Medicine and Health Sciences Commons^™

Human Gucy2c-Targeted Chimeric Antigen Receptor (Car)-Expressing T Cells Eliminate Colorectal Cancer Metastases., Michael S. Magee, Tara S. Abraham, Trevor R. Baybutt, John C. Flickinger, Natalie A. Ridge, Glen P Marszalowicz, Priyanka Prajapati, Adam R. Hersperger, Scott A. Waldman, Adam E. Snook

Department of Pharmacology and Experimental Therapeutics Faculty Papers

One major hurdle to the success of adoptive T-cell therapy is the identification of antigens that permit effective targeting of tumors in the absence of toxicities to essential organs. Previous work has demonstrated that T cells engineered to express chimeric antigen receptors (CAR-T cells) targeting the murine homolog of the colorectal cancer antigen GUCY2C treat established colorectal cancer metastases, without toxicity to the normal GUCY2C-expressing intestinal epithelium, reflecting structural compartmentalization of endogenous GUCY2C to apical membranes comprising the intestinal lumen. Here, we examined the utility of a human-specific, GUCY2C-directed single-chain variable fragment as the basis for a CAR construct targeting …

Decorin-Evoked Paternally Expressed Gene 3 (Peg3) Is An Upstream Regulator Of The Transcription Factor Eb (Tfeb) In Endothelial Cell Autophagy., Thomas Neill, Catherine Sharpe, Rick T. Owens, Renato V. Iozzo

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Macroautophagy is a fundamental and evolutionarily conserved catabolic process that eradicates damaged and aging macromolecules and organelles in eukaryotic cells. Decorin, an archetypical small leucine-rich proteoglycan, initiates a protracted autophagic program downstream of VEGF receptor 2 (VEGFR2) signaling that requires paternally expressed gene 3 (PEG3). We have discovered that PEG3 is an upstream transcriptional regulator of transcription factor EB (TFEB), a master transcription factor of lysosomal biogenesis, for decorin-evoked endothelial cell autophagy. We found a functional requirement of PEG3 for TFEB transcriptional induction and nuclear translocation in human umbilical vein endothelial and PAER2 cells. Mechanistically, inhibiting VEGFR2 or AMP-activated protein …

Calcineurin Dysregulation Underlies Spinal Cord Injury-Induced K(+) Channel Dysfunction In Drg Neurons., Benjamin M. Zemel, Tanziyah Muqeem, Eric V. Brown, Miguel Goulão, Mark W Urban, Stephen R. Tymanskyj, Angelo C. Lepore, Manuel Covarrubias

Department of Neuroscience Faculty Papers

Dysfunction of the fast-inactivating Kv3.4 potassium current in dorsal root ganglion (DRG) neurons contributes to the hyperexcitability associated with persistent pain induced by spinal cord injury (SCI). However, the underlying mechanism is not known. In light of our previous work demonstrating modulation of the Kv3.4 channel by phosphorylation, we investigated the role of the phosphatase calcineurin (CaN) using electrophysiological, molecular, and imaging approaches in adult female Sprague Dawley rats. Pharmacological inhibition of CaN in small-diameter DRG neurons slowed repolarization of the somatic action potential (AP) and attenuated the Kv3.4 current. Attenuated Kv3.4 currents also exhibited slowed inactivation. We observed similar …

Map7 Regulates Axon Collateral Branch Development In Dorsal Root Ganglion Neurons., Stephen R Tymanskyj, Benjamin Yang, Aditi Falnikar, Angelo C Lepore, Le Ma

Department of Neuroscience Faculty Papers

Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. Like axon growth and guidance, formation of collateral branches depends on the regulation of microtubules, but how such regulation is coordinated to ensure proper circuit development is not known. Based on microarray analysis, we have identified a role for microtubule-associated protein 7 (MAP7) during collateral branch development of dorsal root ganglion (DRG) sensory neurons. We show that MAP7 is expressed at the onset of collateral branch formation. Perturbation of its expression by overexpression or shRNA knockdown alters axon branching in …

Identification Of Early Gene Expression Changes In Primary Cultured Neurons Treated With Topoisomerase I Poisons., Sharyn L. Rossi, Casey J. Lumpkin, Ashlee W. Harris, Jennifer Holbrook, Cinsley Gentillon, Suzanne M. Mccahan, Wenlan Wang, Matthew E.R. Butchbach

Department of Pediatrics Faculty Papers

Topoisomerase 1 (TOP1) poisons like camptothecin (CPT) are currently used in cancer chemotherapy but these compounds can have damaging, off-target effects on neurons leading to cognitive, sensory and motor deficits. To understand the molecular basis for the enhanced sensitivity of neurons to CPT, we examined the effects of compounds that inhibit TOP1-CPT, actinomycin D (ActD) and β-lapachone (β-Lap)-on primary cultured rat motor (MN) and cortical (CN) neurons as well as fibroblasts. Neuronal cells expressed higher levels of Top1 mRNA than fibroblasts but transcript levels are reduced in all cell types after treatment with CPT. Microarray analysis was performed to identify …

Extracellular Traps Are Associated With Human And Mouse Neutrophil And Macrophage Mediated Killing Of Larval Strongyloides Stercoralis., Sandra Bonne-Annee, Laura A. Kerepesi, Jessica A. Hess, Jordan Wesolowski, Fabienne Paumet, James B. Lok, Thomas J. Nolan, David Abraham

Department of Microbiology and Immunology Faculty Papers

Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed …

Efficient B Cell Responses To Borrelia Hermsii Infection Depend On Baff And Baffr But Not Taci., Gregory S. Dickinson, Phd, Guizhi Sun, Richard J Bram, Kishore Alugupalli

Department of Microbiology and Immunology Faculty Papers

T cell-independent antibody responses develop rapidly, within 3 to 4 days, and are critical for preventing blood-borne pathogens from evolving into life-threatening infections. The interaction of BAFF, also known as BLyS, with its receptors BAFFR and TACI on B cells is critical for B cell homeostasis and function. Using a synthetic polysaccharide antigen, it has previously been shown that TACI is critical for T cell-independent antibody responses. To examine the role of BAFFR and TACI in T cell-independent antibody responses to an active infection, we utilized the Borrelia hermsii infection system. In this infection system, T cell-independent responses mediated by …

Orai1 Deficiency Leads To Heart Failure And Skeletal Myopathy In Zebrafish., Mirko Völkers, Nima Dolatabadi, Natalie Gude, Patrick Most, Mark A Sussman, David Hassel

Center for Translational Medicine Faculty Papers

Mutations in the store-operated Ca²⁺ entry pore protein ORAI1 have been reported to cause myopathies in human patients but the mechanism involved is not known. Cardiomyocytes express ORAI1 but its role in heart function is also unknown. Using reverse genetics in zebrafish, we demonstrated that inactivation of the highly conserved zebrafish orthologue of ORAI1 resulted in severe heart failure, reduced ventricular systolic function, bradycardia and skeletal muscle weakness. Electron microscopy of Orai1-deficient myocytes revealed progressive skeletal muscle instability with loss of myofiber integrity and ultrastructural abnormalities of the z-disc in both skeletal and cardiac muscle. Isolated Orai1-deficient cardiomyocytes showed loss …

Evidence That The Density Of Self Peptide-Mhc Ligands Regulates T-Cell Receptor Signaling., Nadia Anikeeva, Dimitry Gakamsky, Jørgen Schøller, Yuri Sykulev

Department of Microbiology and Immunology Faculty Papers

Noncognate or self peptide-MHC (pMHC) ligands productively interact with T-cell receptor (TCR) and are always in a large access over the cognate pMHC on the surface of antigen presenting cells. We assembled soluble cognate and noncognate pMHC class I (pMHC-I) ligands at designated ratios on various scaffolds into oligomers that mimic pMHC clustering and examined how multivalency and density of the pMHCs in model clusters influences the binding to live CD8 T cells and the kinetics of TCR signaling. Our data demonstrate that the density of self pMHC-I proteins promotes their interaction with CD8 co-receptor, which plays a critical role …

B1b Lymphocyte-Derived Antibodies Control Borrelia Hermsii Independent Of Fcα/Μ Receptor And In The Absence Of Host Cell Contact., Matthew J. Colombo, David Abraham, Akira Shibuya, Kishore R. Alugupalli

Department of Microbiology and Immunology Faculty Papers

The critical role of IgM in controlling pathogen burden has been demonstrated in a variety of infection models. In the murine model of Borrelia hermsii infection, IgM is necessary and sufficient for the rapid clearance of bacteremia. Convalescent, but not naïve, B1b cells generate a specific IgM response against B. hermsii, but the mechanism of IgM-mediated protection is unknown. Here, we show that neither Fcα/μR, a high-affinity receptor for IgM, nor IgM-dependent complement activation is required for controlling B. hermsii. Bacteria in diffusion chambers with a pore size impermeable to cells were killed when diffusion chambers were implanted into either …

The Interplay Between Nf-Kappab And E2f1 Coordinately Regulates Inflammation And Metabolism In Human Cardiac Cells., Xavier Palomer, David Álvarez-Guardia, Mercy M Davidson, Tung O Chan, Arthur M Feldman, Manuel Vázquez-Carrera

Department of Medicine Faculty Papers

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, …

Ms4a4b, A Cd20 Homologue In T Cells, Inhibits T Cell Propagation By Modulation Of Cell Cycle., Hui Xu, Yaping Yan, Mark S Williams, Gregory B Carey, Jingxian Yang, Hongmei Li, Guang-Xian Zhang, Abdolmohamad Rostami

Department of Neurology Faculty Papers

MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural …

S-Glutathionylation Activates Stim1 And Alters Mitochondrial Homeostasis., Brian J Hawkins, Krishna M Irrinki, Karthik Mallilankaraman, Yu-Chin Lien, Youjun Wang, Cunnigaiper D Bhanumathy, Ramasamy Subbiah, Michael F Ritchie, Jonathan Soboloff, Yoshihiro Baba, Tomohiro Kurosaki, Suresh K Joseph, Donald L Gill, Muniswamy Madesh

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to …

Differential Impact Of Tumor Suppressor Pathways On Dna Damage Response And Therapy-Induced Transformation In A Mouse Primary Cell Model., A Kathleen Mcclendon, Jeffry L Dean, Adam Ertel, Erik S Knudsen

Department of Cancer Biology Faculty Papers

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying …

Disruption Of C-Jun Reduces Cellular Migration And Invasion Through Inhibition Of C-Src And Hyperactivation Of Rock Ii Kinase., Xuanmao Jiao, Sanjay Katiyar, Manran Liu, Susette C Mueller, Michael P. Lisanti, Anping Li, Timothy G Pestell, Kongming Wu, Xiaoming Ju, Zhiping Li, Erwin F Wagner, Tatsuo Takeya, Chenguang Wang, Richard G Pestell

Kimmel Cancer Center Faculty Papers

The spread of metastatic tumors to different organs is associated with poor prognosis. The metastatic process requires migration and cellular invasion. The protooncogene c-jun encodes the founding member of the activator protein-1 family and is required for cellular proliferation and DNA synthesis in response to oncogenic signals and plays an essential role in chemical carcinogenesis. The role of c-Jun in cellular invasion remains to be defined. Genetic deletion of c-Jun in transgenic mice is embryonic lethal; therefore, transgenic mice encoding a c-Jun gene flanked by LoxP sites (c-jun(f/f)) were used. c-jun gene deletion reduced c-Src expression, hyperactivated ROCK II signaling, …

Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance And Lead To Significant Bone Tissue Formation., Antonio Graziano, Riccardo D'Aquino, Maria Gabriella Cusella-De Angelis, Gregorio Laino, Adriano Piattelli, Maurizio Pacifici, Alfredo De Rosa, Gianpaolo Papaccio

Department of Orthopaedic Surgery Faculty Papers

BACKGROUND: Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.

METHODOLOGY AND FINDINGS: In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter …

Signaling Through Galphai2 Protein Is Required For Recruitment Of Neutrophils For Antibody-Mediated Elimination Of Larval Strongyloides Stercoralis In Mice., Udaikumar M. Padigel, Louis Stein, Kevin Redding, James J. Lee, Thomas J. Nolan, Gerhard A. Schad, Lutz Birnbaumer, David Abraham

Department of Microbiology and Immunology Faculty Papers

The heterotrimeric guanine nucleotide-binding protein Galphai2 is involved in regulation of immune responses against microbial and nonmicrobial stimuli. Galphai2-/- mice have a selectively impaired IgM response consistent with a disorder in B cell development yet have augmented T cell effector function associated with increased production of IFN-gamma and IL-4. The goal of the present study was to determine if a deficiency in the Galphai2 protein in mice would affect the protective immune response against Strongyloides stercoralis, which is IL-4-, IL-5-, and IgM-dependent. Galphai2-/- and wild-type mice were immunized and challenged with S. stercoralis larvae and analyzed for protective immune responses …

Selective Role For Superoxide In Insp3 Receptor-Mediated Mitochondrial Dysfunction And Endothelial Apoptosis., Muniswamy Madesh, Brian J Hawkins, Tatyana Milovanova, Cunnigaiper D Bhanumathy, Suresh K Joseph, Satish P Ramachandrarao, Kumar Sharma, Tomohiro Kurosaki, Aron B Fisher

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane …

Functionally Active T1-T1 Interfaces Revealed By The Accessibility Of Intracellular Thiolate Groups In Kv4 Channels., Guangyu Wang, Mohammad Shahidullah, Carmen A Rocha, Candace Strang, Paul J Pfaffinger, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is …

Ocular Surface Epithelia Contain Abcg2-Dependent Side Population Cells Exhibiting Features Associated With Stem Cells., Murat T Budak, Onder S Alpdogan, Mingyuan Zhou, Robert M Lavker, M A Murat Akinci, J Mario Wolosin

Department of Medical Oncology Faculty Papers

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate …