Publications by authors named "David K Flaherty"

26 Publications

  • Page 1 of 1

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

NAR Cancer 2022 Mar 3;4(1):zcac007. Epub 2022 Mar 3.

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of , which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation . These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.
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http://dx.doi.org/10.1093/narcan/zcac007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8892060PMC
March 2022

WIN site inhibition disrupts a subset of WDR5 function.

Sci Rep 2022 02 3;12(1):1848. Epub 2022 Feb 3.

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, 465 21st Avenue South, Nashville, TN, 37232, USA.

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions-that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.
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http://dx.doi.org/10.1038/s41598-022-05947-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8813994PMC
February 2022

Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler.

Oncogene 2021 05 30;40(20):3593-3609. Epub 2021 Apr 30.

Department of Biology, Middle Tennessee State University, Murfreesboro, TN, USA.

The SNF5 subunit of the SWI/SNF chromatin remodeling complex has been shown to act as a tumor suppressor through multiple mechanisms, including impairing the ability of the oncoprotein transcription factor MYC to bind chromatin. Beyond SNF5, however, it is unknown to what extent MYC can access additional SWI/SNF subunits or how these interactions affect the ability of MYC to drive transcription, particularly in SNF5-null cancers. Here, we report that MYC interacts with multiple SWI/SNF components independent of SNF5. We show that MYC binds the pan-SWI/SNF subunit BAF155 through the BAF155 SWIRM domain, an interaction that is inhibited by the presence of SNF5. In SNF5-null cells, MYC binds with remaining SWI/SNF components to essential genes, although for a purpose that is distinct from chromatin remodeling. Analysis of MYC-SWI/SNF target genes in SNF5-null cells reveals that they are associated with core biological functions of MYC linked to protein synthesis. These data reveal that MYC can bind SWI/SNF in an SNF5-independent manner and that SNF5 modulates access of MYC to core SWI/SNF complexes. This work provides a framework in which to interrogate the influence of SWI/SNF on MYC function in cancers in which SWI/SNF or MYC are altered.
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http://dx.doi.org/10.1038/s41388-021-01804-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8141032PMC
May 2021

Coordinated interactions between endothelial cells and macrophages in the islet microenvironment promote β cell regeneration.

NPJ Regen Med 2021 Apr 6;6(1):22. Epub 2021 Apr 6.

Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN, USA.

Endogenous β cell regeneration could alleviate diabetes, but proliferative stimuli within the islet microenvironment are incompletely understood. We previously found that β cell recovery following hypervascularization-induced β cell loss involves interactions with endothelial cells (ECs) and macrophages (MΦs). Here we show that proliferative ECs modulate MΦ infiltration and phenotype during β cell loss, and recruited MΦs are essential for β cell recovery. Furthermore, VEGFR2 inactivation in quiescent ECs accelerates islet vascular regression during β cell recovery and leads to increased β cell proliferation without changes in MΦ phenotype or number. Transcriptome analysis of β cells, ECs, and MΦs reveals that β cell proliferation coincides with elevated expression of extracellular matrix remodeling molecules and growth factors likely driving activation of proliferative signaling pathways in β cells. Collectively, these findings suggest a new β cell regeneration paradigm whereby coordinated interactions between intra-islet MΦs, ECs, and extracellular matrix mediate β cell self-renewal.
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http://dx.doi.org/10.1038/s41536-021-00129-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024255PMC
April 2021

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

Gastroenterology 2021 02 30;160(3):755-770.e26. Epub 2020 Sep 30.

Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee. Electronic address:

Background & Aims: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions.

Methods: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction.

Results: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes.

Conclusions: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.
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http://dx.doi.org/10.1053/j.gastro.2020.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878294PMC
February 2021

WDR5 is a conserved regulator of protein synthesis gene expression.

Nucleic Acids Res 2020 04;48(6):2924-2941

Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37240, USA.

WDR5 is a highly-conserved nuclear protein that performs multiple scaffolding functions in the context of chromatin. WDR5 is also a promising target for pharmacological inhibition in cancer, with small molecule inhibitors of an arginine-binding pocket of WDR5 (the 'WIN' site) showing efficacy against a range of cancer cell lines in vitro. Efforts to understand WDR5, or establish the mechanism of action of WIN site inhibitors, however, are stymied by its many functions in the nucleus, and a lack of knowledge of the conserved gene networks-if any-that are under its control. Here, we have performed comparative genomic analyses to identify the conserved sites of WDR5 binding to chromatin, and the conserved genes regulated by WDR5, across a diverse panel of cancer cell lines. We show that a specific cohort of protein synthesis genes (PSGs) are invariantly bound by WDR5, demonstrate that the WIN site anchors WDR5 to chromatin at these sites, and establish that PSGs are bona fide, acute, and persistent targets of WIN site blockade. Together, these data reveal that WDR5 plays a predominant transcriptional role in biomass accumulation and provide further evidence that WIN site inhibitors act to repress gene networks linked to protein synthesis homeostasis.
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http://dx.doi.org/10.1093/nar/gkaa051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7102967PMC
April 2020

The LRRC8 volume-regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel.

Physiol Rep 2019 12;7(23):e14303

Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.

There has been a resurgence of interest in the volume-regulated anion channel (VRAC) since the recent cloning of the LRRC8A-E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best-in-class VRAC inhibitor, DCPIB, suffers from off-target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A-knockout HAP-1 and HEK-293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP-1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP-linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP-1 and HEK-293 cells which lack LRRC8A expression. Finally, we demonstrate that DCPIB activates ATP-inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I-III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases.
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http://dx.doi.org/10.14814/phy2.14303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900491PMC
December 2019

Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules.

Am J Physiol Renal Physiol 2019 05 13;316(5):F847-F855. Epub 2019 Feb 13.

Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center , Nashville, Tennessee.

Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.
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http://dx.doi.org/10.1152/ajprenal.00354.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580245PMC
May 2019

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis.

Cell Metab 2019 03 15;29(3):745-754.e4. Epub 2018 Nov 15.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37240, USA; Department of Medicine, Division of Diabetes, Endocrinology, and Metabolism, Vanderbilt University Medical Center, Nashville, TN 37232, USA; VA Tennessee Valley Healthcare, Nashville, TN 37212, USA. Electronic address:

Identification of cell-surface markers specific to human pancreatic β cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human β cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet β cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human β cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human β cells. Thus, NTPDase3 is a cell-surface biomarker of adult human β cells, and the antibody directed to this protein should be a useful new reagent for β cell sorting, in vivo imaging, and targeting.
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http://dx.doi.org/10.1016/j.cmet.2018.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402969PMC
March 2019

Alternative splicing of ALCAM enables tunable regulation of cell-cell adhesion through differential proteolysis.

Sci Rep 2018 02 16;8(1):3208. Epub 2018 Feb 16.

Vanderbilt University, Program in Cancer Biology, Nashville, USA.

While many adhesion receptors are known to influence tumor progression, the mechanisms by which they dynamically regulate cell-cell adhesion remain elusive. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a clinically relevant driver of metastasis and hypothesized that a tunable mechanism of ectodomain shedding regulates its contribution to dissemination. To test this hypothesis, we examined an under-explored ALCAM splice variant (ALCAM-Iso2) and demonstrated that loss of the membrane-proximal region of ALCAM (exon 13) increased metastasis four-fold. Mechanistic studies identified a novel MMP14-dependent membrane distal cleavage site in ALCAM-Iso2, which mediated a ten-fold increase in shedding, thereby decreasing cellular cohesion. Importantly, the loss of cohesion is not limited to the cell capable of shedding because the released extracellular domain diminished cohesion of non-shedding cells through disruption of ALCAM-ALCAM interactions. ALCAM-Iso2-dominated expression in bladder cancer tissue, compared to normal bladder, further emphasizes that ALCAM alternative splicing may contribute to clinical disease progression. The requirement for both the loss of exon 13 and the gain of metalloprotease activity suggests that ALCAM shedding and concomitant regulation of tumor cell adhesion is a locally tunable process.
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http://dx.doi.org/10.1038/s41598-018-21467-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816644PMC
February 2018

Obesity Alters B Cell and Macrophage Populations in Brown Adipose Tissue.

Obesity (Silver Spring) 2017 11 18;25(11):1881-1884. Epub 2017 Sep 18.

Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, Tennessee, USA.

Objective: The prevalence of obesity continues to rise, and it is understood that regulation of white adipose tissue (WAT) function is important to systemic metabolic homeostasis. Immune cells play a central role in the maintenance of WAT, and their compositions change in number and inflammatory phenotype with the progression of obesity. Because of its energy-burning capabilities, brown adipose tissue (BAT) has become a focus of obesity research. Although novel studies have focused on the function of brown adipocytes in thermogenesis, the tissue as a whole has not been immunologically characterized.

Methods: BAT immune cell populations were analyzed by flow cytometry and immunohistochemistry in mice with diet-induced obesity (3, 8, or 16 weeks of diet) and in aged mice (1, 6-7, and 10-15 months).

Results: The data confirmed the presence of macrophages and eosinophils, as previously reported, and showed that 20% to 30% of the immune cells in BAT were B cells. The number of B cells and eosinophils increased with diet-induced obesity, whereas macrophages decreased. There was no change in number of any immune cell quantified with age.

Conclusions: These studies reveal a novel finding of B220 + B cells in BAT and show that BAT immune cell populations change in response to diet-induced obesity.
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http://dx.doi.org/10.1002/oby.21982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679082PMC
November 2017

Distributions of Cells and Neurons across the Cortical Sheet in Old World Macaques.

Brain Behav Evol 2016 23;88(1):1-13. Epub 2016 Aug 23.

Department of Psychology, Vanderbilt University, Nashville, Tenn., USA.

According to previous research, cell and neuron densities vary across neocortex in a similar manner across primate taxa. Here, we provide a more extensive examination of this effect in macaque monkeys. We separated neocortex from the underlying white matter in 4 macaque monkey hemispheres (1 Macaca nemestrina, 2 Macaca radiata, and 1 Macaca mulatta), manually flattened the neocortex, and divided it into smaller tissue pieces for analysis. The number of cells and neurons were determined for each piece across the cortical sheet using flow cytometry. Primary visual cortex had the most densely packed neurons and primary motor cortex had the least densely packed neurons. With respect to differences in brain size between cases, there was little variability in the total cell and neuron numbers within specific areas, and overall trends were similar to what has been previously described in Old World baboons and other primates. The average hemispheric total cell number per hemisphere ranged from 2.9 to 3.7 billion, while the average total neuron number ranged from 1.3 to 1.7 billion neurons. The visual cortex neuron densities were predictably higher, ranging from 18.2 to 34.7 million neurons/cm2 in macaques, in comparison to a range of 9.3-17.7 million neurons/cm2 across cortex as a whole. The results support other evidence that neuron surface densities vary across the cortical sheet in a predictable pattern within and across primate taxa.
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http://dx.doi.org/10.1159/000446762DOI Listing
July 2017

Chronic HIV-1 Infection Impairs Superantigen-Induced Activation of Peripheral CD4+CXCR5+PD-1+ Cells, With Relative Preservation of Recall Antigen-Specific Responses.

J Acquir Immune Defic Syndr 2017 01;74(1):72-80

*Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN; †Flow Cytometry Shared Resource, Vanderbilt University Medical Center, Nashville, TN; ‡Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN; and §Seattle Biomedical Research Institute, Seattle, WA.

Peripheral CD4+CXCR5+PD-1+ T cells are a putative circulating counterpart to germinal center T follicular helper (TFH) cells. They show both phenotypic and functional similarities to TFH cells, which provide necessary help for the differentiation of B cells to antibody-secreting plasmablasts. In this study, we evaluated the frequency, phenotypes, and responses of peripheral TFH-like (pTFH) cells to superantigen and recall antigen stimulation in 10 healthy and 34 chronically infected treatment-naive HIV-1+ individuals. There was no difference in the frequency of pTFH cells between HIV+ and HIV- individuals. Surface expression of ICOS, but not CD40L, was higher on pTFH cells at baseline in HIV+ individuals. Compared with HIV- individuals, pTFH cells from HIV+ individuals had decreased maximal expression of ICOS and CD40L in response to in vitro superantigen stimulation. This decreased response did not correlate with viral control, CD4 T-cell count, duration of infection, or the degree of neutralizing antibody breadth. Despite a decreased maximal response, pTFH responses to HIV Gag and tetanus toxoid recall antigens were preserved.
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http://dx.doi.org/10.1097/QAI.0000000000001152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5140753PMC
January 2017

Cortical cell and neuron density estimates in one chimpanzee hemisphere.

Proc Natl Acad Sci U S A 2016 Jan 4;113(3):740-5. Epub 2016 Jan 4.

Department of Psychology, Vanderbilt University, Nashville, TN 37240;

The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.
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http://dx.doi.org/10.1073/pnas.1524208113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725503PMC
January 2016

Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry.

Cytometry A 2016 Mar 24;89(3):271-80. Epub 2015 Nov 24.

Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee.

Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high-dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be indispensable tools for detecting perturbations in protein expression in both health and disease.
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http://dx.doi.org/10.1002/cyto.a.22799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808335PMC
March 2016

Epileptic baboons have lower numbers of neurons in specific areas of cortex.

Proc Natl Acad Sci U S A 2013 Nov 4;110(47):19107-12. Epub 2013 Nov 4.

Department of Psychology, Vanderbilt University, Nashville, TN 37240.

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.
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http://dx.doi.org/10.1073/pnas.1318894110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839763PMC
November 2013

Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system.

Brain Behav Immun 2014 Jan 4;35:33-42. Epub 2013 Jul 4.

Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, United States.

Obesity is associated with chronic low-grade inflammation in peripheral tissues caused, in part, by the recruitment of inflammatory monocytes into adipose tissue. Studies in rodent models have also shown increased inflammation in the central nervous system (CNS) during obesity. The goal of this study was to determine whether obesity is associated with recruitment of peripheral immune cells into the CNS. To do this we used a bone marrow chimerism model to track the entry of green-fluorescent protein (GFP) labeled peripheral immune cells into the CNS. Flow cytometry was used to quantify the number of GFP(+) immune cells recruited into the CNS of mice fed a high-fat diet compared to standard chow fed controls. High-fat feeding resulted in obesity associated with a 30% increase in the number of GFP(+) cells in the CNS compared to control mice. Greater than 80% of the GFP(+) cells recruited to the CNS were also CD45(+) CD11b(+) indicating that the GFP(+) cells displayed characteristics of microglia/macrophages. Immunohistochemistry further confirmed the increase in GFP(+) cells in the CNS of the high-fat fed group and also indicated that 93% of the recruited cells were found in the parenchyma and had a stellate morphology. These findings indicate that peripheral immune cells can be recruited to the CNS in obesity and may contribute to the inflammatory response.
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http://dx.doi.org/10.1016/j.bbi.2013.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858467PMC
January 2014

Use of flow cytometry for high-throughput cell population estimates in brain tissue.

Front Neuroanat 2012 11;6:27. Epub 2012 Jul 11.

Department of Psychology, Vanderbilt University, Nashville TN, USA.

The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis.
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http://dx.doi.org/10.3389/fnana.2012.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394395PMC
October 2012

CBA/J mice generate protective immunity to soluble Ag85 but fail to respond efficiently to Ag85 during natural Mycobacterium tuberculosis infection.

Eur J Immunol 2012 Apr;42(4):870-9

Center for Microbial Interface Biology, The Ohio State University, Columbus, OH, USA.

In CBA/J mice, susceptibility to Mycobacterium tuberculosis (M.tb) is associated with low interferon-gamma (IFN-γ) responses to antigens (Antigen 85 (Ag85) and early secreted antigenic target-6 (ESAT-6)) that have been defined as immunodominant. Here, we asked whether the failure of CBA/J mice to recognize Ag85 is a consequence of M.tb infection or whether CBA/J mice have a general defect in generating specific T-cell responses to this protein antigen. We compared CBA/J mice during primary M.tb infection, Ag85 vaccination followed by M.tb challenge, or M.tb memory immune mice for their capacity to generate Ag85-specific IFN-γ responses and to control M.tb infection. CBA/J mice did not respond efficiently to Ag85 in the context of natural infection or re-infection. In contrast, CBA/J mice could generate Ag85-specific IFN-γ responses and protective immunity when this antigen was delivered as a soluble protein. Our data indicate that although M.tb infection of CBA/J mice does not drive an Ag85 response, these mice can fully and protectively respond to Ag85 if it is delivered as a vaccine. The data from this experimental model suggest that the Ag85-containing vaccines in clinical trials should protect M.tb susceptible humans.
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http://dx.doi.org/10.1002/eji.201142054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641789PMC
April 2012

Helicobacter pylori induction of eosinophil migration is mediated by the cag pathogenicity island via microbial-epithelial interactions.

Am J Pathol 2011 Apr 4;178(4):1448-52. Epub 2011 Mar 4.

Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-2279, USA.

The host immune response directed against Helicobacter pylori is ineffective in eliminating the organism and strains harboring the cag pathogenicity island augment disease risk. Because eosinophils are a prominent component of H. pylori-induced gastritis, we investigated microbial and host mechanisms through which H. pylori regulates eosinophil migration. Our results indicate that H. pylori increases production of the chemokines CCL2, CCL5, and granulocyte-macrophage colony-stimulating factor by gastric epithelial cells and that these molecules induce eosinophil migration. These events are mediated by the cag pathogenicity island and by mitogen-activated protein kinases, suggesting that eosinophil migration orchestrated by H. pylori is regulated by a virulence-related locus.
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http://dx.doi.org/10.1016/j.ajpath.2010.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078468PMC
April 2011

A rapid and reliable method of counting neurons and other cells in brain tissue: a comparison of flow cytometry and manual counting methods.

Front Neuroanat 2010 9;4. Epub 2010 Feb 9.

Department of Psychology, Vanderbilt University School of Medicine Nashville, TN, USA.

It is of critical importance to understand the numbers and distributions of neurons and non-neurons in the cerebral cortex because cell numbers are reduced with normal aging and by diseases of the CNS. The isotropic fractionator method provides a faster way of estimating numbers of total cells and neurons in whole brains and dissected brain parts. Several comparative studies have illustrated the accuracy and utility of the isotropic fractionator method, yet it is a relatively new methodology, and there is opportunity to adjust procedures to optimize its efficiency and minimize error. In the present study, we use 142 samples from a dissected baboon cortical hemisphere to evaluate if isotropic fractionator counts using a Neubauer counting chamber and fluorescence microscopy could be accurately reproduced using flow cytometry methods. We find greater repeatability in flow cytometry counts, and no evidence of constant or proportional bias when comparing microscopy to flow cytometry counts. We conclude that cell number estimation using a flow cytometer is more efficient and more precise than comparable counts using a Neubauer chamber on a fluorescence microscope. This method for higher throughput, precise estimation of cell numbers has the potential to rapidly advance research in post-mortem human brains and vastly improve our understanding of cortical and subcortical structures in normal, injured, aged, and diseased brains.
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http://dx.doi.org/10.3389/neuro.05.005.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841487PMC
July 2011

Interleukin-10 promotes Mycobacterium tuberculosis disease progression in CBA/J mice.

J Immunol 2008 Oct;181(8):5545-50

Center for Microbial Interface Biology, The Ohio State University, Columbus, OH 43210, USA.

IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-gamma production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728584PMC
http://dx.doi.org/10.4049/jimmunol.181.8.5545DOI Listing
October 2008

Peripheral blood gamma interferon release assays predict lung responses and Mycobacterium tuberculosis disease outcome in mice.

Clin Vaccine Immunol 2008 Mar 9;15(3):474-83. Epub 2008 Jan 9.

Center for Microbial Interface Biology, The Ohio State University, Columbus, Ohio 43210, USA.

Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latent Mycobacterium tuberculosis infection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-gamma) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains to M. tuberculosis infection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-gamma in response to M. tuberculosis culture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-gamma secretion in supernatants reflected a reduced frequency of IFN-gamma-responding cells in the lung and blood and not a specific defect in IFN-gamma secretion at the single-cell level. Importantly, detection of antigen-specific IFN-gamma from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-gamma production and low frequencies of IFN-gamma-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains, suggesting that similar results may occur in humans. The development of efficacious predictive diagnostic tests for humans would lead to targeted therapy prior to progression to active TB, reducing transmission, incidence, and prevalence rates while maximizing the use of public health resources.
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http://dx.doi.org/10.1128/CVI.00408-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268254PMC
March 2008

Age dependent increase in early resistance of mice to Mycobacterium tuberculosis is associated with an increase in CD8 T cells that are capable of antigen independent IFN-gamma production.

Exp Gerontol 2006 Nov 6;41(11):1185-94. Epub 2006 Oct 6.

Center for Microbial Interface Biology, Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University Columbus, OH 43210, USA.

The lungs of naïve 18-month-old mice contain an abundant resident population of CD8 T cells that express typical markers of memory, express elevated levels of Th1 cytokine receptors on their surface, and are capable of non-specific IFN-gamma production in response to a Th1 cytokine cocktail. In this study we characterize this population of CD8 T cells in the lungs and spleens of mice with increasing age. In general, the proportion of CD8 T cells expressing markers of memory and Th1 cytokine receptors increased with age. The enhanced ability of CD8 T cells to produce IFN-gamma in an antigen independent manner followed this pattern as well, beginning to increase between 6 and 12 months of age. Interestingly, the phenotypic and functional age-related changes in CD8 T cells were also associated with a progressive age-related increase in early resistance to Mycobacterium tuberculosis. Taken together, these data suggest that as mice age a population of memory CD8 T cells, that are capable of contributing to innate immune responses to M. tuberculosis, gradually emerges and could be relevant for developing strategies to enhance immunity in the elderly.
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http://dx.doi.org/10.1016/j.exger.2006.08.006DOI Listing
November 2006

Exposure to Mycobacterium avium can modulate established immunity against Mycobacterium tuberculosis infection generated by Mycobacterium bovis BCG vaccination.

J Leukoc Biol 2006 Dec 12;80(6):1262-71. Epub 2006 Sep 12.

Department of Internal Medicine, Division of Infectious Diseases, Center for Microbial Interface Biology, 420 West 12th Avenue, Columbus, OH 43210, USA.

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.
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http://dx.doi.org/10.1189/jlb.0606407DOI Listing
December 2006

Th1 cytokines facilitate CD8-T-cell-mediated early resistance to infection with Mycobacterium tuberculosis in old mice.

Infect Immun 2006 Jun;74(6):3314-24

Center for Microbial Interface Biology, The Ohio State University, 420 West 12th Avenue, Columbus, OH 43210, USA.

Numerous immunological defects begin to emerge as an individual ages, the consequence of which is heightened susceptibility to infectious diseases. Despite this decline in immune function, old mice display an early transient resistance to Mycobacterium tuberculosis infection in the lung, which is dependent on CD8 T cells and gamma interferon (IFN-gamma) production. In this study, we investigated the mechanism of resistance by examining the CD8-T-cell phenotype and function in old naïve and M. tuberculosis-infected mice. Pulmonary CD8 T cells from naïve old mice expressed cell surface markers of memory in addition to receptors for several Th1 cytokines. Stimulation of lung cells from naïve old mice with a combination of Th1 cytokines (interleukin-2 [IL-2], IL-12, and IL-18) resulted in nonspecific production of IFN-gamma by memory CD8 T cells. Following aerosol infection with M. tuberculosis, the lungs of old mice contained significantly more IL-12, IL-18, and IFN-gamma than the lungs of young mice contained. Together, these data demonstrate that the increased and early production of Th1 cytokines in the lungs of M. tuberculosis-infected old mice, in combination with CD8 T cells that can nonspecifically produce IFN-gamma, leads to transient control of M. tuberculosis growth in the lungs of old mice. Further characterization of this mechanism should provide essential information regarding the aging immune system and should contribute to the development of novel strategies to decrease the morbidity and mortality of the aging population associated with infectious diseases.
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http://dx.doi.org/10.1128/IAI.01475-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1479270PMC
June 2006
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