Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network

Significance Statement

In the manuscript “Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network” a combination therapy comprising chemotherapeutics and death receptor agonists was analyzed in an in vitro model of pancreatic cancer.  This co-treatment led to a strong synergistic effect, killing a high amount of cancer cells. More detailed analysis revealed a sensitization mechanism via down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, which are typically overexpressed in cancer cells thereby causing defects in the cell death machinery. This gemcitabine-mediated decrease of c-FLIP protein levels changed the composition of the death-inducing signaling complex (DISC) after CD95L-treatment towards a higher amount of the apoptosis-inducing proteins Caspase-8 and Caspase-10. This modified protein complex composition is linked to the higher rate of cell death induction. Cell death assays in presence of inhibitors of apoptosis (zVAD-fmk) and necroptosis (Necrostatin-1) indicated that the combination of gemcitabine and CD95L induced both cell death modes, apoptosis and necroptosis, while the stimulation with CD95L alone led solely to apoptosis. Additionally, the usage of the cutting edge technology “imaging flow cytometry” confirmed that the combinatorial treatment not only sensitized cells to death, but also switched them to an alternative cell death pathway – necroptosis via RIPK1. These results demonstrate that this cell death network is regulated via caspase-dependent and caspase-independent pathways. The combined therapy of chemotherapeutics and death receptor agonists might offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-mediated cell death.  These findings might improve treatment strategies and are important for the development of the systems biology-based approaches for the personalized medicine.

 

About The Author

Dr. Sabine Pietkiewicz studied Biology at the Heinrich-Heine-University in Düsseldorf, Germany and already started focusing on apoptosis and necroptosis after death receptor stimulation during her diploma thesis in the Institute of Molecular Medicine, under the supervision of Prof. Schulze-Osthoff. In Düsseldorf she obtained her Dr. rer. nat. at the Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, and has characterized the role of JNK isoforms in cell death pathways after treatment with the proteasomal inhibitor and chemotherapeutic Bortezomib. She currently conducts research in the department Translational Inflammation Research of Prof. Inna N. Lavrik at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany, analyzing the CD95/APO-1/Fas signaling networks.  

About The Author

Prof. Inna N. Lavrik is a head of department Translational Inflammation Research at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany. She did her Postdoctoral training in Heidelberg, at German Cancer Research Center, at the Division of Immunogenetics, Head Prof. Dr. Peter H. Krammer.  Afterwards she had a group leader position at DKFZ/Bioquant, Heidelberg, Germany, which she held before moving to Magdeburg.

Journal Reference

Exp Cell Res. 2015;339(1):1-9.

Pietkiewicz S¹, Eils R², Krammer PH³, Giese N⁴, Lavrik IN⁵. 

Show Affiliations

1. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany.
2. Bioquant, Heidelberg University, 69120 Heidelberg, Germany; Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany; Division of Theoretical Bioinformatics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
3. Division of Immunogenetics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
4. Department of General Surgery, University of Heidelberg, Germany (g)Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia.
5. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany; Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia. Electronic address: inna.lavrik@med.ovgu.de.

Abstract

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Electrospun Polycaprolactone 3D Nanofibrous Scaffold with Interconnected and Hierarchically Structured Pores for Bone Tissue Engineering.

Significance Statement

Ultrathin fibers (with diameters from tens to hundreds of nanometers, commonly known as electrospun nanofibers) prepared via the electrospinning method are of great interests for tissue engineering applications, because electrospun nanofibers have diameters similar to those of fibrous structures in natural extracellular matrix (ECM).  The major limitation of electrospun scaffolds is owing to their morphological structure of overlaid nanofiber mats with apparent/equivalent pore sizes in sub-micrometers; in other words, electrospun nanofibrous mats lack the needed macropores (with sizes from tens to hundreds of micrometers) for cell growth and tissue formation.

In the reported studies, novel electrospun three-dimensional (3D) nanofibrous scaffold has been developed by an innovative and convenient approach (i.e., thermally induced nanofiber self-agglomeration followed by freeze drying) from a biopolymer of polycaprolactone (PCL) for the first time, and the scaffold possesses interconnected and hierarchically structured pores (in specific, the macropores with sizes in hundreds of micrometers would maintain the structural stability of scaffold, support cell proliferation, ECM deposition, and tissue formation; the medium pores with sizes in tens of micrometers or smaller would facilitate the diffusion of nutrients and promote the formation of vascularization, while the small pores with sizes in micrometers or smaller would have impacts on some cell behaviors such as seeding and genes expressions).  The novel polycaprolactone 3D scaffold is soft and elastic with very high porosity of ~96.4%, thus it is morphologically/structurally similar to natural ECM and well-suited for cell functions (e.g., adhesion, proliferation, migration, and differentiation) and tissue formation.

The in vitro studies reveal that the novel scaffold can lead to high cell viability; more importantly, it is able to promote more potent bone morphogenetic protein 2 (BMP2) induced chondrogenic (rather than osteogenic) differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs).  Consistent to the in vitro findings, the in vivo results indicate that electrospun polycaprolactone 3D scaffold acts as a favorable synthetic ECM for functional bone regeneration through the physiological endochondral ossification process (i.e., through formation of cartilage intermediate), which is a new strategy of “developmental tissue engineering” to mimic natural endochondral bone repair process.  Hence, it is envisioned that the developed electrospun polycaprolactone 3D nanofibrous scaffold would be very promising for tissue engineering applications including the regenerations of bone, cartilage, and their composite tissue of osteochondral (an especially challenged tissue repair in clinics).

About The Author

Dr. Hongli Sun is an Assistant Professor in the Biomedical Engineering program at the University of South Dakota (USD).  Prior to joining the faculty at the USD, he worked as a Research Fellow in the Department of Biologic and Materials Sciences at the University of Michigan.  Dr. Sun earned his Ph.D. in Cell Biology from the Chinese Academy of Sciences (Shanghai, China) in 2007.  Inspired by the fundamental findings in stem cell and developmental biology, Dr. Sun’s research has been focusing on the development of novel stem cell/nano-biomaterials based translational strategies for challenged bone regeneration.  Dr. Sun has authored many articles in peer-reviewed journals including Biomaterials, Stem Cells, and Tissue Engineering.  In addition, he has been serving as a manuscript/grant reviewer in the fields related to bone, stem cells, and tissue engineering.

 

About The Author

Dr. Hao Fong is one of the pioneers and renowned scientists worldwide in the field of “Electrospinning and Nanofibers”.  Presently, he is a tenured Full Professor in the Department of Chemistry and Applied Biological Sciences at the South Dakota School of Mines and Technology (SDSM&T); and he is also an important faculty member in the SDSM&T’s multidisciplinary graduate programs of Materials Engineering and Science (MES), Nanoscience and Nanoengineering (NANO), and Biomedical Engineering (BME).  His highest degree is a Ph.D. earned in 1999 from the Department of Polymer Science at the University of Akron (in Ohio, USA).  Prior to joining the faculty at the SDSM&T in 2003, he worked as a guest research scientist in the Polymer Branch of the Air Force Research Laboratory (AFRL) in the Wright-Patterson Air Force Base, and as a staff research scientist in the Paffenbarger Research Center of the American Dental Association (PRC-ADA) and/or the Polymer Division at the National Institute of Standards and Technology (NIST) in Maryland, for a total of three years.  In the recent years, Dr. Hao Fong’s research interests have been focused on “The Materials-processing Technique of Electrospinning and Various Applications of Electrospun Polymer, Ceramic, Carbon/Graphite, Metallic, Composite, and Hierarchically-structured Nanofibers and/or Nanofibrous Materials”.  The applications include, but not limited to, (1) filtration/separation applications (e.g., separation of biopharmaceutical therapeutics such as proteins, purification of air/water, microfiltration, ultrafiltration, nanofiltration, and reverse osmosis), (2) energy-related applications (e.g., solar cells, batteries, fuel cells, and supercapacitors), (3) biomedical applications (e.g., tissue engineering, drug delivery, and antimicrobial wound dressing), (4) microelectronics-related applications (e.g., sensors/detectors and transistors), (5) composite applications (e.g., hybrid multi-scale composites and dental restorative composites).

Journal Reference

Adv Healthc Mater. 2015;4(15):2238-46.

Xu T¹, Miszuk JM², Zhao Y¹, Sun H², Fong H¹.

Show Affiliations

1. Program of BiomedicalEngineering, South Dakota School of Mines and Technology, Rapid City, SD, 57701, USA.
2. Program of BiomedicalEngineering, University of South Dakota, Sioux Falls, SD, 57107, USA.

Abstract

For the first time, electrospun polycaprolactone (PCL) 3D nanofibrous scaffold has been developed by an innovative and convenient approach (i.e., thermally induced nanofiber self-agglomeration followed by freeze drying), and the scaffold possesses interconnected and hierarchically structured pores including macropores with sizes up to ≈300 μm. The novel polycaprolactone 3D scaffold is soft and elastic with very high porosity of ≈96.4%, thus it is morphologically/structurally similar to natural extracellular matrix and well suited for cell functions and tissue formation. The in vitro studies reveal that the scaffold can lead to high cell viability; more importantly, it is able to promote more potent BMP2-induced chondrogenic than osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Consistent to the in vitro findings, the in vivo results indicate that the electrospun polycaprolactone 3D scaffold acts as a favorable synthetic extracellular matrix for functional bone regeneration through the physiological endochondral ossification process.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Design and synthesis of 2-phenylnaphthalenoids and 2-phenylbenzofuranoids as DNA topoisomerase inhibitors and antitumor agents

Significance Statement

Human DNA topoisomerase IIα (TopoIIα), an Achilles’ heel of cancer, is a well-validated target for antitumor drugs. The inhibitors of TopoIIα are among the most effective and commonly used antitumor drugs in clinic, e. g. etoposide and doxorubicin. However, clinically used v inhibitors exhibited serious side effects including cardiotoxicity, development of secondary malignancies and multidrug resistance, thus that it is urgent to explore novel DNA topoisomerase IIα inhibitors for the development of antitumor drugs.

In the recent work of Shen laboratory, a group of 2-phenylnaphthalenoids (2PNs) and 2-phenylbenzofuranoids (2PBFs) were designed and synthesized for the sake of innovation of DNA topoisomerase IIα inhibitors. These 2PNs and 2PBFs were proved to display favorable TopoIIα inhibitory as well as antiproliferative activities. In particular, 2PBFs possess divergent mechanism of action on inhibiting DNA topoisomerase IIα from 2PNs, switching Topo poisons 2PNs to Topo catalytic inhibitors 2PBFs. These results suggest that the chromophore scaffold replacement drug design strategy used in this study may result in a change of the binding sites of inhibitors to TopoIIα. This study provides insights to the structure optimization of DNA topoisomerase IIα inhibitors.

About The Author

Dr. Yuemao Shen received his B. Sc. degree in Chemistry from Anhui Normal University in 1986, M.Sc. in natural products chemistry from Kunming Institute of Botany (KIB) of Chinese Academy of Sciences in 1989. He went to Floss Lab at the University of Washington (UW) as a visiting Scientist and became a joint Ph. D. student of KIB and UW in 1995, and received his Ph. D. degree in Botany from KIB of Chinese Academy of Sciences in 1999. Before joining the faculty of Shandong University in 2010, he was an intern Researcher (1989-1991), assistant Researcher (1991-1995), associate Researcher (1998-1999) and Researcher (1999-2004) in KIB, and Professor in Xiamen University (2004-2010). Being a professor of Shandong University, Dr. Shen’s research interests focus on isolation, structure elucidation, and biosynthesis of natural products with anti-infective or antitumor activities.

About The Author

Miss Huilin Hao received her B. Sc. degree in Pharmacy from Shandong University in 2013. She is currently a graduate student under the supervision of Prof. Yuemao Shen at Shandong University School of Pharmaceutical Sciences. Her research interest includes antitumor mechanisms of topoisomerase inhibitors, endocytic vesicle trafficking and cancer cell metabolism.

About The Author

Dr. Wang Chen received his B. Sc. degree in Pharmacy from Xi’an Jiaotong University Health Sciences Center in 2010, and obtained his Ph. D. degree in Medicine from Shandong University School of Pharmaceutical Sciences in 2015. He is currently a lecturer in Shaanxi University of Technology Vitamin D Research Institute. His research interest is the synthesis of Vitamin D derivatives.

Journal Reference

Eur J Med Chem. 2015;102:277-87.

Hao H¹, Chen W¹, Zhu J², Lu C¹, Shen Y³.

Show Affiliations

1. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China.
2. State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China.
3. Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, 250012, PR China; State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, Shandong, 250100, PR China. Electronic address: yshen@sdu.edu.cn.

Abstract

Eight 2-phenylnaphthalenoids (2PNs) (3a-h) and twenty four 2-phenylbenzofuranoids (2PBFs) (4a–4j, 5a-5j, 6a, 6f-6h) were successfully designed, synthesized and their antiproliferative and in vitro DNA topoisomerase inhibitory activities were evaluated. Nine compounds (four 2PNs and five 2PBFs) showed either TopoI or TopoIIα inhibitory activities. Six compounds (four 2PNs and two 2PBFs) exhibited potent cytotoxicity with IC50 values for 72 h exposure ranging from 0.3 to above 20 μM against MDA-MB-231, MDA-MB-435, HepG2 and PC3 cell lines. The two 2PBFs displayed comparable and even better antiproliferative as well as TopoIIα inhibitory activities than 2PNs. Interestingly, the active 2PBFs displayed different mechanisms of TopoIIα inhibition from that of 2PNs, suggesting that the chromophore scaffold replacement may result in a change of the binding site of inhibitors to TopoIIα. Furthermore, the mechanisms of antiproliferation on MDA-MB-231 cells indicate that compounds 5a and 5f are promising for further development of anticancer agents. The results of this study reveal that the evolutionary strategy of medicinal chemistry through scaffold hopping is a promising strategy for structure optimization of TopoIIα inhibitors.

Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility

Significance Statement

SREBP signaling regulates transcriptions of genes necessary for the homeostasis of fatty acids and cholesterol in the cell, which is a key factor for cellular wellbeing and proliferation. Accordingly, the abrogation of SREBP signaling is associated with numerous pathological conditions ranging from cardiometabolic disorders to metabolic syndrome and cancer. Representing many other signaling processes from homeostasis to proliferation, compartmentalization, and differentiation like Wnt and Notch, the signal cascade is elicited via a regulated intramembrane proteolysis (RIP), i. e. the proteolytic cleavage of a membrane-integral transcription factor. In this work, Linser et al. find that the membrane-spanning part of the SREBP transcriptional activator precursor molecule, which is the substrate in the RIP process, comprises interrupted structural stability. This enables conformational flexibility and maybe one of the factors explaining the enigmatic specificity and selectivity of the eminent and evolutionarily conserved RIP process, which is poorly understood to-date.

About The Author

Dr. Rasmus Linser obtained his PhD in Biophysics at the Leibniz Institute for Molecular Pharmacology in Berlin, Germany, in 2010. During his postdoctoral studies, he worked at the University of New South Wales in Sydney, Australia, and Harvard Medical School, Boston, MA, developing and using Nuclear Magnetic Resonance (NMR) spectroscopic methods both in the solution and solid state. His independent group, which he started in 2014 at the Max Planck Institute for biophysical chemistry in Göttingen, is concerned with the development and application of NMR methods mostly for solid proteins. With these, the molecular mechanisms of disease-related or functional amyloids, membrane proteins, and enzymes are characterized from a structural point of view and concerning protein dynamics. This helps to shed light on the various mechanisms that cells have invented to survive. Dr. Linser’s lab is currently funded by the German Research Association (DFG, Emmy Noether program and SFB 803, project A03), the Association of the Chemical Industries (VCI), and the Michael J. Fox Foundation. 

 

About The Author

 Prof. Dr. Gerhard Wagner obtained his PhD in Biophysics at the Eidgenössische Technische Hochschule (ETH) in Zürich, Switzerland, in 1977. After postdoctoral work at the Massachusetts Institute of Technology (MIT) in Cambridge, MA and the ETH, he accepted an Associate Professor position at the University of Michigan in 1987. Since 1990, he has been a Professor (since 1992 Elkan Rogers Blout Professor) at Harvard Medical School, Boston, MA, in the Department of Biological Chemistry and Molecular Pharmacology. He has been awarded with numerous high-rank honors and has been elected a member of various associations, including the German Academy of Sciences (Leopoldina), the National Academy of Sciences of the United States and the American Academy of Arts and Sciences. His lab has been pivotal regarding the development and application of solution NMR methods. The approaches developed in the Wagner lab have been leading the way to characterization of structure and function of proteins in solution  by NMR spectroscopy, for example with regard to the elucidation of eukaryotic translation initiation. Dr. Wagner’s lab is funded by the National Institute of Health (NIH) with grants GM046476 and HL116391. 

Figure Legend:  Dynamics of the substrate, enabled by interrupted structural definition, may play a role for the specificity in Regulated Intramembrane Proteolysis. Many molecular details of this evolutionarily conserved mechanism are still enigmatic, even though it is a constituent of numerous signaling cascades. This work was pursued in the case of SREBP signaling, which is a hallmark of numerous pathological conditions from cardiometabolic disorders to cancer.

Journal Reference

Proc Natl Acad Sci U S A. 2015;112(40):12390-5.

Linser R¹, Salvi N², Briones R³, Rovó P⁴, de Groot BL³, Wagner G⁵.

Show Affiliations

1. Department NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; rali@nmr.mpibpc.mpg.de gerhard_wagner@hms.harvard.edu.
2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; Université Grenoble Alpes, Centre National de la Recherche Scientifique, and Commissariat à l’Énergie Atomique et aux Énergies Alternatives, Institut de Biologie Structurale, F-38044 Grenoble, France;
3. Biomolecular Dynamics Group, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
4. Department NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany;
5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; rali@nmr.mpibpc.mpg.de gerhard_wagner@hms.harvard.edu. 

Abstract

Regulated intramembrane proteolysis (RIP) is a conserved mechanism crucial for numerous cellular processes, including signaling, transcriptional regulation, axon guidance, cell adhesion, cellular stress responses, and transmembrane protein fragment degradation. Importantly, it is relevant in various diseases including Alzheimer’s disease, cardiovascular diseases, and cancers. Even though a number of structures of different intramembrane proteases have been solved recently, fundamental questions concerning mechanistic underpinnings of RIP and therapeutic interventions remain. In particular, this includes substrate recognition, what properties render a given substrate amenable for RIP, and how the lipid environment affects the substrate cleavage. Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors are critical regulators of genes involved in cholesterol/lipid homeostasis. After site-1 protease cleavage of the inactive SREBP transmembrane precursor protein, RIP of the anchor intermediate by site-2 protease generates the mature transcription factor. In this work, we have investigated the labile anchor intermediate of SREBP-1 using NMR spectroscopy. Surprisingly, NMR chemical shifts, site-resolved solvent exposure, and relaxation studies show that the cleavage site of the lipid-signaling protein intermediate bears rigid α-helical topology. An evolutionary conserved motif, by contrast, interrupts the secondary structure ∼9-10 residues C-terminal of the scissile bond and acts as an inducer of conformational flexibility within the carboxyl-terminal transmembrane region. These results are consistent with molecular dynamics simulations. Topology, stability, and site-resolved dynamics data suggest that the cleavage of the α-helical substrate in the case of RIP may be associated with a hinge motion triggered by the molecular environment.

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The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

Journal Reference

Oncotarget. 2015;6(33):34910-23.

Eike LM¹, Yang N², Rekdal Ø^1,3, Sveinbjørnsson B¹.

Show Affiliations

1. Department of Molecular Inflammation Research and Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway.
2. Department of Community Medicine, Faculty of Health University of Tromsø, Tromsø, Norway.
3. Lytix Biopharma, Oslo, Norway.

Abstract

Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models.

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Adipose-derived stromal cells for the reconstruction of a human vesical equivalent

Journal Reference

J Tissue Eng Regen Med. 2015;9(11):E135-43.

Rousseau A¹, Fradette J¹, Bernard G¹, Gauvin R¹, Laterreur V¹, Bolduc S^1,2

Show Affiliations

1. Centre LOEX de l’Université Laval, Génie Tissulaire et Régénération, LOEX du Centre de Recherche FRSQ du CHU de Québec, Département de Chirurgie, Faculté de Médecine, Université Laval, Québec, QC, Canada.
2. CMDGT/LOEX-ÉquipeReconstruction Vésicale, Centre de Recherche FRQS du CHU de Québec, Aile-R, Hôpital de l’Enfant-Jésus, Centre Hospitalier Affilié Universitaire de Québec, QC, Canada.

Abstract

Despite a wide panel of tissue-engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self-assembly method. Moreover, we tested the contribution of adipose-derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical  equivalent, adipose-derived stromal cell vesical equivalent and hybrid vesical equivalent – the latter containing both adipose-derived  stromal  cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO-1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue-engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright © 2013 John Wiley & Sons, Ltd.

Copyright © 2013 John Wiley & Sons, Ltd.

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Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

Journal Reference

PLoS One. 2015;10(9):e0138215.

Yang B¹, Rajput PS², Kumar U², Sastry BR¹.

Show Affiliations

1. Neuroscience Research Laboratory, Department of Anesthesiology, Pharmacology & Therapeutics, Faculty of Medicine, The University of British Columbia, Vancouver, Canada.
2. Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, Canada.

Abstract

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.

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Lignocellulose-based analytical devices: bamboo as an analytical platform for chemical detection.

Journal Reference

Sci Rep. 2015 Dec 21;5:18570. doi: 10.1038/srep18570.

Kuan CM¹, York RL^2,3, Cheng CM⁴.

Show Affiliations

1. Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 30013, Taiwan.
2. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
3. Department of Anesethesiology, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
4. Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.

 

Abstract

This article describes the development of lignocellulose-based analytical devices (LADs) for rapid bioanalysis in low-resource settings. LADs are constructed using either a single lignocellulose or a hybrid design consisting of multiple types of lignocellulose. LADs are simple, low-cost, easy to use, provide rapid response, and do not require external instrumentation during operation. Here, we demonstrate the implementation of LADs for food and water safety (i.e., nitrite assay in hot-pot soup, bacterial detection in water, and resazurin assay in milk) and urinalysis (i.e., nitrite, urobilinogen, and pH assays in human urine). Notably, we created a unique approach using simple chemicals to achieve sensitivity similar to that of commercially available immunochromatographic strips that is low-cost, and provides on-site, rapid detection, for instance, of Eschericia coli (E. coli) in water.

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Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection.

Journal Reference

Am J Respir Crit Care Med. 2015 Dec 15;192(12):1490-503.

Gregson AL¹, Hoji A², Injean P³, Poynter ST³, Briones C³, Palchevskiy V³, Sam Weigt S³, Shino MY³, Derhovanessian A³, Sayah D³, Saggar R³, Ross D³,Ardehali A⁴, Lynch JP 3rd³, Belperio JA³.

Show Affiliations

1. Division of Infectious Diseases, Department of Medicine.
2. Department of Transplantation, University of Pittsburgh, Pittsburgh, Pennsylvania.
3. Division of Pulmonary, Critical Care, Allergy, and Immunology, Department of Medicine, and.
4. Division of Cardiothoracic Surgery, Department of Surgery, University of California, Los Angeles, California; and.

Abstract

RATIONALE:

The mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejectionprocess. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes.

OBJECTIVES:

To provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence.

METHODS:

Serial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed.

MEASUREMENTS AND MAIN RESULTS:

AR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets.

CONCLUSIONS:

Our findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation.

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Control of calcium oxalate morphology through electrocrystallization as an electrochemical approach for preventing pathological disease

Journal Reference

Ionics, November 2015, Volume 21, Issue 11, pp 3141-3149.

Andrónico Neira-Carrillo ^1,2, Patricio Vásquez-Quitral¹, Marianela Sánchez¹, Andrés Vargas-ernández¹, Juan Francisco Silva³ 

Show Affiliations

1. Faculty of Veterinary and Animal Sciences, University of Chile, Av. Santa Rosa, 11735, Santiago, Chile
2. Advanced Center for Chronic Diseases (ACCDiS), University of Chile, Santiago, Chile
3. Departamento de Química de los Materiales, Laboratorio de Electrocatálisis, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile

Abstract

Pathological crystallization of calcium oxalate (CaOx) inside the urinary tract is called calculi or kidney stone (Urolithiasis). CaOx exhibits three crystalline types in nature: CaOx monohydrate COM, dihydrate COD and trihydrate COT. COD and COM are often found in urinary calculi, particularly COM. Electrocrystallization has been recently used to perform oriented crystallization of inorganic compounds such as Ca-salts. Although many mineralization methods exist, the mechanisms involved in the control of CaOx polymorphism still remain unclear. Herein, we induced selective electrocrystallization of COD by modifying the electrical current, time and electrochemical cell type. By combining above factors, we established an efficient method without the use of additives for stabilizing non-pathological CaOx crystals. We found notorious stabilization of CaOx polymorphisms with hierarchically complex shape with nano-organization assembly, size and aggregated crystalline particles. Our results demonstrated that, by using an optimized electrochemical approach, this technique could have great potential for studying the nucleation and crystal growth of CaOx through functionalized synthetic polymers, and to develop a novel pathway to evaluate new calculi preventing-compound inhibitors.

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Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor

Significance Statement

The article features a novel bioreactor device, called CellTank, for the culture of animal cells at high cell density. This system is a compact perfusion bioreactor for manufacturing of biologics. It consists of a non-woven fiber matrix continuously perfused with the culture medium, using an integrated centrifugal pump. The matrix, immersed in a reservoir, harbors the cells, which are either anchorage dependent or adapted for suspension.

The purpose of the article study was to evaluate the CellTank potentials to support very high cell densities and biologics production. The work has been performed and supervised in the group of Cell Technology at KTH, Royal Institute of Technology (Sweden) in collaboration with CerCell (Denmark) for the CellTank design and manufacturing, and with Belach (Sweden) for the control system of the culture process. Three perfusion runs of Chinese Hamster Ovary cells producing an IgG1 antibody in CellTank of 150 mL matrix volume are presented. The results include a perfusion run with a cell concentration up to 200 millions cells per mL and two runs with cell arrest by mild hypothermia at densities around 100 to 130 millions cells per mL. The cell density was monitored by dielectric spectroscopy, i.e. bioimpedance.

One of the advantages of the CellTank system is the homogeneous distribution of the liquid flow in the whole bioreactor: homogeneous concentrations of the metabolites in the different parts of the bioreactor were observed despite the very high cell densities. In comparison, other bioreactor technologies, where the cells are immobilized such as hollow fiber bioreactor, are typically subject to nutrient and by-product gradients, creating differential and sub-optimal environments. The other advantages are the compactness, the easiness of operation, and the minimal need of tuning the system for operations.

The 150 mL matrix CellTank has the capacity to support the production of hundreds milligrams protein (for low producing cell lines) to several grams protein (for high producing lines). Larger matrices, up to 2.2 liter using the same flow design, can be used to increase the production capacity if needed. This technology could also be used for virus production. The CellTank is very suitable for the manufacturing of protein for diagnostics, research, early stage development or production. 

About The Author

Dr. Veronique Chotteau [M. Sc. Electrical Engineering, M. Sc. Molecular Biology and Biotechnology, Ph.D. Biotechnology/Automatic Control (Université Catholique de Louvain, Belgium)] has ≥ 25 years of experience in mammalian cell culture including ≥ 10 years in biopharmaceutical industry at Pharmacia Upjohn, Biovitrum (nowadays Swedish Orphan Biovitrum). Her expertise covers process development: perfusion (e.g. recombinant factor VIII ReFacto), fed-batch, stem cell bioprocessing, small-, pilot- and commercial scale, GMP, project management, evaluation of new projects. Since 2008, Dr. Chotteau is Principal Investigator, leading the group of Cell Technology at the Biotechnology Faculty, KTH, Royal Institute of Technology, Stockholm, Sweden. Her group is world-leader in high cell density perfusion of mammalian cell-based processes for biopharmaceutical production, and is highly active in mathematical modeling of culture process, as well as in human stem cell bioprocessing.  

About The Author

Ye Zhang [M. Sc. Biotechnology Eng.] is PhD student in Chotteau’s group.  

About The Author

Per Stobbe [M. Sc. Mechanical Eng.] is CEO of CerCell (Denmark). He has 30 years experience of filtration techniques and mechanical devices.  

About The Author

Christian Orrego Silvander [M. Sc. Biotechnology Eng.], former business and bioprocess application engineer at Belach (Sweden), is currently business developer at Serendipity Innovation, Sweden.

 

Journal Reference

J Biotechnol. 2015;213:28-41.

Zhang Y¹, Stobbe P², Silvander CO³, Chotteau V⁴.

Show Affiliations

¹School of Biotechnology, Dept. Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden. Electronic address: yezhang@kth.se.

²PerfuseCell, Malmmosevej 19C, DK-2840 Holte, Denmark.

³Belach Bioteknik, Dumpervägen 8, SE-14250 Skogås, Sweden(1).

⁴School of Biotechnology, Dept. Industrial Biotechnology/Bioprocess Design, Cell Technology Group (CETEG), Royal Institute of Technology, KTH, SE-10691 Stockholm, Sweden. Electronic address: chotteau@kth.se.

Abstract

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200pF/cm (where 1pF/cm is equivalent to 1×10(6) viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37°C to 29°C was studied during 13 days at cell densities above 100pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31°C was applied at cell density 100pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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Changes in corneal endothelial cell density and the cumulative risk of corneal decompensation after Ahmed glaucoma valve implantation

Significance Statement

Since the introduction of the glaucoma drainage device (GDD), the success rate of surgical treatment for refractory glaucoma, such as failed trabeculectomy, neovascular glaucoma, uveitic glaucoma, and glaucoma with wide conjunctival scarring, has improved significantly, compared with conventional trabeculectomy.

Corneal damage after surgery, such as cataract surgery, trabeculectomy, vitrectomy, laser iridotomy, and cyclophotocoagulation, are thought to occur only during treatment. The amount of change is greater and more variable in glaucoma drainage device group than the trabeculectomy group (figure). Moreover, corneal endothelial cell loss after glaucoma drainage device surgery is particularly significant due to its continuity.

Previous studies have reported corneal damage after use of the GDD. McDermott et al. reported endothelial cell loss averaging two cells per mm² per postoperative month after Molteno implant drainage procedures, but no clinically significant progressive trend in endothelial cell loss was seen in patients undergoing uncomplicated procedures.[1] However, Topouzis et al. reported that the most frequent complication was corneal decompensation or corneal graft failure, and most failures after 12 months of follow-up from Ahmed glaucoma valve (AGV) implantation resulted from corneal complications.[2] The frequency of corneal decompensation after glaucoma implant surgery with long-term (≥ 2 years) follow-up ranged from 5–27%.

A longitudinal study by this team using a baseline preoperative specular microscopic examination reported a cell loss of 10.6% a year after AGV implantation.[3] The second study by this group reported a 15.3% cell loss at 1 year and an 18.6% cell loss at 2 years after AGV implantation. The cell losses at 1 and 2 years after surgery were significantly greater than those of the controls.[4]

This is the third 5-year report stating the endothelial cell loss was significant only up to 2 years after surgery, compared with control eyes without AGV.[5] Although there was a more rapid loss of endothelial cells in the AGV group compared with the control group for 5 years (−7.0%/year and −0.1%/year, respectively; p < 0.001), the average cell loss, measured at the central cornea, decreased with time from −10.7% during the first year to −2.7%/year from 3 years to the final follow up (45.3 ± 20.6 months), and the statistically significant difference compared with the control group was maintained only during the first 2 years after surgery. However, although there was no statistical significance compared to the control eyes, the endothelial cell density decreased 2 years after surgery: −4.2% 2–3 years after surgery and −2.7% 3 years after surgery. Nevertheless, there is a possibility that a prospective study with a larger number of cases may yield different results.

After excluding previous penetrating keratoplasty cases, the cumulative risk of corneal decompensation was 3.3% at 5 years after surgery in uncomplicated cases that did not involve apparent contact between the tube and the cornea. Because the rate of endothelial cell loss remained the same according to this study, regardless of the baseline cell count, patients with low endothelial cell densities before surgery were at a higher risk of corneal decompensation.

Therefore, surgeons must be very careful not to damage the cornea during GDD surgery for the treatment of refractory glaucoma, and all patients treated with the GDD procedure should be monitored for possible corneal problems for more than 2 years.

For patients with low corneal endothelial cell counts or previous penetrating keratoplasty, surgical options other than GDD should first be considered, and the patient warned of possible complications from corneal decompensation.

McDermott ML, Swendris RP, Shin DH, et al. Corneal endothelial cell counts after Molteno implantation. Am J Ophthalmol 1993;115:93–6.

Topouzis F, Coleman AL, Choplin N, et al. Follow-up of the original cohort with the Ahmed glaucoma valve implant. Am J Ophthalmol 1999;128:198–204.

Kim CS, Yim JH, Lee EK, et al. Changes in corneal endothelial cell density and morphology after Ahmed glaucoma valve implantation during the first year of follow up. Clin Experiment Ophthalmol 2008;36:142–7.

Lee EK, Yun YJ, Lee JE, et al. Changes in corneal endothelial cells after Ahmed glaucoma valve implantation: 2-year follow-up. Am J Ophthalmol 2009;148:361–7.

Kim KN, Lee SB, Lee YH, et al. Changes in corneal endothelial cell density and the cumulative risk of corneal decompensation after Ahmed glaucoma valve implantation. Br J Ophthalmol Published Online First: [3/10/2015] doi:10.1136/bjophthalmol-2015-306894

Figure Legend: Scatter plot of the corneal endothelial cell density at baseline and 12 months after trabeculectomy and Ahmed glaucoma valve implantation for treating glaucoma. The amount of change was greater and more variable in Ahmed glaucoma valve implant group than the trabeculectomy group.

TRAB: trabeculectomy, AGV: Ahmed glaucoma valve, CE: corneal endothelial cell (unpublished data)

 

About The Author

Kyoung Nam Kim, MD is an assistant professor at Chungnam national University Hospital, Daejeon, South Korea and a member of the Korean Glaucoma Society. Dr Kim graduated from Chungnam National University College of Medicine and completed an internship and residency in Chungnam national University Hospital. She completed glaucoma fellowship in Seoul National University Hospital. Her current research interests include the study of treatment outcomes in glaucoma and the pathophysiology of glaucoma. 

About The Author

Chang-sik Kim, MD & PhD is a professor of Opthalmology at Chungnam National University Hospital, Daejeon, south Korea. He graduated Chungnam National University College of Medicine and completed a residency in Chungnam national University Hospital. He appointed as a full time faculty of the department of Ophthalmology, Chungnam National University Hospital in 1996. He completed an international glaucoma research fellowship in Hospitals and Clinics of University of Iowa, from 1999 to 2000.

He is a member of Korean Ophthalmology Society (since 1990), Korean Glaucoma Society (since 1997), and the Association for Research in Vision and Ophthalmology (ARVO) since 1988. His research interests include glaucoma surgery and wound healing, Glaucoma drainage device, epidemiology and genetics in glaucoma.  

Journal Reference

Br J Ophthalmol. 2015. pii: bjophthalmol-2015-306894.  

Kim KN¹, Lee SB², Lee YH², Lee JJ¹, Lim HB¹, Kim CS².

Show Affiliations

¹Department of Ophthalmology, Chungnam National University Hospital, Daejeon, Korea.

²Department of Ophthalmology, Chungnam National University Hospital, Daejeon, Korea Department of Ophthalmology, Chungnam National University College of Medicine, Daejeon, Korea

Abstract

AIMS:

To evaluate changes in the corneal endothelial cell density (ECD) and corneal decompensation following Ahmed glaucoma valve (AGV) implantation.

METHODS:

This study was retrospective and observational case series. Patients with refractory glaucoma who underwent AGV implantation and were followed >5 years were consecutively enrolled. We reviewed the medical records, including the results of central corneal specular microscopy. Of the 127 enrolled patients, the annual change in endothelial cell density (%) was determined using linear regression for 72 eyes evaluated at least four times using serial specular microscopic examination and compared with 31 control eyes (fellow glaucomatous eyes under medical treatment). The main outcome measures were cumulative risk of corneal decompensation and differences in the endothelial cell density loss rates between subjects and controls.

RESULTS:

The mean follow-up after AGV implantation was 43.1 months. There were no cases of postoperative tube-corneal touch. The cumulative risk of corneal decompensation was 3.3%, 5 years after AGV implantation. There was a more rapid loss of endothelial cell density in the 72 subject eyes compared with the 31 controls (-7.0% and -0.1%/year, respectively; p<0.001). However, the rate of loss decreased over time and statistical significance compared with control eyes disappeared after 2 years postoperatively: -10.7% from baseline to 1 year (p<0.01), -7.0% from 1 year to 2 years (p=0.037), -4.2% from 2 years to 3 years (p=0.230) and -2.7% from 3 years to the final follow-up (p=0.111).

CONCLUSIONS:

In case of uncomplicated AGV implantation, the cumulative risk of corneal decompensation was 3.3%, 5 years after the operation. The endothelial cell density loss was statistically greater in eyes with AGV than in control eyes without AGV, but the difference was significant only up to 2 years post surgery.

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Blood-Derived CD4 T Cells Naturally Resist Pyroptosis during Abortive HIV-1 Infection

Significance Statement

Blood-Derived CD4 T cells are Resistant to Pyroptosis During HIV-1 Infection

The progressive loss of CD4 T cells lies at the heart of HIV/AIDS. HIV infects and kills activated CD4 T cells by apoptosis, but these permissive cells are too few in number to explain the massive CD4 T cell losses observed in untreated, HIV-infected people.  We recently reported that most CD4 T cells residing in lymphoid tissues correspond to bystander cells that die by abortive HIV infection involving pyroptosis, a highly inflammatory form of programmed cell death. For pyroptosis to occur, the virus must be transmitted to quiescent CD4 T cells through virological synapses (cell-to-cell transmission is required)(Galloway et al., 2015), but in these resting cells, the viral life cycle is arrested during the elongation step of reverse transcription.  Consequently, incomplete HIV DNA transcripts accumulate in the cytosol.  These viral DNAs are detected by the IFI16 DNA sensor (Monroe et al., 2014), which can recognize both single and double stranded DNA targets. Detection of these transcripts triggers an innate immune response involving the assembly of the IFI16 inflammasome, induction of type I interferon and pyroptosis involving caspase-1 activation.

Pyroptosis of spleen, tonsil, and gut-associated-lymphoid tissue-derived CD4 T cells infected with CXCR4 or CCR5 tropic HIV has been shown (Doitsh et al., 2010; Doitsh et al., 2014; Steele et al., 2014) but the susceptibility of blood-derived CD4 T cells to this mechanism of depletion was unknown. We now report that CD4 T cells are resistant to this death pathway while circulating in the blood stream. This resistance is, at least in part, due to a deeper resting state for these cells resulting in the accumulation of less HIV DNA transcripts and lower expression of the DNA sensor, IFI16. Surprisingly, co-culture allowing direct contact with lymphoid tissue-derived CD4 T, CD8 T, or B cells renders blood-derived CD4 T cells sensitive to HIV mediated pyroptosis. Reverse transcription improves and IFI16 levels rise. Continuous interaction between the tissue and blood cells are required to maintain the sensitivity to pyroptosis. The natural resistance of blood cells to HIV-elicited pyroptosis extends beyond virally infected cells––exposure of these cells to nigericin, an activator of the NLRP3 inflammasome also does not result in pyroptosis(Munoz-Arias et al., 2015)

Our work highlights how the tissue micro-environment shapes the response of CD4 T cells to HIV infection. Indeed, if we had begun our studies with blood instead of lymphoid tissue-derived CD4 T cells, we would have not detected the pyroptotic death pathway, which appears to account for depletion of a significant fraction of CD4 T cells during HIV infection. Further studies are now underway to identify the lymphoid tissue factor(s) involved in sensitizing CD4 T cells to HIV-mediated pyroptosis. Inhibition of the interplay of these factors could lead to host directed therapy that would prevent CD4 T cell death but it will be important to exclude detrimental effects of this treatment on the normal immune response.

 

References

Doitsh, G., Cavrois, M., Lassen, K.G., Zepeda, O., Yang, Z., Santiago, M.L., Hebbeler, A.M., and Greene, W.C. (2010). Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue. Cell 143, 789-801.

Doitsh, G., Galloway, N., Geng, X., Yang, Z., Monroe, K., Zepeda, O., Hunt, P.W., Hatano, H., Sowinski, S., Munoz-Arias, I., et al. (2014). Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection. Nature 505, 509-514.

Galloway, N.L., Doitsh, G., Monroe, K.M., Yang, Z., Munoz-Arias, I., Levy, D.N., and Greene, W.C. (2015). Cell-to-Cell Transmission of HIV-1 Is Required to Trigger Pyroptotic Death of Lymphoid-Tissue-Derived CD4 T Cells. Cell reports 12, 1555-1563.

Monroe, K.M., Yang, Z., J.R., J., Geng, X., Doitsh, G., Krogan, N.J., and Greene, W.C. (2014). IFI16 DNA Sensor is Required for Death of Lymphoid CD4 T cells Abortively Infected with HIV. Science (New York, NY) 343, 428-432.

Munoz-Arias, I., Doitsh, G., Yang, Z., Sowinski, S., Ruelas, D., and Greene, W.C. (2015). Blood-Derived CD4 T Cells Naturally Resist Pyroptosis during Abortive HIV-1 Infection. Cell host & microbe 18, 463-470.

Steele, A.K., Lee, E.J., Manuzak, J.A., Dillon, S.M., Beckham, J.D., McCarter, M.D., Santiago, M.L., and Wilson, C.C. (2014). Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways ex vivo. Retrovirology 11, 14.

Journal Reference

Cell Host Microbe. 2015;18(4):463-70.

Isa Muñoz-Arias,^1,2 Gilad Doitsh,² Zhiyuan Yang,² Stefanie Sowinski,² Debbie Ruelas² and Warner C. Greene^2,3,

Show Affiliations

1. School of Public Health, Division of Infectious Diseases and Virology, University of California, Berkeley, Berkeley, California 94720 USA.
2. Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, California 94158 USA.
3. Departments of Medicine and ⁴Microbiology and Immunology, University of California, San Francisco, San Francisco, California 94158 USA. Correspondence: wgreene@gladstone.ucsf.edu

Abstract

Progression to AIDS is driven by CD4 T cell depletion, mostly involving pyroptosis elicited by abortive HIV infection of CD4 T cells in lymphoid tissues. Inefficient reverse transcription in these cells leads to cytoplasmic accumulation of viral DNAs that are detected by the DNA sensor IFI16, resulting in inflammasome assembly, caspase-1 activation, and pyroptosis. Unexpectedly, we found that peripheral blood-derived CD4 T cells naturally resist pyroptosis. This resistance is partly due to their deeper resting state, resulting in fewer HIV-1 reverse transcripts and lower IFI16 expression. However, when co-cultured with lymphoid-derived cells, blood-derived CD4 T cells become sensitized to pyroptosis, likely recapitulating interactions occurring within lymphoid tissues. Sensitization correlates with higher levels of activated NF-κB, IFI16 expression, and reverse transcription. Blood-derived lymphocytes purified from co-cultures lose sensitivity to pyroptosis. These differences highlight how the lymphoid tissue microenvironment encountered by trafficking CD4 T lymphocytes dynamically shapes their biological response to HIV.

Copyright © 2015 Elsevier Inc. All rights reserved.

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