizing potentially harmful insults. Disruption of central nervous system (CNS) homeostasis results in transition of microglia to a reactive state characterized by morphological changes and production of cytokines to prevent further damage to CNS tissue. Immunoproteasome levels are elevated in activated microglia in models of stroke, infection and traumatic brain injury, though the exact role of the immunoproteasome in neuropathology remains poorly defined. Using gene expression analysis and native gel electrophoresis we characterize the expression and assembly of the immunoproteasome in microglia following interferon-gamma exposure. Transcriptome analysis suggests that the immunoproteasome regulates multiple features of microglial activation including nitric oxide production and phagocytosis. We show that inhibiting the immunoproteasome attenuates expression of pro-inflammatory cytokines and suppresses interferon-gamma-dependent priming of microglia. These results imply that targeting immunoproteasome function following CNS injury may attenuate select microglial activity to improve the pathophysiology of neurodegenerative conditions or the progress of inflammation-mediated secondary injury following neurotrauma.
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http://dx.doi.org/10.1038/s41598-017-09715-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571106PMC
August 2017

Intrinsic Plasma Cell Differentiation Defects in B Cell Expansion with NF-κB and T Cell Anergy Patient B Cells.

Front Immunol 2017 2;8:913. Epub 2017 Aug 2.

Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.

B cell Expansion with NF-κB and T cell Anergy (BENTA) disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDCD38 plasmablasts or CD138 long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.
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http://dx.doi.org/10.3389/fimmu.2017.00913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5539167PMC
August 2017

Salsalate treatment following traumatic brain injury reduces inflammation and promotes a neuroprotective and neurogenic transcriptional response with concomitant functional recovery.

Brain Behav Immun 2017 Mar 7;61:96-109. Epub 2016 Dec 7.

Department of Microbiology and Immunology, Uniformed Services University, Bethesda, MD, USA; Center for Neuroscience and Regenerative Medicine, Uniformed Services University, Bethesda, MD, USA. Electronic address:

Neuroinflammation plays a critical role in the pathogenesis of traumatic brain injury (TBI). TBI induces rapid activation of astrocytes and microglia, infiltration of peripheral leukocytes, and secretion of inflammatory cytokines. In the context of modest or severe TBI, such inflammation contributes to tissue destruction and permanent brain damage. However, it is clear that the inflammatory response is also necessary to promote post-injury healing. To date, anti-inflammatory therapies, including the broad class of non-steroidal anti-inflammatory drugs (NSAIDs), have met with little success in treatment of TBI, perhaps because these drugs have inhibited both the tissue-damaging and repair-promoting aspects of the inflammatory response, or because inhibition of inflammation alone is insufficient to yield therapeutic benefit. Salsalate is an unacetylated salicylate with long history of use in limiting inflammation. This drug is known to block activation of NF-κB, and recent data suggest that salsalate has a number of additional biological activities, which may also contribute to its efficacy in treatment of human disease. Here, we show that salsalate potently blocks pro-inflammatory gene expression and nitrite secretion by microglia in vitro. Using the controlled cortical impact (CCI) model in mice, we find that salsalate has a broad anti-inflammatory effect on in vivo TBI-induced gene expression, when administered post-injury. Interestingly, salsalate also elevates expression of genes associated with neuroprotection and neurogenesis, including the neuropeptides, oxytocin and thyrotropin releasing hormone. Histological analysis reveals salsalate-dependent decreases in numbers and activation-associated morphological changes in microglia/macrophages, proximal to the injury site. Flow cytometry data show that salsalate changes the kinetics of CCI-induced accumulation of various populations of CD11b-positive myeloid cells in the injured brain. Behavioral assays demonstrate that salsalate treatment promotes significant recovery of function following CCI. These pre-clinical data suggest that salsalate may show promise as a TBI therapy with a multifactorial mechanism of action to enhance functional recovery.
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http://dx.doi.org/10.1016/j.bbi.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316369PMC
March 2017

MicroRNA Expression Profiling of the Armed Forces Health Surveillance Branch Cohort for Identification of "Enviro-miRs" Associated With Deployment-Based Environmental Exposure.

J Occup Environ Med 2016 08;58(8 Suppl 1):S97-S103

Department of Anatomy, Physiology & Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland (Dr Dalgard, Mr Polston, Mr Sukumar, Drs Wilkerson, Pollard); Collaborative Health Initiative Research Program, Uniformed Services University of the Health Sciences, Bethesda, Maryland (Drs Dalgard, Wilkerson, Pollard); The American Genome Center, Uniformed Services University of the Health Sciences, Bethesda, Maryland (Drs Dalgard, Wilkerson, Pollard); and Professor, Department of Preventive Medicine and Biostatistics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (Dr Mallon). The opinions expressed are those of the authors and do not necessarily reflect the official positions of the Uniformed Services University, the U.S. Departments of Defense, the Army and the Air Force, the 779th Aerospace Medical Squadron, the U.S. Army Public Health Center (Provisional), Emory University, Clarkson University, or the University of Rochester.

Objective: The aim of this study was to identify serum microRNA (miRNA) biomarkers that indicate deployment-associated exposures in service members at military installations with open burn pits. Another objective was to determine detection rates of miRNAs in Department of Defense Serum Repository (DoDSR) samples with a high-throughput methodology.

Methods: Low-volume serum samples (n = 800) were profiled by miRNA-capture isolation, pre-amplification, and measurement by a quantitative PCR-based OpenArray platform. Normalized quantitative cycle values were used for differential expression analysis between groups.

Results: Assay specificity, dynamic range, reproducibility, and detection rates by OpenArray passed target desired specifications. Serum abundant miRNAs were consistently measured in study specimens. Four miRNAs were differentially expressed in the case deployment group subjects.

Conclusions: miRNAs are suitable RNA species for biomarker discovery in the DoDSR serum specimens. Serum miRNAs are candidate biomarkers for deployment and environmental exposure in military service members.
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http://dx.doi.org/10.1097/JOM.0000000000000764DOI Listing
August 2016

Inhibition of the histone demethylase Kdm5b promotes neurogenesis and derepresses Reln (reelin) in neural stem cells from the adult subventricular zone of mice.

Mol Biol Cell 2016 Feb 6;27(4):627-39. Epub 2016 Jan 6.

Center for Neuroscience and Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814 Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions, Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion, notably the up-regulation of reelin (Reln), the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln, and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ.
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http://dx.doi.org/10.1091/mbc.E15-07-0513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750923PMC
February 2016

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Front Pharmacol 2015 13;6:168. Epub 2015 Aug 13.

Department of Anatomy, Physiology and Genetics, F. Edward Hebert School of Medicine, Uniformed Services University of the Health Sciences Bethesda, MD, USA.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic β-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.
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http://dx.doi.org/10.3389/fphar.2015.00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534802PMC
August 2015

Germline CARD11 Mutation in a Patient with Severe Congenital B Cell Lymphocytosis.

J Clin Immunol 2015 Jan 29;35(1):32-46. Epub 2014 Oct 29.

Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD, 20814, USA.

Purpose: Activating germline mutations in CARD11 have recently been linked to a rare genetic disorder associated with congenital B cell lymphocytosis. We describe a patient with a similar clinical phenotype who had a de novo germline G123D CARD11 mutation.

Methods: Whole exome sequencing was performed on DNA from the patient and his biological parents. Laboratory studies examined characteristics of the patient's B and T lymphocytes. A CARD11 cDNA containing the mutation was transfected into a lymphocyte cell line to gain an understanding of its function. RNA sequencing was performed on samples from the patient and from patients with alternate germline CARD11 mutations and differential gene expression analysis was performed.

Results: The patient had a decade-long history of severe polyclonal B lymphocytosis in the 20,000-90,000 lymphocytes/mm(3) range, which was markedly exacerbated by EBV infection and splenectomy at different times. He had a heterozygous germline CARD11 mutation causing a G123D amino acid substitution, which was demonstrated to induce NF-κB activation in unstimulated lymphocytes. In contrast to previous patients with CARD11 mutations, this patient's B cells exhibited higher expression of several cell cycle progression genes, as well as enhanced proliferation and improved survival following B cell receptor stimulation.

Conclusions: This is the third reported germline and first de novo CARD11 mutation shown to cause congenital B cell lymphocytosis. The mutation was associated with a dramatically greater lymphocytosis than in previously described cases, disproportionate to the level of constitutive NF-κB activation. However, comparative review of the patient's clinical history, combined with additional genomic and functional analyses, underscore other important variables that may affect pathophysiology or regulate mutant CARD11 function in B cell proliferation and disease. We now refer to these patients as having BENTA disease (B cell Expansion with NF-κB and T cell Anergy).
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http://dx.doi.org/10.1007/s10875-014-0106-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466218PMC
January 2015

Oncogenic herpesvirus HHV-8 promotes androgen-independent prostate cancer growth.

Cancer Res 2013 Sep 4;73(18):5695-708. Epub 2013 Sep 4.

Authors' Affiliation: Emerging Infectious Diseases Program, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland.

Mechanisms underlying progression to androgen-independent prostate cancer following radical ablation therapy remain poorly defined. Although intraprostatic infections have been highlighted as potential cofactors, pathogen influences on pathways that support tumor regrowth are not known. To explore this provocative concept, we derived androgen-sensitive and -insensitive prostate epithelial cells persistently infected with human herpesvirus 8 (HHV-8), an oncogenic herpesvirus that has been detected in normal prostate epithelium, prostate adenocarcinoma, and biologic fluids of patients with prostate cancer, to explore its effects on transition to hormone-refractory disease. Strikingly, we found that HHV-8 infection of androgen-sensitive prostate cancer cells conferred the capacity for androgen-independent growth. This effect was associated with altered expression and transcriptional activity of the androgen receptor (AR). However, HHV-8 infection bypassed AR signaling by promoting enhancer of zeste homolog 2 (EZH2)-mediated epigenetic silencing of tumor-suppressor genes, including MSMB and DAB2IP that are often inactivated in advanced disease. Furthermore, we found that HHV-8 triggered epithelial-to-mesenchymal transition. Although HHV-8 has not been linked etiologically to prostate cancer, virologic outcomes revealed by our study provide mechanistic insight into how intraprostatic infections could constitute risk for progression to androgen-independent metastatic disease where EZH2 has been implicated. Taken together, our findings prompt further evaluations of the relationship between HHV-8 infections and risk of advanced prostate cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-4196DOI Listing
September 2013

Controlled cortical impact and craniotomy induce strikingly similar profiles of inflammatory gene expression, but with distinct kinetics.

Front Neurol 2012 31;3:155. Epub 2012 Oct 31.

Department of Microbiology and Immunology, Uniformed Services University Bethesda, MD, USA ; Center for Neuroscience and Regenerative Medicine, Uniformed Services University Bethesda, MD, USA.

An immediate consequence of traumatic brain injury (TBI) is the induction of an inflammatory response. Mounting data suggest that inflammation is a major contributor to TBI-induced brain damage. However, much remains unknown regarding the induction and regulation of the inflammatory response to TBI. In this study we compared the TBI-induced inflammatory response to severe parenchymal injury (controlled cortical impact) vs. mild brain injury (craniotomy) over a 21-day period. Our data show that both severe and mild brain injury induce a qualitatively similar inflammatory response, involving highly overlapping sets of effector molecules. However, kinetic analysis revealed that the inflammatory response to mild brain injury is of much shorter duration than the response to severe TBI. Specifically, the inflammatory response to severe brain injury persists for at least 21 days, whereas the response to mild brain injury returns to near baseline values within 10 days post-injury. Our data therefore imply that the development of accurate diagnostic tests of TBI severity that are based on imaging or biomarker analysis of the inflammatory response may require repeated measures over at least a 10-day period, post-injury.
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http://dx.doi.org/10.3389/fneur.2012.00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484408PMC
November 2012

The cytokine temporal profile in rat cortex after controlled cortical impact.

Front Mol Neurosci 2012 25;5. Epub 2012 Jan 25.

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda MD, USA.

Cerebral inflammatory responses may initiate secondary cascades following traumatic brain injury (TBI). Changes in the expression of both cytokines and chemokines may activate, regulate, and recruit innate and adaptive immune cells associated with secondary degeneration, as well as alter a host of other cellular processes. In this study, we quantified the temporal expression of a large set of inflammatory mediators in rat cortical tissue after brain injury. Following a controlled cortical impact (CCI) on young adult male rats, cortical and hippocampal tissue of the injured hemisphere and matching contralateral material was harvested at early (4, 12, and 24 hours) and extended (3 and 7 days) time points post-procedure. Naïve rats that received only anesthesia were used as controls. Processed brain homogenates were assayed for chemokine and cytokine levels utilizing an electrochemiluminescence-based multiplex ELISA platform. The temporal profile of cortical tissue samples revealed a multi-phasic injury response following brain injury. CXCL1, IFN-γ, TNF-α levels significantly peaked at four hours post-injury compared to levels found in naïve or contralateral tissue. CXCL1, IFN-γ, and TNF-α levels were then observed to decrease at least 3-fold by 12 hours post-injury. IL-1β, IL-4, and IL-13 levels were also significantly elevated at four hours post-injury although their expression did not decrease more than 3-fold for up to 24 hours post-injury. Additionally, IL-1β and IL-4 levels displayed a biphasic temporal profile in response to injury, which may suggest their involvement in adaptive immune responses. Interestingly, peak levels of CCL2 and CCL20 were not observed until after four hours post-injury. CCL2 levels in injured cortical tissue were significantly higher than peak levels of any other inflammatory mediator measured, thus suggesting a possible use as a biomarker. Fully elucidating chemokine and cytokine signaling properties after brain injury may provide increased insight into a number of secondary cascade events that are initiated or regulated by inflammatory responses.
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http://dx.doi.org/10.3389/fnmol.2012.00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265961PMC
October 2012