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Archive for the ‘Proteomics’ Category


Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Once herpes simplex infects a person, the virus goes into hiding inside nerve cells, hibernating there for life, periodically waking up from its sleep to reignite infection, causing cold sores or genital lesions to recur. Research from Harvard Medical School showed that the virus uses a host protein called CTCF, or cellular CCCTC-binding factor, to display this type of behavior. Researchers revealed with experiments on mice that CTCF helps herpes simplex regulate its own sleep-wake cycle, enabling the virus to establish latent infections in the body’s sensory neurons where it remains dormant until reactivated. Preventing that latency-regulating protein from binding to the virus’s DNA, weakened the virus’s ability to come out of hiding.

 

Herpes simplex virus’s ability to go in and out of hiding is a key survival strategy that ensures its propagation from one host to the next. Such symptom-free latency allows the virus to remain out of the reach of the immune system most of the time, while its periodic reactivation ensures that it can continue to spread from one person to the next. On one hand, so-called latency-associated transcript genes, or LAT genes, turn off the transcription of viral RNA, inducing the virus to go into hibernation, or latency. On the other hand, a protein made by a gene called ICP0 promotes the activity of genes that stimulate viral replication and causes active infection.

 

Based on these earlier findings, the new study revealed that this balancing act is enabled by the CTCF protein when it binds to the viral DNA. Present during latent or dormant infections, CTCF is lost during active, symptomatic infections. The researchers created an altered version of the virus that lacked two of the CTCF binding sites. The absence of the binding sites made no difference in early-stage or acute infections. Similar results were found in infected cultured human nerve cells (trigeminal ganglia) and infected mice model. The researchers concluded that the mutant virus was found to have significantly weakened reactivation capacity.

 

Taken together, the experiments showed that deleting the CTCF binding sites weakened the virus’s ability to wake up from its dormant state thereby establishing the evidence that the CTCF protein is a key regulator of sleep-wake cycle in herpes simplex infections.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/29437926

 

https://hms.harvard.edu/news/viral-hideout?utm_source=Silverpop

 

https://www.ncbi.nlm.nih.gov/pubmed/30110885

 

https://www.ncbi.nlm.nih.gov/pubmed/30014861

 

https://www.ncbi.nlm.nih.gov/pubmed/18264117

 

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  1. Lungs can supply blood stem cells and also produce platelets: Lungs, known primarily for breathing, play a previously unrecognized role in blood production, with more than half of the platelets in a mouse’s circulation produced there. Furthermore, a previously unknown pool of blood stem cells has been identified that is capable of restoring blood production when bone marrow stem cells are depleted.

 

  1. A new drug for multiple sclerosis: A new multiple sclerosis (MS) drug, which grew out of the work of UCSF (University of California, San Francisco) neurologist was approved by the FDA. Ocrelizumab, the first drug to reflect current scientific understanding of MS, was approved to treat both relapsing-remitting MS and primary progressive MS.

 

  1. Marijuana legalized – research needed on therapeutic possibilities and negative effects: Recreational marijuana will be legal in California starting in January, and that has brought a renewed urgency to seek out more information on the drug’s health effects, both positive and negative. UCSF scientists recognize marijuana’s contradictory status: the drug has proven therapeutic uses, but it can also lead to tremendous public health problems.

 

  1. Source of autism discovered: In a finding that could help unlock the fundamental mysteries about how events early in brain development lead to autism, researchers traced how distinct sets of genetic defects in a single neuronal protein can lead to either epilepsy in infancy or to autism spectrum disorders in predictable ways.

 

  1. Protein found in diet responsible for inflammation in brain: Ketogenic diets, characterized by extreme low-carbohydrate, high-fat regimens are known to benefit people with epilepsy and other neurological illnesses by lowering inflammation in the brain. UCSF researchers discovered the previously undiscovered mechanism by which a low-carbohydrate diet reduces inflammation in the brain. Importantly, the team identified a pivotal protein that links the diet to inflammatory genes, which, if blocked, could mirror the anti-inflammatory effects of ketogenic diets.

 

  1. Learning and memory failure due to brain injury is now restorable by drug: In a finding that holds promise for treating people with traumatic brain injury, an experimental drug, ISRIB (integrated stress response inhibitor), completely reversed severe learning and memory impairments caused by traumatic brain injury in mice. The groundbreaking finding revealed that the drug fully restored the ability to learn and remember in the brain-injured mice even when the animals were initially treated as long as a month after injury.

 

  1. Regulatory T cells induce stem cells for promoting hair growth: In a finding that could impact baldness, researchers found that regulatory T cells, a type of immune cell generally associated with controlling inflammation, directly trigger stem cells in the skin to promote healthy hair growth. An experiment with mice revealed that without these immune cells as partners, stem cells cannot regenerate hair follicles, leading to baldness.

 

  1. More intake of good fat is also bad: Liberal consumption of good fat (monounsaturated fat) – found in olive oil and avocados – may lead to fatty liver disease, a risk factor for metabolic disorders like type 2 diabetes and hypertension. Eating the fat in combination with high starch content was found to cause the most severe fatty liver disease in mice.

 

  1. Chemical toxicity in almost every daily use products: Unregulated chemicals are increasingly prevalent in products people use every day, and that rise matches a concurrent rise in health conditions like cancers and childhood diseases, Thus, researcher in UCSF is working to understand the environment’s role – including exposure to chemicals – in health conditions.

 

  1. Cytomegalovirus found as common factor for diabetes and heart disease in young women: Cytomegalovirus is associated with risk factors for type 2 diabetes and heart disease in women younger than 50. Women of normal weight who were infected with the typically asymptomatic cytomegalovirus, or CMV, were more likely to have metabolic syndrome. Surprisingly, the reverse was found in those with extreme obesity.

 

References:

 

https://www.ucsf.edu/news/2017/12/409241/most-popular-science-stories-2017

 

https://www.ucsf.edu/news/2017/03/406111/surprising-new-role-lungs-making-blood

 

https://www.ucsf.edu/news/2017/03/406296/new-multiple-sclerosis-drug-ocrelizumab-could-halt-disease

 

https://www.ucsf.edu/news/2017/06/407351/dazed-and-confused-marijuana-legalization-raises-need-more-research

 

https://www.ucsf.edu/news/2017/01/405631/autism-researchers-discover-genetic-rosetta-stone

 

https://www.ucsf.edu/news/2017/09/408366/how-ketogenic-diets-curb-inflammation-brain

 

https://www.ucsf.edu/news/2017/07/407656/drug-reverses-memory-failure-caused-traumatic-brain-injury

 

https://www.ucsf.edu/news/2017/05/407121/new-hair-growth-mechanism-discovered

 

https://www.ucsf.edu/news/2017/06/407536/go-easy-avocado-toast-good-fat-can-still-be-bad-you-research-shows

 

https://www.ucsf.edu/news/2017/06/407416/toxic-exposure-chemicals-are-our-water-food-air-and-furniture

 

https://www.ucsf.edu/news/2017/02/405871/common-virus-tied-diabetes-heart-disease-women-under-50

 

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Medical Scientific Discoveries for the 21st Century & Interviews with Scientific Leaders at https://www.amazon.com/dp/B078313281 – electronic Table of Contents 

Author, Curator and Editor: Larry H Bernstein, MD, FCAP

Available on Kindle Store @ Amazon.com since 12/9/2017

List of Contributors & Contributors’ Biographies

Volume Author, Curator and Editor

Larry H Bernstein, MD, FCAP

Preface, all Introductions, all Summaries and Epilogue

Part One:

1.4, 1.5, 1.6, 2.1.1, 2.1.2, 2.1.3, 2.1.4, 2.2.1, 2.2.2, 2.2.3, 2.3, 2.4, 2.4.1, 2.4.2, 2.5, 2.6.1, 2.6.2, 2.6.3, 2.6.4, 2.7, 2.8, 2.9, 2.10, 3.1, 3.2, 3.3, 3.4, 4.1, 4.2, 4.3

Part Two:

5.2, 5.3, 5.6, 6.1.2, 6.1.4, 6.2.1, 6.2.2, 6.3.2, 6.3.4, 6.3.5, 6.3.6, 6.3.8, 6.3.10, 6.4.1, 6.4.2, 6.5.1.2, 6.5.1.3, 6.5.2.2, 7.1, 7.2, 7.3, 7.4, 7.5, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 8.9.1, 8.9.3, 8.9.4, 8.9.5, 8.9.6, 8.10.1, 8.10.2, 8.10.3, 8.10.4, 9.2, 9.3, 9.5, 9.6, 9.7, 9.8, 9.9, 9.10, 9.11, 9.12, 9.13, 9.14, 9.15, 9.16, 10.2, 10.5, 10.6, 10.7, 10.8, 10.10, 10.11, 11.1, 11.2, 11.3, 11.5, 11.6, 11.7, 12.1, 12.2, 12.3, 12.4, 12.5, 12.7, 12.8, 12.9, 12.10, 12.11, 12.12, 13.1, 13.2, 13.3, 13.6, 13.12, 13.13, 14.1, 14.2

Guest Authors:

Pnina Abir-Am, PhD Part Two: 6.1.1

Stephen J Williams, PhDPart Two: 6.2.6, 6.5.2.2, 10.4, 10.9, 13.4

Aviva Lev-Ari, PhD, RN:

Part One:

1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 2.2.1, 2.3

Part Two:

5.1, 5.4, 5.5, 5.7, 5.8, 5.9, 5.10, 5.11, 6.1.3, 6.2.3, 6.2.4, 6.2.5, 6.3.1, 6.3.3, 6.3.7, 6.3.9, 6.4.3, 6.5.1.1, 6.5.2.1, 6.5.2.2, 6.5.3.1, 6.5.4, 6.5.5, 6,5,6, 8.9.2, 8.10.2, 9.1, 9.4, 10.1, 10.3, 11.4, 12.6, 13.5, 13.7, 13.8, 13.9, 13.10, 13.11

Adam Sonnenberg, BSC, MSc(c)Part Two: 13.9

 

electronic Table of Contents

PART ONE:

Physician as Authors, Writers in Medicine and Educator in Public Health

 

Chapter 1: Physicians as Authors

Introduction

1.1  The Young Surgeon and The Retired Pathologist: On Science, Medicine and HealthCare Policy – Best writers Among the WRITERS

1.2 Atul Gawande: Physician and Writer

1.3 Editorial & Publication of Articles in e-Books by  Leaders in Pharmaceutical Business Intelligence:  Contributions of Larry H Bernstein, MD, FCAP

1.4 Abraham Verghese, MD, Physician and Notable Author

1.5 Eric Topol, M.D.

1.6 Gregory House, MD

1.7 Peter Mueller, MD  Professor of Radiology @MGH & HMS – 2015 Synergy’s Honorary Award Recipient

Summary

Chapter 2: Professional Recognition

Introduction

2.1 Proceedings

2.1.1 Research Presentations

2.1.2 Proceedings of the NYAS

2.1.3 Cold Spring Harbor Conference Meetings

2.1.4 Young Scientist Seminars

2.2 Meet Great Minds

2.2.1 Meet the Laureates

2.2.2 Richard Feynman, Genius and Laureate

2.2.3 Fractals and Heat Energy

2.3 MacArthur Foundation Awards

2.4 Women’s Contributions went beyond Rosie the Riveter

2.4.1 Secret Maoist Chinese Operation Conquered Malaria

2.4.2 Antiparasite Drug Developers Win Nobel

2.5 Impact Factors and Achievement

2.6   RAPsodisiac Medicine

2.6.1 Outstanding-achievements-in-radiology-or-radiotherapy

2.6.2 Outstanding-achievement-in-anesthesiology

2.6.3 Outstanding-achievement-in-pathology

2.6.4 Topics in Pathology – Special Issues from Medscape Pathology

2.7 How to win the Nobel Prize

2.8 Conversations about Medicine

2.9 Current Advances in Medical Technology

2.10 Atul Butte, MD, PhD

Summary

Chapter 3:  Medical and Allied Health Sciences Education

Introduction

3.1 National Outstanding Medical Student Award Winners

3.2 Outstanding Awards in Medical Education

3.3 Promoting Excellence in Physicians and Nurses

3.4 Excellence in mentoring

Summary

Chapter 4: Science Teaching in Math and Technology (STEM)

Introduction

4.1 Science Teaching in Math and Technology

4.2 Television as a Medium for Science Education

4.2.1 Science Discovery TV

4.3 From Turing to Watson

Summary

PART TWO:

Medical Scientific Discoveries Interviews with Scientific Leaders

Chapter 5: Cardiovascular System

Introduction

5.1 Physiologist, Professor Lichtstein, Chair in Heart Studies at The Hebrew University elected Dean of the Faculty of Medicine at The Hebrew University of Jerusalem

5.2 Mitochondrial Dysfunction and Cardiac Disorders

5.3 Notable Contributions to Regenerative Cardiology

5.4 For Accomplishments in Cardiology and Cardiovascular Diseases: The Arrigo Recordati International Prize for Scientific Research

5.5 Becoming a Cardiothoracic Surgeon: An Emerging Profile in the Surgery Theater and through Scientific Publications

5.6 Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions and Historical Scientific Leaders Memoirs

5.7 CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin

5.8 2013 as A Year of Revolutionizing Medicine and Top 11 Cardiology Stories

5.9 Bridging the Gap in Medical Innovations – Elazer Edelman @ TEDMED 2013

5.10 Development of a Pancreatobiliary Chemotherapy Eluting Stent for Pancreatic Ductal Adenocarcinoma PIs: Jeffrey Clark (MGH), Robert Langer (Koch), Elazer Edelman (Harvard:MIT HST Program)

5.11 Publications on Heart Failure by Prof. William Gregory Stevenson, M.D., BWH

Summary

Chapter 6: Genomics

Introduction
6.1 Genetics before the Human Genome Project

6.1.1 Why did Pauling Lose the “Race” to James Watson and Francis Crick? How Crick Describes his Discovery in a Letter to his Son

6.1.2 John Randall’s MRC Research Unit and Rosalind Franklin’s role at Kings College

6.1.3 Interview with the co-discoverer of the structure of DNA: Watson on The Double Helix and his changing view of Rosalind Franklin

6.1.4 The Initiation and Growth of Molecular Biology and Genomics, Part I

6.2 The Human Genome Project: Articles of Note  @ pharmaceuticalintelligence.com by multiple authors

6.2.1 CRACKING THE CODE OF HUMAN LIFE: The Birth of BioInformatics & Computational Genomics

6.2.2 What comes after finishing the Euchromatic Sequence of the Human Genome?

6.2.3 Human Genome Project – 10th Anniversary: Interview with Kevin Davies, PhD – The $1000 Genome

6.2.4 University of California Santa Cruz’s Genomics Institute will create a Map of Human Genetic Variations

6.2.5 Exceptional Genomes: The Process to find them

6.2.6 Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

6.3 The Impact of Genome Sequencing on Biology and Medicine

6.3.1 Genomics in Medicine – Establishing a Patient-Centric View of Genomic Data

6.3.2 Modification of genes by homologous recombination – Mario Capecchi, Martin Evans, Oliver Smithies

6.3.3 AAAS February 14-18, 2013, Boston: Symposia – The Science of Uncertainty in Genomic Medicine

6.3.4 The Metabolic View of Epigenetic Expression

6.3.5  Pharmacogenomics

6.3.6 Neonatal Pathophysiology

6.3.7 Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

6.3.8 3D mapping of genome in combine FISH and RNAi

6.3.9 Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence

6.3.10 DNA mutagenesis and DNA repair

6.4 Scientific Leadership Recognition for Contributions to Genomics

6.4.1 Interview with Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak (44 minutes)

6.4.2 DNA Repair Pioneers Win Nobel – Tomas Lindahl, Paul Modrich, and Aziz Sancar 2015 Nobel Prize in Chemistry for the mechanisms of DNA repair

6.4.3  Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

6.5 Contemporary Field Leaders in Genomics

6.5.1 ROBERT LANGER

6.5.1.1 2014 Breakthrough Prizes Awarded in Fundamental Physics and Life Sciences for a Total of $21 Million – MIT’s Robert Langer gets $3 Million

6.5.1.2 National Medal of Science – 2006 Robert S. Langer

6.5.1.3  Confluence of Chemistry, Physics, and Biology

6.5.2 JENNIFER DOUDNA

6.5.2.1 Jennifer Doudna, cosmology teams named 2015 Breakthrough Prize winners

6.5.2.2 UPDATED – Medical Interpretation of the Genomics Frontier – CRISPR – Cas9: Gene Editing Technology for New Therapeutics

6.5.3 ERIC LANDER

6.5.3.1  2012 Harvey Prize in April 30: at the Technion-Israel Institute of Technology to Eric S. Lander @MIT & Eli Yablonovitch @UC, Berkeley

6.5.4 2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

6.5.5 Recognitions for Contributions in Genomics by Dan David Prize Awards

6.5.6   65 Nobel Laureates meet 650 young scientists covering the fields of physiology and medicine, physics, and chemistry, 28 June – 3 July, 2015, Lindau & Mainau Island, Germany

Summary

Chapter 7: The RNAs

Introduction

7.1 RNA polymerase – molecular basis for DNA transcription – Roger Kornberg, MD

7.2  One gene, one protein – Charles Yanofsky

7.3 Turning genetic information into working proteins – James E. Darnell Jr.

7.4 Small but mighty RNAs – Victor Ambros, David Baulcombe, and Gary Ruvkun, Phillip A. Sharp

7.5 Stress-response gene networks – Nina V. Fedoroff

Summary

Chapter 8: Proteomics, Protein-folding, and Cell Regulation
Introduction.

8.1 The Life and Work of Allan Wilson

8.2 Proteomics

8.3 More Complexity in Protein Evolution

8.4 Proteins: An evolutionary record of diversity and adaptation

8.5 Heroes in Basic Medical Research – Leroy Hood

8.6 Ubiquitin researchers win Nobel – Ciechanover, Hershko, and Rose awarded for discovery of ubiquitin-mediated proteolysis

8.7 Buffering of genetic modules involved in tricarboxylic acid cycle metabolism provides homeostatic regulation

8.8 Dynamic Protein Profiling

8.9 Protein folding

8.9.1 Protein misfolding and prions – Susan L. Lindquist, Stanley B. Prusiner

8.9.2 A Curated Census of Autophagy-Modulating Proteins and Small Molecules Candidate Targets for Cancer Therapy

8.9.3 Voluntary and Involuntary S-Insufficiency

8.9.4 Transthyretin and Lean Body Mass in Stable and Stressed State

8.9.5 The matter of stunting in the Ganges Plains

8.9.6 Proteins, Imaging and Therapeutics

8.10 Protein Folding and Vesicle Cargo

8.10.1 Heat Shock Proteins (HSP) and Molecular Chaperones

8.10.2 Collagen-binding Molecular Chaperone HSP47: Role in Intestinal Fibrosis – colonic epithelial cells and sub epithelial myofibroblasts

8.10.3 Biology, Physiology and Pathophysiology of Heat Shock Proteins

8.10.4 The Role of Exosomes in Metabolic Regulation 


Summary

Chapter 9:  Neuroscience

Introduction

9.1 Nobel Prize in Physiology or Medicine 2013 for Cell Transport: James E. Rothman of Yale University; Randy W. Schekman of the University of California, Berkeley; and Dr. Thomas C. Südhof of Stanford University

9.2 Proteins that control neurotransmitter release – Richard H. Scheller

9.3 Heroes in Basic Medical Research – Robert J. Lefkowitz

9.4 MIND AND MEMORY: BIOLOGICAL AND DIGITAL – 2014 Dan David Prize Symposium

9.5 A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

9.6 Role the basal ganglia

9.7 The Neurogenetics of Language – Patricia Kuhl – 2015 George A. Miller Award

9.8 The structure of our visual system

9.9 Outstanding Achievement in Schizophrenia Research

9.10 George A. Miller, a Pioneer in Cognitive Psychology, Is Dead at 92

9.11 – To understand what happens in the brain to cause mental illness

9.12 Brain and Cognition

9.13 – To reduce symptoms of mental illness and retrain the brain

9.14 Behavior

9.15 Notable Papers in Neurosciences

9.16 Pyrroloquinoline quinone (PQQ) – an unproved supplement

Summary

Chapter 10: Microbiology & Immunology

Introduction

10.1 Reference Genes in the Human Gut Microbiome: The BGI Catalogue

10.2 Malnutrition in India, high newborn death rate and stunting of children age under five years

10.3 In His Own Words: Leonard Herzenberg, The Immunologist Who Revolutionized Research, Dies at 81

10.4 Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

10.5 Tang Prize for 2014: Immunity and Cancer

10.6 Halstedian model of cancer progression

10.7 The History of Hematology and Related Sciences

10.8 Pathology Emergence in the 21st Century

10.9 Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

10.10  T cell-mediated immune responses & signaling pathways activated by TLRs – Bruce A. Beutler, Jules A. Hoffmann, Ralph M. Steinman

10.11 Roeder – the coactivator OCA-B, the first cell-specific coactivator, discovered by Roeder in 1992, is unique to immune system B cells

Summary

Chapter 11: Endocrine Hormones

Introduction

11.1 Obesity – 2010 Douglas L. ColemanJeffrey M. Friedman

11.2 Lonely Receptors: RXR – Jensen, Chambon, and Evans – Nuclear receptors provoke RNA production in response to steroid hormones

11.3 The Fred Conrad Koch Lifetime Achievement Award—the Society’s highest honor—recognizes the lifetime achievements and exceptional contributions of an individual to the field of endocrinology

11.4 Gerald D Aurbach Award for Outstanding Translational Research

11.5 Roy O. Greep Award for Outstanding Research in Endocrinology – Martin M. Matzuk

11.6 American Physiology Society Awards

11.7 Solomon Berson and Rosalyn Yalow

Summary

Chapter 12. Stem Cells

Introduction

12.1 Mature cells can be reprogrammed to become pluripotent – John Gurdon and Shinya Yamanaka

12.2 Observing the spleen colonies in mice and proving the existence of stem cells – Till and McCulloch

12.3 McEwen Award for Innovation: Irving Weissman, M.D., Stanford School of Medicine, and Hans Clevers, M.D., Ph.D., Hubrecht Institute

12.4 Developmental biology

12.5  CRISPR/Cas-mediated genome engineering – Rudolf Jaenisch

12.6 Ribozymes and RNA Machines –  Work of Jennifer A. Doudna

12.7 Ralph Brinster, ‘Father of Transgenesis’

12.8 Targeted gene modification

12.9 Stem Cells and Cancer

12.10 ALPSP Awards

12.11 Eppendorf Award for Young European Investigators

12.12 Breaking news about genomic engineering, T2DM and cancer treatments

Summary
Chapter 13: 3D Printing and Medical Application

Introduction

13.1 3D Printing

13.2 What is 3D printing?

13.3 The Scientist Who Is Making 3D Printing More Human

13.4 Join These Medical 3D Printing Groups on Twitter and LinkedIn for great up to date news

13.5 Neri Oxman and her Mediated Matter group @MIT Media Lab have developed a technique for 3D-printing Molten Glass

13.6 The ‘chemputer’ that could print out any drug

13.7 3-D-Bioprinting in use to Create Cardiac Living Tissue: Print your Heart out

13.8 LPBI’s Perspective on Medical and Life Sciences Applications – 3D Printing: BioInks, BioMaterials-BioPolymer

13.9 Medical MEMS, Sensors and 3D Printing: Frontier in Process Control of BioMaterials

13.10 NIH and FDA on 3D Printing in Medical Applications: Views for On-demand Drug Printing, in-Situ direct Tissue Repair and Printed Organs for Live Implants

13.11 ‘Pop-up’ fabrication technique trumps 3D printing

13.12 Augmentation of the ONTOLOGY of the 3D Printing Research

13.13 Superresolution Microscopy

Summary

Chapter 14: Synthetic Medicinal Chemistry

Introduction

14.1 Insights in Biological and Synthetic Medicinal Chemistry

14.2 Breakthrough work in cancer

Summary to Part Two

Volume Summary and Conclusions

EPILOGUE

 

 

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Familial transthyretin amyloid polyneuropathy

Curator: Larry H. Bernstein, MD, FCAP

LPBI

 

First-Ever Evidence that Patisiran Reduces Pathogenic, Misfolded TTR Monomers and Oligomers in FAP Patients

We reported data from our ongoing Phase 2 open-label extension (OLE) study of patisiran, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis) patients with familial amyloidotic polyneuropathy (FAP). Alnylam scientists and collaborators from The Scripps Research Institute and Misfolding Diagnostics, Inc. were able to measure the effects of patisiran on pathogenic, misfolded TTR monomers and oligomers in FAP patients. Results showed a rapid and sustained reduction in serum non-native conformations of TTR (NNTTR) of approximately 90%. Since NNTTR is pathogenic in ATTR amyloidosis and the level of NNTTR reduction correlated with total TTR knockdown, these results provide direct mechanistic evidence supporting the therapeutic hypothesis that TTR knockdown has the potential to result in clinical benefit. Furthermore, complete 12-month data from all 27 patients that enrolled in the patisiran Phase 2 OLE study showed sustained mean maximum reductions in total serum TTR of 91% for over 18 months and a mean 3.1-point decrease in mNIS+7 at 12 months, which compares favorably to an estimated increase in mNIS+7 of 13 to 18 points at 12 months based upon analysis of historical data sets in untreated FAP patients with similar baseline characteristics. Importantly, patisiran administration continues to be generally well tolerated out to 21 months of treatment.

Read our press release

View the non-native TTR poster (480 KB PDF)

View the complete 12-month patisiran Phase 2 OLE data presentation (620 KB PDF)

We are encouraged by these new data that provide continued support for our hypothesis that patisiran has the potential to halt neuropathy progression in patients with FAP. If these results are replicated in a randomized, double-blind, placebo-controlled study, we believe that patisiran could emerge as an important treatment option for patients suffering from this debilitating, progressive and life-threatening disease.

 

Hereditary ATTR Amyloidosis with Polyneuropathy (hATTR-PN)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge

 

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with polyneuropathy (hATTR-PN), also known as familial amyloidotic polyneuropathy (FAP), is an inherited, progressive disease leading to death within 5 to 15 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils predominantly in peripheral nerves and other organs. hATTR-PN can cause sensory, motor, and autonomic dysfunction, resulting in significant disability and death.

It is estimated that hATTR-PN, also known as FAP, affects approximately 10,000 people worldwide.  Patients have a life expectancy of 5 to 15 years from symptom onset, and the only treatment options for early stage disease are liver transplantation and TTR stabilizers such as tafamidis (approved in Europe) and diflunisal.  Unfortunately liver transplantation has limitations, including limited organ availability as well as substantial morbidity and mortality. Furthermore, transplantation eliminates the production of mutant TTR but does not affect wild-type TTR, which can further deposit after transplantation, leading to cardiomyopathy and worsening of neuropathy. There is a significant need for novel therapeutics to treat patients who have inherited mutations in the TTR gene.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing patisiran (ALN-TTR02), an intravenously administered RNAi therapeutic, to treat the hATTR-PN form of the disease.

Patisiran for the Treatment hATTR-PN

APOLLO Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a significantly broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam will lead development and commercialization of patisiran in North America and Europe while Genzyme will develop and commercialize the product in the rest of world.

 

Hereditary ATTR Amyloidosis with Cardiomyopathy (hATTR-CM)

ATTR amyloidosis is a progressive, life-threatening disease caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in multiple organs, but primarily in the peripheral nerves and heart. ATTR amyloidosis can lead to significant morbidity, disability, and mortality. The TTR protein is produced primarily in the liver and is normally a carrier for retinol binding protein – one of the vehicles used to transport vitamin A around the body.  Mutations in the TTR gene cause misfolding of the protein and the formation of amyloid fibrils that typically contain both mutant and wild-type TTR that deposit in tissues such as the peripheral nerves and heart, resulting in intractable peripheral sensory neuropathy, autonomic neuropathy, and/or cardiomyopathy.

Click to Enlarge                            http://www.alnylam.com/web/assets/tetramer.jpg

ATTR represents a major unmet medical need with significant morbidity and mortality. There are over 100 reported TTR mutations; the particular TTR mutation and the site of amyloid deposition determine the clinical manifestations of the disease, whether it is predominantly symptoms of neuropathy or cardiomyopathy.

Specifically, hereditary ATTR amyloidosis with cardiomyopathy (hATTR-CM), also known as familial amyloidotic cardiomyopathy (FAC), is an inherited, progressive disease leading to death within 2 to 5 years. It is due to a mutation in the transthyretin (TTR) gene, which causes misfolded TTR proteins to accumulate as amyloid fibrils primarily in the heart. Hereditary ATTR amyloidosis with cardiomyopathy can result in heart failure and death.

While the exact numbers are not known, it is estimated hATTR-CM, also known as FAC affects at least 40,000 people worldwide.  hATTR-CM is fatal within 2 to 5 years of diagnosis and treatment is currently limited to supportive care.  Wild-type ATTR amyloidosis (wtATTR amyloidosis), also known as senile systemic amyloidosis, is a nonhereditary, progressive disease leading to death within 2 to 5 years. It is caused by misfolded transthyretin (TTR) proteins that accumulate as amyloid fibrils in the heart. Wild-type ATTR amyloidosis can cause cardiomyopathy and result in heart failure and death. There are no approved therapies for the treatment of hATTR-CM or SSA; hence there is a significant unmet need for novel therapeutics to treat these patients.

Our ATTR program is the lead effort in our Genetic Medicine Strategic Therapeutic Area (STAr) product development and commercialization strategy, which is focused on advancing innovative RNAi therapeutics toward genetically defined targets for the treatment of rare diseases with high unmet medical need.  We are developing revusiran (ALN-TTRsc), a subcutaneously administered RNAi therapeutic for the treatment of hATTR-CM.

Revusiran for the Treatment of hATTR-CM

ENDEAVOUR Phase 3 Trial

In 2012, Alnylam entered into an exclusive alliance with Genzyme, a Sanofi company, to develop and commercialize RNAi therapeutics, including patisiran and revusiran, for the treatment of ATTR amyloidosis in Japan and the broader Asian-Pacific region. In early 2014, this relationship was extended as a broader alliance to advance RNAi therapeutics as genetic medicines. Under this new agreement, Alnylam and Genzyme have agreed to co-develop and co-commercialize revusiran in North America and Europe, with Genzyme developing and commercializing the product in the rest of world. This broadened relationship on revusiran is aimed at expanding and accelerating the product’s global value.

Pre-Clinical Data and Advancement of ALN-TTRsc02 for Transthyretin-Mediated Amyloidosis

We presented pre-clinical data with ALN-TTRsc02, an investigational RNAi therapeutic targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis).  In pre-clinical studies, including those in non-human primates (NHPs), ALN-TTRsc02 achieved potent and highly durable knockdown of serum TTR of up to 99% with multi-month durability achieved after just a single dose, supportive of a potentially once quarterly dose regimen. Results from studies comparing TTR knockdown activity of ALN-TTRsc02 to that of revusiran showed that ALN-TTRsc02 has a markedly superior TTR knockdown profile.  Further, in initial rat toxicology studies, ALN-TTRsc02 was found to be generally well tolerated with no significant adverse events at doses as high as 100 mg/kg.

Read our press release

View the presentation

http://www.alnylam.com/product-pipeline/hereditary-attr-amyloidosis-with-cardiomyopathy/

 

Emerging Therapies for Transthyretin Cardiac Amyloidosis Could Herald a New Era for the Treatment of HFPEF

Oct 14, 2015   |  Adam Castano, MDDavid Narotsky, MDMathew S. Maurer, MD, FACC

http://www.acc.org/latest-in-cardiology/articles/2015/10/13/08/35/emerging-therapies-for-transthyretin-cardiac-amyloidosis#sthash.9xzc0rIe.dpuf

Heart failure with a preserved ejection fraction (HFPEF) is a clinical syndrome that has no pharmacologic therapies approved for this use to date. In light of failed medicines, cardiologists have refocused treatment strategies based on the theory that HFPEF is a heterogeneous clinical syndrome with different etiologies. Classification of HFPEF according to etiologic subtype may, therefore, identify cohorts with treatable pathophysiologic mechanisms and may ultimately pave the way forward for developing meaningful HFPEF therapies.1

A wealth of data now indicates that amyloid infiltration is an important mechanism underlying HFPEF. Inherited mutations in transthyretin cardiac amyloidosis (ATTRm) or the aging process in wild-type disease (ATTRwt) cause destabilization of the transthyretin (TTR) protein into monomers or oligomers, which aggregate into amyloid fibrils. These insoluble fibrils accumulate in the myocardium and result in diastolic dysfunction, restrictive cardiomyopathy, and eventual congestive heart failure (Figure 1). In an autopsy study of HFPEF patients, almost 20% without antemortem suspicion of amyloid had left ventricular (LV) TTR amyloid deposition.2 Even more resounding evidence for the contribution of TTR amyloid to HFPEF was a study in which 120 hospitalized HFPEF patients with LV wall thickness ≥12 mm underwent technetium-99m 3,3-diphosphono-1,2-propranodicarboxylic acid (99mTc-DPD) cardiac imaging,3,4 a bone isotope known to have high sensitivity and specificity for diagnosing TTR cardiac amyloidosis.5,6 Moderate-to-severe myocardial uptake indicative of TTR cardiac amyloid deposition was detected in 13.3% of HFPEF patients who did not have TTR gene mutations. Therefore, TTR cardiac amyloid deposition, especially in older adults, is not rare, can be easily identified, and may contribute to the underlying pathophysiology of HFPEF.

Figure 1

As no U.S. Food and Drug Administration-approved drugs are currently available for the treatment of HFPEF or TTR cardiac amyloidosis, the development of medications that attenuate or prevent TTR-mediated organ toxicity has emerged as an important therapeutic goal. Over the past decade, a host of therapies and therapeutic drug classes have emerged in clinical trials (Table 1), and these may herald a new direction for treating HFPEF secondary to TTR amyloid.

Table 1

TTR Silencers (siRNA and Antisense Oligonucleotides)

siRNA

Ribonucleic acid interference (RNAi) has surfaced as an endogenous cellular mechanism for controlling gene expression. Small interfering RNAs (siRNAs) delivered into cells can disrupt the production of target proteins.7,8 A formulation of lipid nanoparticle and triantennary N-acetylgalactosamine (GalNAc) conjugate that delivers siRNAs to hepatocytes is currently in clinical trials.9 Prior research demonstrated these GalNAc-siRNA conjugates result in robust and durable knockdown of a variety of hepatocyte targets across multiple species and appear to be well suited for suppression of TTR gene expression and subsequent TTR protein production.

The TTR siRNA conjugated to GalNAc, ALN-TTRSc, is now under active investigation as a subcutaneous injection in phase 3 clinical trials in patients with TTR cardiac amyloidosis.10 Prior phase 2 results demonstrated that ALN-TTRSc was generally well tolerated in patients with significant TTR disease burden and that it reduced both wild-type and mutant TTR gene expression by a mean of 87%. Harnessing RNAi technology appears to hold great promise for treating patients with TTR cardiac amyloidosis. The ability of ALN-TTRSc to lower both wild-type and mutant proteins may provide a major advantage over liver transplantation, which affects the production of only mutant protein and is further limited by donor shortage, cost, and need for immunosuppression.

Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are under clinical investigation for their ability to inhibit hepatic expression of amyloidogenic TTR protein. Currently, the ASO compound, ISIS-TTRRx, is under investigation in a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial in patients with familial amyloid polyneuropathy (FAP).11 The primary objective is to evaluate its efficacy as measured by change in neuropathy from baseline relative to placebo. Secondary measures will evaluate quality of life (QOL), modified body mass index (mBMI) by albumin, and pharmacodynamic effects on retinol binding protein. Exploratory objectives in a subset of patients with LV wall thickness ≥13 mm without a history of persistent hypertension will examine echocardiographic parameters, N-terminal pro–B-type natriuretic peptide (NT-proBNP), and polyneuropathy disability score relative to placebo. These data will facilitate analysis of the effect of antisense oligonucleotide-mediated TTR suppression on the TTR cardiac phenotype with a phase 3 trial anticipated to begin enrollment in 2016.

TTR Stabilizers (Diflunisal, Tafamidis)

Diflunisal

Several TTR-stabilizing agents are in various stages of clinical trials. Diflunisal, a traditionally used and generically available nonsteroidal anti-inflammatory drug (NSAID), binds and stabilizes familial TTR variants against acid-mediated fibril formation in vitro and is now in human clinical trials.12,13 The use of diflunisal in patients with TTR cardiac amyloidosis is controversial given complication of chronic inhibition of cyclooxygenase (COX) enzymes, including gastrointestinal bleeding, renal dysfunction, fluid retention, and hypertension that may precipitate or exacerbate heart failure in vulnerable individuals.14-17 In TTR cardiac amyloidosis, an open-label cohort study suggested that low-dose diflunisal with careful monitoring along with a prophylactic proton pump inhibitor could be safely administered to compensated patients.18 An association was observed, however, between chronic diflunisal use and adverse changes in renal function suggesting that advanced kidney disease may be prohibitive in diflunisal therapy.In FAP patients with peripheral or autonomic neuropathy randomized to diflunisal or placebo, diflunisal slowed progression of neurologic impairment and preserved QOL over two years of follow-up.19 Echocardiography demonstrated cardiac involvement in approximately 50% of patients.20 Longer-term safety and efficacy data over an average 38 ± 31 months in 40 Japanese patients with hereditary ATTR amyloidosis who were not candidates for liver transplantation showed that diflunisal was mostly well tolerated.12 The authors cautioned the need for attentive monitoring of renal function and blood cell counts. Larger multicenter collaborations are needed to determine diflunisal’s true efficacy in HFPEF patients with TTR cardiac amyloidosis.

Tafamidis

Tafamidis is under active investigation as a novel compound that binds to the thyroxine-binding sites of the TTR tetramer, inhibiting its dissociation into monomers and blocking the rate-limiting step in the TTR amyloidogenesis cascade.21 The TTR compound was shown in an 18-month double-blind, placebo-controlled trial to slow progression of neurologic symptoms in patients with early-stage ATTRm due to the V30M mutation.22 When focusing on cardiomyopathy in a phase 2, open-label trial, tafamidis also appeared to effectively stabilize TTR tetramers in non-V30M variants, wild-type and V122I, as well as biochemical and echocardiographic parameters.23,24 Preliminary data suggests that clinically stabilized patients had shorter disease duration, lower cardiac biomarkers, less myocardial thickening, and higher EF than those who were not stabilized, suggesting early institution of therapy may be beneficial. A phase 3 trial has completed enrollment and will evaluate the efficacy, safety, and tolerability of tafamidis 20 or 80 mg orally vs. placebo.25 This will contribute to long-term safety and efficacy data needed to determine the therapeutic effects of tafamidis among ATTRm variants.

Amyloid Degraders (Doxycycline/TUDCA and Anti-SAP Antibodies)

Doxycycline/TUDCA

While silencer and stabilizer drugs are aimed at lowering amyloidogenic precursor protein production, they cannot remove already deposited fibrils in an infiltrated heart. Removal of already deposited fibrils by amyloid degraders would be an important therapeutic strategy, particularly in older adults with heavily infiltrated hearts reflected by thick walls, HFPEF, systolic heart failure, and restrictive cardiomyopathy. Combined doxycycline and tauroursodeoxycholic acid (TUDCA) disrupt TTR amyloid fibrils and appeared to have an acceptable safety profile in a small phase 2 open-label study among 20 TTR patients. No serious adverse reactions or clinical progression of cardiac or neuropathic involvement was observed over one year.26 An active phase 2, single-center, open-label, 12-month study will assess primary outcome measures including mBMI, neurologic impairment score, and NT-proBNP.27 Another phase 2 study is examining the tolerability and efficacy of doxycycline/TUDCA over an 18-month period in patients with TTR amyloid cardiomyopathy.28 Additionally, a study in patients with TTR amyloidosis is ongoing to determine the effect of doxycycline alone on neurologic function, cardiac biomarkers, echocardiographic parameters, modified body mass index, and autonomic neuropathy.29

Anti-SAP Antibodies

In order to safely clear established amyloid deposits, the role of the normal, nonfibrillar plasma glycoprotein present in all human amyloid deposits, serum amyloid P component (SAP), needs to be more clearly understood.30 In mice with amyloid AA type deposits, administration of antihuman SAP antibody triggered a potent giant cell reaction that removed massive visceral amyloid deposits without adverse effects.31 In humans with TTR cardiac amyloidosis, anti-SAP antibody treatments could be feasible because the bis-D proline compound, CPHPC, is capable of clearing circulating human SAP, which allow anti-SAP antibodies to reach residual deposited SAP. In a small, open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis, none of whom had clinical evidence of cardiac amyloidosis, were treated with CPHPC followed by human monoclonal IgG1 anti-SAP antibody.32 No serious adverse events were reported and amyloid deposits were cleared from the liver, kidney, and lymph node. Anti-SAP antibodies hold promise as a potential amyloid therapy because of their potential to target all forms of amyloid deposits across multiple tissue types.

Mutant or wild-type TTR cardiac amyloidoses are increasingly recognized as a cause of HFPEF. Clinicians need to be aware of this important HFPEF etiology because the diverse array of emerging disease-modifying agents for TTR cardiac amyloidosis in human clinical trials has the potential to herald a new era for the treatment of HFPEF.

References

  1. Maurer MS, Mancini D. HFpEF: is splitting into distinct phenotypes by comorbidities the pathway forward? J Am Coll Cardiol 2014;64:550-2.
  2. Mohammed SF, Mirzoyev SA, Edwards WD, et al. Left ventricular amyloid deposition in patients with heart failure and preserved ejection fraction. JACC Heart Fail 2014;2:113-22.
  3. González-López E, Gallego-Delgado M, Guzzo-Merello G, et al. Wild-type transthyretin amyloidosis as a cause of heart failure with preserved ejection fraction. Eur Heart J 2015.
  4. Castano A, Bokhari S, Maurer MS. Unveiling wild-type transthyretin cardiac amyloidosis as a significant and potentially modifiable cause of heart failure with preserved ejection fraction. Eur Heart J 2015 Jul 28. [Epub ahead of print]
  5. Rapezzi C, Merlini G, Quarta CC, et al. Systemic cardiac amyloidoses: disease profiles and clinical courses of the 3 main types. Circulation 2009;120:1203-12.
  6. Bokhari S, Castano A, Pozniakoff T, Deslisle S, Latif F, Maurer MS. (99m)Tc-pyrophosphate scintigraphy for differentiating light-chain cardiac amyloidosis from the transthyretin-related familial and senile cardiac amyloidoses. Circ Cardiovasc Imaging 2013;6:195-201.
  7. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-11.
  8. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-8.
  9. Kanasty R, Dorkin JR, Vegas A, Anderson D. Delivery materials for siRNA therapeutics. Nature Mater 2013;12:967-77.
  10. U.S. National Institutes of Health. Phase 2 Study to Evaluate ALN-TTRSC in Patients With Transthyretin (TTR) Cardiac Amyloidosis (ClinicalTrials.gov website). 2014. Available at: https://www.clinicaltrials.gov/ct2/show/NCT01981837. Accessed 8/19/2015.
  11. U.S. National Institutes of Health. Efficacy and Safety of ISIS-TTRRx in Familial Amyloid Polyneuropathy (Clinical Trials.gov Website. 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01737398. Accessed 8/19/2015.
  12. Sekijima Y, Dendle MA, Kelly JW. Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis. Amyloid 2006;13:236-49.
  13. Tojo K, Sekijima Y, Kelly JW, Ikeda S. Diflunisal stabilizes familial amyloid polyneuropathy-associated transthyretin variant tetramers in serum against dissociation required for amyloidogenesis. Neurosci Res 2006;56:441-9.
  14. Epstein M. Non-steroidal anti-inflammatory drugs and the continuum of renal dysfunction. J Hypertens Suppl 2002;20:S17-23.
  15. Wallace JL. Pathogenesis of NSAID-induced gastroduodenal mucosal injury. Best Pract Res Clin Gastroenterol 2001;15:691-703.
  16. Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 2001;286:954-9.
  17. Page J, Henry D. Consumption of NSAIDs and the development of congestive heart failure in elderly patients: an underrecognized public health problem. Arch Intern Med 2000;160:777-84.
  18. Castano A, Helmke S, Alvarez J, Delisle S, Maurer MS. Diflunisal for ATTR cardiac amyloidosis. Congest Heart Fail 2012;18:315-9.
  19. Berk JL, Suhr OB, Obici L, et al. Repurposing diflunisal for familial amyloid polyneuropathy: a randomized clinical trial. JAMA 2013;310:2658-67.
  20. Quarta CCF, Solomon RH Suhr SD, et al. The prevalence of cardiac amyloidosis in familial amyloidotic polyneuropathy with predominant neuropathy: The Diflunisal Trial. International Symposium on Amyloidosis 2014:88-9.
  21. Hammarstrom P, Jiang X, Hurshman AR, Powers ET, Kelly JW. Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity. Proc Natl Acad Sci U S A 2002;99 Suppl 4:16427-32.
  22. Coelho T, Maia LF, Martins da Silva A, et al. Tafamidis for transthyretin familial amyloid polyneuropathy: a randomized, controlled trial. Neurology 2012;79:785-92.
  23. Merlini G, Plante-Bordeneuve V, Judge DP, et al. Effects of tafamidis on transthyretin stabilization and clinical outcomes in patients with non-Val30Met transthyretin amyloidosis. J Cardiovasc Transl Res 2013;6:1011-20.
  24. Maurer MS, Grogan DR, Judge DP, et al. Tafamidis in transthyretin amyloid cardiomyopathy: effects on transthyretin stabilization and clinical outcomes. Circ Heart Fail 2015;8:519-26.
  25. U.S. National Institutes of Health. Safety and Efficacy of Tafamidis in Patients With Transthyretin Cardiomyopathy (ATTR-ACT) (ClinicalTrials.gov website). 2014. Available at: http://www.clinicaltrials.gov/show/NCT01994889. Accessed 8/19/2015.
  26. Obici L, Cortese A, Lozza A, et al. Doxycycline plus tauroursodeoxycholic acid for transthyretin amyloidosis: a phase II study. Amyloid 2012;19 Suppl 1:34-6.
  27. U.S. National Institutes of Health. Safety, Efficacy and Pharmacokinetics of Doxycycline Plus Tauroursodeoxycholic Acid in Transthyretin Amyloidosis (ClinicalTrials.gov website). 2011. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01171859. Accessed 8/19/2015.
  28. U.S. National Institutes of Health. Tolerability and Efficacy of a Combination of Doxycycline and TUDCA in Patients With Transthyretin Amyloid Cardiomyopathy (ClinicalTrials.gov website). 2013. Available at: http://www.clinicaltrials.gov/ct2/show/NCT01855360. Accessed 8/19/2015.
  29. U.S. National Institutes of Health. Safety and Effect of Doxycycline in Patients With Amyloidosis (ClinicalTrials.gov website).2015. Available at: https://clinicaltrials.gov/ct2/show/NCT01677286. Accessed 8/19/2015.
  30. Pepys MB, Dash AC. Isolation of amyloid P component (protein AP) from normal serum as a calcium-dependent binding protein. Lancet 1977;1:1029-31.
  31. Bodin K, Ellmerich S, Kahan MC, et al. Antibodies to human serum amyloid P component eliminate visceral amyloid deposits. Nature 2010;468:93-7.
  32. Richards DB, Cookson LM, Berges AC, et al. Therapeutic Clearance of Amyloid by Antibodies to Serum Amyloid P Component. N Engl J Med 2015;373:1106-14.

 

The Acid-Mediated Denaturation Pathway of Transthyretin Yields a Conformational Intermediate That Can Self-Assemble into Amyloid

Zhihong Lai , Wilfredo Colón , and Jeffery W. Kelly *
Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255
Biochemistry199635 (20), pp 6470–6482   http://dx.doi.org:/10.1021/bi952501g
Publication Date (Web): May 21, 1996  Copyright © 1996 American Chemical Society

Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0−3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand−loop−D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS−PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly β-sheet structure having a well-defined tertiary structure.

 

Prevention of Transthyretin Amyloid Disease by Changing Protein Misfolding Energetics

Per Hammarström*, R. Luke Wiseman*, Evan T. Powers, Jeffery W. Kelly   + Author Affiliations

Science  31 Jan 2003; 299(5607):713-716   http://dx.doi.org:/10.1126/science.1079589

Genetic evidence suggests that inhibition of amyloid fibril formation by small molecules should be effective against amyloid diseases. Known amyloid inhibitors appear to function by shifting the aggregation equilibrium away from the amyloid state. Here, we describe a series of transthyretin amyloidosis inhibitors that functioned by increasing the kinetic barrier associated with misfolding, preventing amyloidogenesis by stabilizing the native state. The trans-suppressor mutation, threonine 119 → methionine 119, which is known to ameliorate familial amyloid disease, also functioned through kinetic stabilization, implying that this small-molecule strategy should be effective in treating amyloid diseases.

 

Rational design of potent human transthyretin amyloid disease inhibitors

Thomas Klabunde1,2, H. Michael Petrassi3, Vibha B. Oza3, Prakash Raman3, Jeffery W. Kelly3 & James C. Sacchettini1

Nature Structural & Molecular Biology 2000; 7: 312 – 321.                http://dx.doi.org:/10.1038/74082

The human amyloid disorders, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and senile systemic amyloidosis, are caused by insoluble transthyretin (TTR) fibrils, which deposit in the peripheral nerves and heart tissue. Several nonsteroidal anti-inflammatory drugs and structurally similar compounds have been found to strongly inhibit the formation of TTR amyloid fibrils in vitro. These include flufenamic acid, diclofenac, flurbiprofen, and resveratrol. Crystal structures of the protein–drug complexes have been determined to allow detailed analyses of the protein–drug interactions that stabilize the native tetrameric conformation of TTR and inhibit the formation of amyloidogenic TTR. Using a structure-based drug design approach ortho-trifluormethylphenyl anthranilic acid and N-(meta-trifluoromethylphenyl) phenoxazine 4,6-dicarboxylic acid have been discovered to be very potent and specific TTR fibril formation inhibitors. This research provides a rationale for a chemotherapeutic approach for the treatment of TTR-associated amyloid diseases.

 

First European consensus for diagnosis, management, and treatment of transthyretin familial amyloid polyneuropathy

Adams, Davida; Suhr, Ole B.b; Hund, Ernstc; Obici, Laurad; Tournev, Ivailoe,f; Campistol, Josep M.g; Slama, Michel S.h; Hazenberg, Bouke P.i; Coelho, Teresaj; from the European Network for TTR-FAP (ATTReuNET)

Current Opin Neurol: Feb 2016; 29 – Issue – p S14–S26      http://dx.doi.org:/10.1097/WCO.0000000000000289

Purpose of review: Early and accurate diagnosis of transthyretin familial amyloid polyneuropathy (TTR-FAP) represents one of the major challenges faced by physicians when caring for patients with idiopathic progressive neuropathy. There is little consensus in diagnostic and management approaches across Europe.

Recent findings: The low prevalence of TTR-FAP across Europe and the high variation in both genotype and phenotypic expression of the disease means that recognizing symptoms can be difficult outside of a specialized diagnostic environment. The resulting delay in diagnosis and the possibility of misdiagnosis can misguide clinical decision-making and negatively impact subsequent treatment approaches and outcomes.

Summary: This review summarizes the findings from two meetings of the European Network for TTR-FAP (ATTReuNET). This is an emerging group comprising representatives from 10 European countries with expertise in the diagnosis and management of TTR-FAP, including nine National Reference Centres. The current review presents management strategies and a consensus on the gold standard for diagnosis of TTR-FAP as well as a structured approach to ongoing multidisciplinary care for the patient. Greater communication, not just between members of an individual patient’s treatment team, but also between regional and national centres of expertise, is the key to the effective management of TTR-FAP.

http://images.journals.lww.com/co-neurology/Original.00019052-201602001-00003.FF1.jpeg

Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a highly debilitating and irreversible neurological disorder presenting symptoms of progressive sensorimotor and autonomic neuropathy [1▪,2▪,3]. TTR-FAP is caused by misfolding of the transthyretin (TTR) protein leading to protein aggregation and the formation of amyloid fibrils and, ultimately, to amyloidosis (commonly in the peripheral and autonomic nervous system and the heart) [4,5]. TTR-FAP usually proves fatal within 7–12 years from the onset of symptoms, most often due to cardiac dysfunction, infection, or cachexia [6,7▪▪].

The prevalence and disease presentation of TTR-FAP vary widely within Europe. In endemic regions (northern Portugal, Sweden, Cyprus, and Majorca), patients tend to present with a distinct genotype in large concentrations, predominantly a Val30Met substitution in the TTR gene [8–10]. In other areas of Europe, the genetic footprint of TTR-FAP is more varied, with less typical phenotypic expression [6,11]. For these sporadic or scattered cases, a lack of awareness among physicians of variable clinical features and limited access to diagnostic tools (i.e., pathological studies and genetic screening) can contribute to high rates of misdiagnosis and poorer patient outcomes [1▪,11]. In general, early and late-onset variants of TTR-FAP, found within endemic and nonendemic regions, present several additional diagnostic challenges [11,12,13▪,14].

Delay in the time to diagnosis is a major obstacle to the optimal management of TTR-FAP. With the exception of those with a clearly diagnosed familial history of FAP, patients still invariably wait several years between the emergence of first clinical signs and accurate diagnosis [6,11,14]. The timely initiation of appropriate treatment is particularly pertinent, given the rapidity and irreversibility with which TTR-FAP can progress if left unchecked, as well as the limited effectiveness of available treatments during the later stages of the disease [14]. This review aims to consolidate the existing literature and present an update of the best practices in the management of TTR-FAP in Europe. A summary of the methods used to achieve a TTR-FAP diagnosis is presented, as well as a review of available treatments and recommendations for treatment according to disease status.

Patients with TTR-FAP can present with a range of symptoms [11], and care should be taken to acquire a thorough clinical history of the patient as well as a family history of genetic disease. Delay in diagnosis is most pronounced in areas where TTR-FAP is not endemic or when there is no positive family history [1▪]. TTR-FAP and TTR-familial amyloid cardiomyopathy (TTR-FAC) are the two prototypic clinical disease manifestations of a broader disease spectrum caused by an underlying hereditary ATTR amyloidosis [19]. In TTR-FAP, the disease manifestation of neuropathy is most prominent and definitive for diagnosis, whereas cardiomyopathy often suggests TTR-FAC. However, this distinction is often superficial because cardiomyopathy, autonomic neuropathy, vitreous opacities, kidney disease, and meningeal involvement all may be present with varying severity for each patient with TTR-FAP.

Among early onset TTR-FAP with usually positive family history, symptoms of polyneuropathy present early in the disease process and usually predominate throughout the progression of the disease, making neurological testing an important diagnostic aid [14]. Careful clinical examination (e.g., electromyography with nerve conduction studies and sympathetic skin response, quantitative sensation test, quantitative autonomic test) can be used to detect, characterize, and scale the severity of neuropathic abnormalities involving small and large nerve fibres [10]. Although a patient cannot be diagnosed definitively with TTR-FAP on the basis of clinical presentation alone, symptoms suggesting the early signs of peripheral neuropathy, autonomic dysfunction, and cardiac conduction disorders or infiltrative cardiomyopathy are all indicators that further TTR-FAP diagnostic investigation is warranted. Late-onset TTR-FAP often presents as sporadic cases with distinct clinical features (e.g., milder autonomic dysfunction) and can be more difficult to diagnose than early-onset TTR-FAP (Table 2) [1▪,11,12,13▪,14,20].

http://images.journals.lww.com/co-neurology/LargeThumb.00019052-201602001-00003.TT2.jpeg

Genetic testing is carried out to allow detection of specific amyloidogenic TTR mutations (Table 1), using varied techniques depending on the expertise and facilities available in each country (Table S2, http://links.lww.com/CONR/A39). A targeted approach to detect a specific mutation can be used for cases belonging to families with previous diagnosis. In index cases of either endemic and nonendemic regions that do not have a family history of disease, are difficult to confirm, and have atypical symptoms, TTR gene sequencing is required for the detection of both predicted and new amyloidogenic mutations [26,27].

Following diagnosis, the neuropathy stage and systemic extension of the disease should be determined in order to guide the next course of treatment (Table 4) [3,30,31]. The three stages of TTR-FAP severity are graded according to a patient’s walking disability and degree of assistance required [30]. Systemic assessment, especially of the heart, eyes, and kidney, is also essential to ensure all aspects of potential impact of the disease can be detected [10].

Table 4

http://images.journals.lww.com/co-neurology/LargeThumb.00019052-201602001-00003.TT4.jpeg

Image Tools

The goals of cardiac investigations are to detect serious conduction disorders with the risk of sudden death and infiltrative cardiomyopathy. Electrocardiograms (ECG), Holter-ECG, and intracardiac electrophysiology study are helpful to detect conduction disorders. Echocardiograms, cardiac magnetic resonance imaging, scintigraphy with bone tracers, and biomarkers (e.g., brain natriuretic peptide, troponin) can all help to diagnose infiltrative cardiomyopathy[10]. An early detection of cardiac abnormalities has obvious benefits to the patient, given that the prophylactic implantation of pacemakers was found to prevent 25% of major cardiac events in TTR-FAP patients followed up over an average of 4 years [32▪▪]. Assessment of cardiac denervation with 123-iodine meta-iodobenzylguanidine is a powerful prognostic marker in patients diagnosed with FAP [33].

…..

Tafamidis

Tafamidis is a first-in-class therapy that slows the progression of TTR amyloidogenesis by stabilizing the mutant TTR tetramer, thereby preventing its dissociation into monomers and amyloidogenic and toxic intermediates [55,56]. Tafamidis is currently indicated in Europe for the treatment of TTR amyloidosis in adult patients with stage I symptomatic polyneuropathy to delay peripheral neurological impairment [57].

In an 18-month, double-blind, placebo-controlled study of patients with early-onset Val30Met TTR-FAP, tafamidis was associated with a 52% lower reduction in neurological deterioration (P = 0.027), a preservation of nerve function, and TTR stabilization versus placebo [58▪▪]. However, only numerical differences were found for the coprimary endpoints of neuropathy impairment [neuropathy impairment score in the lower limb (NIS-LL) responder rates of 45.3% tafamidis vs 29.5% placebo; P = 0.068] and quality of life scores [58▪▪]. A 12-month, open-label extension study showed that the reduced rates of neurological deterioration associated with tafamidis were sustained over 30 months, with earlier initiation of tafamidis linking to better patient outcomes (P = 0.0435) [59▪]. The disease-slowing effects of tafamidis may be dependent on the early initiation of treatment. In an open-label study with Val30Met TTR-FAP patients with late-onset and advanced disease (NIS-LL score >10, mean age 56.4 years), NIS-LL and disability scores showed disease progression despite 12 months of treatment with tafamidis, marked by a worsening of neuropathy stage in 20% and the onset of orthostatic hypotension in 22% of patients at follow-up [60▪].

Tafamidis is not only effective in patients exhibiting the Val30Met mutation; it also has proven efficacy, in terms of TTR stabilization, in non-Val30Met patients over 12 months [61]. Although tafamidis has demonstrated safe use in patients with TTR-FAP, care should be exercised when prescribing to those with existing digestive problems (e.g., diarrhoea, faecal incontinence) [60▪].

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Diflunisal

Diflunisal is a nonsteroidal anti-inflammatory drug (NSAID) that, similar to tafamidis, slows the rate of amyloidogenesis by preventing the dissociation, misfolding, and misassembly of the mutated TTR tetramer [62,63]. Off-label use has been reported for patients with stage I and II disease, although diflunisal is not currently licensed for the treatment of TTR-FAP.

Evidence for the clinical effectiveness of diflunisal in TTR-FAP derives from a placebo-controlled, double-blind, 24-month study in 130 patients with clinically detectable peripheral or autonomic neuropathy[64▪]. The deterioration in NIS scores was significantly more pronounced in patients receiving placebo compared with those taking diflunisal (P = 0.001), and physical quality of life measures showed significant improvement among diflunisal-treated patients (P = 0.001). Notable during this study was the high rate of attrition in the placebo group, with 50% more placebo-treated patients dropping out of this 2-year study as a result of disease progression, advanced stage of the disease, and varied mutations.

One retrospective analysis of off-label use of diflunisal in patients with TTR-FAP reported treatment discontinuation in 57% of patients because of adverse events that were largely gastrointestinal [65]. Conclusions on the safety of diflunisal in TTR-FAP will depend on further investigations on the impact of known cardiovascular and renal side-effects associated with the NSAID drug class [66,67].

 

 

 

 

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Signaling through the T Cell Receptor (TCR) Complex and the Co-stimulatory Receptor CD28

Curator: Larry H. Bernstein, MD, FCAP

 

 

New connections: T cell actin dynamics

Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems.

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Triple-Color FRET Analysis Reveals Conformational Changes in the WIP-WASp Actin-Regulating Complex

 

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T cell activation by antigens involves the formation of a complex, highly dynamic, yet organized signaling complex at the site of the T cell receptors (TCRs). Srikanth et al. found that the lymphocyte-specific large guanosine triphosphatase of the Rab family CRACR2A-a associated with vesicles near the Golgi in unstimulated mouse and human CD4+ T cells. Upon TCR activation, these vesicles moved to the immunological synapse (the contact region between a T cell and an antigen-presenting cell). The guanine nucleotide exchange factor Vav1 at the TCR complex recruited CRACR2A-a to the complex. Without CRACR2A-a, T cell activation was compromised because of defective calcium and kinase signaling.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed that CRACR2A (Ca2+ release–activated Ca2+ channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca2+ and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

 

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Novel Discoveries in Molecular Biology and Biomedical Science

Curator: Larry H. Bernstein, MD, FCAP

LPBI

Updated June 1, 2016

The following is a collection of current articles on noncoding DNA, synthetic genome engineering, protein regulation of apoptosis, drug design, and geometrics.

 

No longer ‘junk DNA’ — shedding light on the ‘dark matter’ of the genome

A new tool called “LIGR-Seq” enables scientists to explore in depth what non-coding RNAs actually do in human cells   May 23, 2016

http://www.kurzweilai.net/no-longer-junk-dna-shedding-light-on-the-dark-matter-of-the-genome

http://www.kurzweilai.net/images/LIGR-seq-method.png

he LIGR-seq method for global-scale mapping of RNA-RNA interactions in vivo to reveal unexpected functions for uncharacterized RNAs that act via base-pairing interactions (credit: University of Toronto)

What used to be dismissed by many as “junk DNA” has now become vitally important, as accelerating genomic data points to the importance of non-coding RNAs (ncRNAs) — a genome’s messages that do not specifically code for proteins — in development and disease.

But our progress in understanding these molecules has been slow because of the lack of technologies that allow for systematic mapping of their functions.

Now, professor Benjamin Blencowe’s team at the University of Toronto’s Donnelly Centre has developed a method called “LIGR-seq” that enables scientists to explore in depth what ncRNAs do in human cells.

The study, described in Molecular Cell, was published on May 19, along with two other papers, in Molecular Cell and Cell, respectively, from Yue Wan’s group at the Genome Institute of Singapore and Howard Chang’s group at Stanford University in California, who developed similar methods to study RNAs in different organisms.

So what exactly do ncRNAs do?

http://www.kurzweilai.net/images/ncRNA.png

mRNAs vs. ncRNAs (credit: Thomas Shafee/CC)

Of the 3 billion letters in the human genome, only two per cent make up the protein-coding genes. The genes are copied, or transcribed, into messenger RNA (mRNA) molecules, which provide templates for building proteins that do most of the work in the cell. Much of the remaining 98 per cent of the genome was initially considered by some as lacking in functional importance. However, large swaths of the non-coding genome — between half and three quarters of it — are also copied into RNA.

So then what might the resulting ncRNAs do? That depends on whom you ask. Some researchers believe that most ncRNAs have no function, that they are just a by-product of the genome’s powerful transcription machinery that makes mRNA. However, it is emerging that many ncRNAs do have important roles in gene regulation — some ncRNAs act as carriages for shuttling the mRNAs around the cell, or provide a scaffold for other proteins and RNAs to attach to and do their jobs.

But the majority of available data has trickled in piecemeal or through serendipitous discovery. And with emerging evidence that ncRNAs could drive disease progression, such as cancer metastasis, there was a great need for a technology that would allow a systematic functional analysis of ncRNAs.

Up until now, with existing methods, you had to know what you are looking for because they all require you to have some information about the RNA of interest. The power of our method is that you don’t need to preselect your candidates; you can see what’s occurring globally in cells, and use that information to look at interesting things we have not seen before and how they are affecting biology,” says Eesha Sharma, a PhD candidate in Blencowe’s group who, along with postdoctoral fellow Tim Sterne-Weiler, co-developed the method.

A new ncRNA identification tool

http://www.kurzweilai.net/images/rna-rna-interactions.jpg

The human RNA-RNA interactome, showing interactions detected by LIGR-seq (credit: University of Toronto)

The new ‘‘LIGation of interacting RNA and high-throughput sequencing’’ (LIGR-seq) tool captures interactions between different RNA molecules. When two RNA molecules have matching sequences — strings of letters copied from the DNA blueprint — they will stick together like Velcro. With LIGR-seq, the paired RNA structures are removed from cells and analyzed by state-of-the-art sequencing methods to precisely identify the RNAs that are stuck together.

Most researchers in the life sciences agree that there’s an urgent need to understand what ncRNAs do. This technology will open the door to developing a new understanding of ncRNA function,” says Blencowe, who is also a professor in the Department of Molecular Genetics.

Not having to rely on pre-existing knowledge will boost the discovery of RNA pairs that have never been seen before. Scientists can also, for the first time, look at RNA interactions as they occur in living cells, in all their complexity, unlike in the juices of mashed up cells that they had to rely on before. This is a bit like moving on to explore marine biology from collecting shells on the beach to scuba-diving among the coral reefs, where the scope for discovery is so much bigger.

Actually, ncRNAs come in multiple flavors: there’s rRNA, tRNA, snRNA, snoRNA, piRNA, miRNA, and lncRNA, to name a few, where prefixes reflect the RNA’s place in the cell or some aspect of its function. But the truth is that no one really knows the extent to which these ncRNAs control what goes on in the cell, or how they do this.

Discoveries

Nonetheless, the new technology developed by Blencowe’s group has been able to pick up new interactions involving all classes of RNAs and has already revealed some unexpected findings.

The team discovered new roles for small nucleolar RNAs (snoRNAs), which normally guide chemical modifications of other ncRNAs. It turns out that some snoRNAs can also regulate stability of a set of protein-coding mRNAs. In this way, snoRNAs can also directly influence which proteins are made, as well as their abundance, adding a new level of control in cell biology.

And this is only the tip of the iceberg; the researchers plan to further develop and apply their technology to investigate the ncRNAs in different settings.

“We would like to understand how ncRNAs function during development. We are particularly interested in their role in the formation of neurons. But we will also use our method to discover and map changes in RNA-RNA interactions in the context of human diseases,” says Blencowe.

Abstract of Global Mapping of Human RNA-RNA Interactions

The majority of the human genome is transcribed into non-coding (nc)RNAs that lack known biological functions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked in vivo, “LIGation of interacting RNA followed by high-throughput sequencing” (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions.

references:

 

Venter’s Research Team Creates an Artificial Cell and Reports That 32% of Genes Are Life-Essential but Contain Unknown Functions
http://www.radmailer.com/t/r-l-sttullk-ykogyktt-k/
May 27, 2016

Understanding the unknown functions of these genes may lead to the creation of new diagnostic tests for clinical laboratories and anatomic pathology groups

Once again, J. Craig Venter, PhD, is charting new ground in gene sequencing andgenomic science. This time his research team has built upon the first synthetic cell they created in 2010 to build a more sophisticated synthetic cell. Their findings from this work may give pathologists and medical laboratory scientists new tools to diagnose disease.

Recently the research team at the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) published their latest findings. Among the things they learned is that science still does not understand the functions of about a third of the genes required for their synthetic cells to function.

JCVI-syn3.0 Could Radically Alter Understanding of Human Genome

Based in La Jolla, Calif., and Rockville, Md., JCVI is a not-for-profit research institute aiming to advance genomics. Building upon its first synthetic cell—Mycoplasma mycoides (M. mycoides) JCVI-syn1.0, which JCVI constructed in 2010—the same team of scientists created the first minimal synthetic bacterial cell, which they calledJCVI-syn3.0. This new artificial cell contains 531,560 base pairs and just 473 genes, which means it is the smallest genome of any organism that can be grown in laboratory media, according to a JCVI-SGI statement.

For pathologists and medical laboratory leaders, the creation of a synthetic life form is a milestone toward better understanding genome sequencing and how this new knowledge may help advance both diagnostics and therapeutics.

“What we’ve done is important because it is a step toward completely understanding how a living cell works,” Clyde Hutchison III, PhD, told New Scientist. “If we can really understand how the cell works, then we will be able to design cells efficiently for the production of pharmaceutical and other useful products.” Hutchison is Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill, Distinguished Professor at the J. Craig Venter Institute, a member of the National Academy of Sciences, and a fellow of the American Academy of Arts and Sciences.

Click here to see images

Clyde Hutchison, III, PhD (above), Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill and Distinguished Professor at the J. Craig Venter Institute, stated that his team’s “goal is to have a cell for which the precise biological function of every gene is known.” (Photo credit: JCVI.)

Understanding a Gene’s True Purpose

According to the JCVI researchers, 149 genes have no known purpose. They are, however, necessary for life and health.

“We know about two-thirds of the essential biology, and we’re missing a third,” stated J. Craig Venter, PhD, Founder and CEO of JCVI, in a story published by MedPage Today.

This knowledge is based upon decades of research. JCVI seeks to create a minimal cell operating system to understand biology, while also providing what the JCVI statement called a “chassis for use in industrial applications.”

What Do these Genes Do Anyway?

The JCVI team found that among most genes’ biological functions:

“JCVI-syn3.0 is a working approximation of a minimal cellular genome—a compromise between a small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life,” the researchers told the journal Science.

More research is needed, the scientists say, into the 149 genes that appear to lack specific biologic functions.

Unlocking Mystery of the 149 Genes Could Lead to Advances in Genomic Science

“Finding so many genes without a known function is unsettling, but it’s exciting because it’s left us with much still to learn. It’s like the ‘dark matter’ of biology,” said Alistair Elfick, PhD, Chair of Synthetic Biological Engineering, University of Edinburgh, UK, in the New Scientist article.

Studies such as JCVI’s research is key to broadening understanding and framing appropriate questions about scientific, ethical, and economic implications of synthetic biology.

The creation of a synthetic cell will have a profound and positive impact on understanding of biology and how life works, JCVI said.

Such research may inspire new whole genome synthesis tools and semi-automated processes that could dramatically affect clinical laboratory procedures. It also could lead to new techniques and tools for advanced vaccine and pharmaceuticals, JCVI pointed out.

—Donna Marie Pocius

Related Information:

First Minimal Synthetic Bacterial Cell Designed and Constructed by Scientists at Venter Institute and Synthetic Genomics, Inc.

 

CRISPR Versatility Inspires Molecular Biology Innovation

GEN Tech Focus: CRISPR/Gene Editing
No single technique has set the molecular biology field ablaze with excitement and potential like the CRISPR-Cas9 genome editing system has following its introduction only a few short years ago. The following articles represent the flexibility of this technique to potentially treat a host of genetic disorders and possibly even prevent the onset of disease.

 

CRISPR Moves from Butchery to Surgery

Scientists recently convened at the CRISPR Precision Gene Editing Congress, held in Boston, to discuss the new technology. As with any new technique, scientists have discovered that CRISPR comes with its own set of challenges, and the Congress focused its discussion around improving specificity, efficiency, and delivery.

 

New CRISPR System Targets Both DNA and RNA

With a staggering number of papers published in the past several years involving the characterization and use of the CRISPR/Cas9 gene editing system, it is surprising that researchers are still finding new features of the versatile molecular scissor enzyme.

 

High-Fidelity CRISPR-Cas9 Nucleases Virtually Free of Off-Target Noise

If a Cas9 nuclease variant could be engineered that was less grabby, it might loosen its grip on DNA sequences throughout the genome—except those sequences representing on-target sites. That’s the assumption that guided a new investigation by researchers at Massachusetts General Hospital.

 

CRISPR Works Well but Needs Upgrades

The gene-editing technology known as CRISPR-Cas9 is starting to raise expectations in the therapeutic realm. In fact, CRISPR-Cas9 and other CRISPR systems are moving so close to therapeutic uses that the technology’s ethical implications are starting to attract notice.

 

A Guide to CRISPR Gene Activation
http://www.technologynetworks.com/rnai/news.aspx?ID=191776

Published: Tuesday, May 24, 2016
A comparison of synthetic gene-activating Cas9 proteins can help guide research and development of therapeutic approaches.

The CRISPR-Cas9 system has come to be known as the quintessential tool that allows researchers to edit the DNA sequences of many organisms and cell types. However, scientists are also increasingly recognizing that it can be used to activate the expression of genes. To that end, they have built a number of synthetic gene activating Cas9 proteins to study gene functions or to compensate for insufficient gene expression in potential therapeutic approaches.

“The possibility to selectively activate genes using various engineered variants of the CRISPR-Cas9 system left many researchers questioning which of the available synthetic activating Cas9 proteins to use for their purposes. The main challenge was that all had been uniquely designed and tested in different settings; there was no side-by-side comparison of their relative potentials,” said George Church, Ph.D., who is Core Faculty Member at the Wyss Institute for Biologically Inspired Engineering at Harvard University, leader of its Synthetic Biology Platform, and Professor of Genetics at Harvard Medical School. “We wanted to provide that side-by-side comparison to the biomedical research community.”

In a study published on 23 May in Nature Methods, the Wyss Institute team reports how it rigorously compared and ranked the most commonly used artificial Cas9 activators in different cell types from organisms including humans, mice and flies. The findings provide a valuable guide to researchers, allowing them to streamline their endeavors.

The team also included Wyss Core Faculty Member James Collins, Ph.D., who also is the Termeer Professor of Medical Engineering & Science and Professor of Biological Engineering at the Massachusetts Institute of Technology (MIT)’s Department of Biological Engineering and Norbert Perrimon, Ph.D., a Professor of Genetics at Harvard Medical School.

Gene activating Cas9 proteins are fused to variable domains borrowed from proteins with well-known gene activation potentials and engineered so that the DNA editing ability is destroyed. In some cases, the second component of the CRISPR-Cas9 system, the guide RNA that targets the complex to specific DNA sequences, also has been engineered to bind gene-activating factors.

“We first surveyed seven advanced Cas9 activators, comparing them to each other and the original Cas9 activator that served to provide proof-of-concept for the gene activation potential of CRISPR-Cas9. Three of them, provided much higher gene activation than the other candidates while maintaining high specificities toward their target genes,” said Marcelle Tuttle, Research Fellow at the Wyss and a co-lead author of the study.

The team went on to show that the three top candidates were comparable in driving the highest level of gene expression in cells from humans, mice and fruit flies, irrespective of their tissue and developmental origins. The researchers also pinpointed ways to further maximize gene activation employing the three leading candidates.

“In some cases, maximum possible activation of a target gene is necessary to achieve a cellular or therapeutic effect. We managed to cooperatively enhance expression of specific genes when we targeted them with three copies of a top performing activator using three different guide RNAs,” said Alejandro Chavez, Ph.D., a Postdoctoral Fellow and the study’s co-first author.

“The ease of use of CRISPR-Cas9 offers enormous potential for development of genome therapeutics. This study provides valuable new design criteria that will help enable synthetic biologists and bioengineers to develop more effective targeted genome engineering technologies in the future,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital, and also Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

 

Engineering T Cells to Functionally Cure HIV-1 Infection

Rachel S Leibman and James L Riley
Molecular Therapy (21 April 2015) |    http://dx.doi.org:/10.1038/mt.2015.70

Despite the ability of antiretroviral therapy to minimize human immunodeficiency virus type 1 (HIV-1) replication and increase the duration and quality of patients’ lives, the health consequences and financial burden associated with the lifelong treatment regimen render a permanent cure highly attractive. Although T cells play an important role in controlling virus replication, they are themselves targets of HIV-mediated destruction. Direct genetic manipulation of T cells for adoptive cellular therapies could facilitate a functional cure by generating HIV-1–resistant cells, redirecting HIV-1–specific immune responses, or a combination of the two strategies. In contrast to a vaccine approach, which relies on the production and priming of HIV-1–specific lymphocytes within a patient’s own body, adoptive T-cell therapy provides an opportunity to customize the therapeutic T cells prior to administration. However, at present, it is unclear how to best engineer T cells so that sustained control over HIV-1 replication can be achieved in the absence of antiretrovirals. This review focuses on T-cell gene-engineering and gene-editing strategies that have been performed in efforts to inhibit HIV-1 replication and highlights the requirements for a successful gene therapy–mediated functional cure.

 

Automated top-down design technique simplifies creation of DNA origami nanostructures

http://www.kurzweilai.net/automated-top-down-design-technique-simplifies-creation-of-dna-origami-nanostructures

Nanoparticles for drug delivery and cell targeting, nanoscale robots, custom-tailored optical devices, and DNA as a storage medium are among the possible applications

May 27, 2016

The boldfaced line, known as a spanning tree, follows the desired geometric shape of the target DNA origami design method, touching each vertex just once. A spanning tree algorithm is used to map out the proper routing path for the DNA strand. (credit: Public Domain)

MITBaylor College of Medicine, and Arizona State University Biodesign Institute researchers have developed a radical new top-down DNA origami* design method based on a computer algorithm that allows for creating designs for DNA nanostructures by simply inputting a target shape.

DNA origami (using DNA to design and build geometric structures) has already proven wildly successful in creating myriad forms in 2- and 3- dimensions, which conveniently self-assemble when the designed DNA sequences are mixed together. The tricky part is preparing the proper DNA sequence and routing design for scaffolding and staple strands to achieve the desired target structure. Typically, this is painstaking work that must be carried out manually.

The new algorithm, which is reported together with a novel synthesis approach in the journal Science, promises to eliminate all that and expands the range of possible applications of DNA origami in biomolecular science and nanotechnology. Think nanoparticles for drug delivery and cell targeting, nanoscale robots in medicine and industry, custom-tailored optical devices, and most interesting: DNA as a storage medium, offering retention times in the millions of years.**

 

Shape-shifting, top-down software

Unlike traditional DNA origami, in which the structure is built up manually by hand, the team’s radical top-down autonomous design method begins with an outline of the desired form and works backward in stages to define the required DNA sequence that will properly fold to form the finished product.

“The Science paper turns the problem around from one in which an expert designs the DNA needed to synthesize the object, to one in which the object itself is the starting point, with the DNA sequences that are needed automatically defined by the algorithm,” said Mark Bathe, an associate professor of biological engineering at MIT, who led the research. “Our hope is that this automation significantly broadens participation of others in the use of this powerful molecular design paradigm.”

The algorithm, which is known as DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined Structures) after the Greek craftsman and artist who designed labyrinths that resemble origami’s complex scaffold structures, can build any type of 3-D shape, provided it has a closed surface. This can include shapes with one or more holes, such as a torus.

A simplified version of the  top-down procedure used to design scaffolded DNA origami nanostructures. It starts with a polygon corresponding to the target shape. Software translates a wireframe version of this structure into a plan for routing DNA scaffold and staple strands. That enables a 3D DNA-based atomic-level structural model that is then validated using 3D cryo-EM reconstruction. (credit: adapted from Biodesign Institute images)

With the new technique, the target geometric structure is first described in terms of a wire mesh made up of polyhedra, with a network of nodes and edges. A DNA scaffold using strands of custom length and sequence is generated, using a “spanning tree” algorithm — basically a map that will automatically guide the routing of the DNA scaffold strand through the entire origami structure, touching each vertex in the geometric form once. Complementary staple strands are then assigned and the final DNA structural model or nanoparticle self-assembles, and is then validated using 3D cryo-EM reconstruction.

The software allows for fabricating a variety of geometric DNA objects, including 35 polyhedral forms (Platonic, Archimedean, Johnson and Catalan solids), six asymmetric structures, and four polyhedra with nonspherical topology, using inverse design principles — no manual base-pair designs needed.

To test the method, simpler forms known as Platonic solids were first fabricated, followed by increasingly complex structures. These included objects with nonspherical topologies and unusual internal details, which had never been experimentally realized before. Further experiments confirmed that the DNA structures produced were potentially suitable for biological applications since they displayed long-term stability in serum and low-salt conditions.

Biological research uses

The research also paves the way for designing nanoscale systems mimicking the properties of viruses, photosynthetic organisms, and other sophisticated products of natural evolution. One such application is a scaffold for viral peptides and proteins for use as vaccines. The surface of the nanoparticles could be designed with any combination of peptides and proteins, located at any desired location on the structure, in order to mimic the way in which a virus appears to the body’s immune system.

The researchers demonstrated that the DNA nanoparticles are stable for more than six hours in serum, and are now attempting to increase their stability further.

The nanoparticles could also be used to encapsulate the CRISPR-Cas9 gene editing tool. The CRISPR-Cas9 tool has enormous potential in therapeutics, thanks to its ability to edit targeted genes. However, there is a significant need to develop techniques to package the tool and deliver it to specific cells within the body, Bathe says.

This is currently done using viruses, but these are limited in the size of package they can carry, restricting their use. The DNA nanoparticles, in contrast, are capable of carrying much larger gene packages and can easily be equipped with molecules that help target the right cells or tissue.

The most exciting aspect of the work, however, is that it should significantly broaden participation in the application of this technology, Bathe says, much like 3-D printing has done for complex 3-D geometric models at the macroscopic scale.

Hao Yan directs the Biodesign Center for Molecular Design and Biomimetics at Arizona State University and is the Milton D. Glick Distinguished Professor, College of Liberal Arts and Sciences, School of Molecular Sciences at ASU.

DNA origami brings the ancient Japanese method of paper folding down to the molecular scale. The basics are simple: Take a length of single-stranded DNA and guide it into a desired shape, fastening the structure together using shorter “staple strands,” which bind in strategic places along the longer length of DNA. The method relies on the fact that DNA’s four nucleotide letters—A, T, C, & G stick together in a consistent manner — As always pairing with Ts and Cs with Gs.

The DNA molecule in its characteristic double stranded form is fairly stiff, compared with single-stranded DNA, which is flexible. For this reason, single stranded DNA makes for an ideal lace-like scaffold material. Further, its pairing properties are predictable and consistent (unlike RNA).

https://vimeo.com/22349631

** A single gram of DNA can store about 700 terabytes of information — an amount equivalent to 14,000 50-gigabyte Blu-ray disks — and could potentially be operated with a fraction of the energy required for other information storage options.

 

Essential role of miRNAs in orchestrating the biology of the tumor microenvironment

Jamie N. Frediani and Muller Fabbri
Molecular Cancer (2016) 15:42   http://dx.doi.org:/10.1186/s12943-016-0525-3

MicroRNAs (miRNAs) are emerging as central players in shaping the biology of the Tumor Microenvironment (TME). They do so both by modulating their expression levels within the different cells of the TME and by being shuttled among different cell populations within exosomes and other extracellular vesicles. This review focuses on the state-of-the-art knowledge of the role of miRNAs in the complexity of the TME and highlights limitations and challenges in the field. A better understanding of the mechanisms of action of these fascinating micro molecules will lead to the development of new therapeutic weapons and most importantly, to an improvement in the clinical outcome of cancer patients. Keywords: Exosomes, microRNAs, Tumor microenvironment, Cancer

While cancer treatment and survival have improved worldwide, the need for further understanding of the underlying tumor biology remains. In recent years, there has been a significant shift in scientific focus towards the role of the tumor microenvironment (TME) on the development, growth, and metastatic spread of malignancies. The TME is defined as the surrounding cellular environment enmeshed around the tumor cells including endothelial cells, lymphocytes, macrophages, NK cells, other cells of the immune system, fibroblasts, mesenchymal stem cells (MSCs), and the extracellular matrix (ECM). Each of these components interacts with and influences the tumor cells, continually shifting the balance between pro- and anti-tumor phenotype. One of the predominant methods of communication between these cells is through extracellular vesicles and their microRNA (miRNA) cargo. Extracellular vesicles (EVs) are between 30 nm to a few microns in diameter, are surrounded by a phospholipid bilayer membrane, and are released from a variety of cell types into the local environment. There are three well characterized groups of EVs: 1) exosomes, typically 30–100 nm, 2) microvesicles (or ectosomes), typically 100–1000 nm, and 3) large oncosomes, typically 1–10 μm. Each of these categories has a distinctly unique biogenesis and purpose in cellcell communication despite the fact that current laboratory methods do not always allow precise differentiation. EVs are found to be enriched with membrane-bound proteins, lipid raft-associated and cytosolic proteins, lipids, DNA, mRNAs, and miRNAs, all of which can be transferred to the recipient cell upon fusion to allow cell-cell communications [1]. Of these, miRNAs have been of particular interest in cancer research, both as modifiers of transcription and translation as well as direct inhibitors or enhancers of key regulatory proteins. These miRNAs are a large family of small non-coding RNAs (19–24 nucleotides) and are known to be aberrantly expressed, both in terms of content as well as number, in both the tumor cells and the cells of the TME. Synthesis of these mature miRNA is a complex process, starting with the transcription of long, capped, and polyadenylated pri-miRNA by RNA polymerase II. These are cropped into a 60–100 nucleotide hairpinstructure pre-miRNA by the microprocessor, a heterodimer of Drosha (a ribonuclease III enzyme) and DGCR8 (DiGeorge syndrome critical region gene 8). The premiRNA is then exported to the cytoplasm by exportin 5, cleaved by Dicer, and separated into single strands by helicases. The now mature miRNA are incorporated into the RNA-induced silencing complex (RISC), a cytoplasmic effector machine of the miRNA pathway. The primary mechanism of action of the mature miRNA-RISC complex is through their binding to the 3’ untranslated region, or less commonly the 5’ untranslated region, of target mRNA, leading to protein downregulation either via translational repression or mRNA degradation. More recently, it has been shown that miRNAs can also upregulate the expression of target genes [2]. MiRNA genes are mostly intergenic and are transcribed by independent promoters [3] but can also be encoded by introns, sharing the same promoter of their host gene [4]. MiRNAs undergo the same regulatory mechanisms of any other protein coding gene (promoter methylation, histone modifications, etc.…) [5, 6]. Interestingly, each miRNA may have contradictory effects both within varying tumor cell lines and within different cells of the TME. In this review, we provide a state-of-the-art description of the key role that miRNAs have in the communication between tumor cells and the TME and their subsequent effects on the malignant phenotype. Finally, this review has made every effort to clarify, whenever possible, whether the reference is to the −3p or the -5p miRNA. Whenever such clarification has not been provided, this indicates that it was not possible to infer such information from the cited bibliography.

Angiogenesis and miRNAs Cellular plasticity, critical in the development of malignancy, includes the many diverse mechanisms elicited by cancer cells to increase their malignant potential and develop increasing treatment resistance. One such mechanism, angiogenesis, is critical to the development of metastatic disease, affecting both the growth of malignant cells locally and their survival at distant sites. In the last ten years, miRNAs, often packaged in tumor cell-derived exosomes, have emerged as important contributors to the complicated regulation and balance of pro- and anti-angiogenic factors.

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7].

Fig. 1   https://static-content.springer.com/image/art%3A10.1186%2Fs12943-016-0525-3/MediaObjects/12943_2016_525_Fig1_HTML.gif

Central role of the miR-17-92 cluster in the biology of the TME. The miR-17-92 cluster encoding miR-17, −18a, −19b, −20a, and -92a is upregulated in multiple tumor types and interacts with various components of the TME to finely “tune” the TME through a complex combination of pro- and anti-tumoral effects

Most commonly, miRNAs derived from cancer cells have oncogenic activity, promoting angiogenesis and tumor growth and survival. The most-well characterized of the pro-angiogenic miRNAs, the miR-17-92 cluster encoding six miRNAs (miR-17, −18a, −19a, −19b, −20a, and −92a), is found on chromosome 13, and is highly conserved among vertebrates [7]. The complex and multifaceted functions of the miR-17-92 cluster are summarized in Fig. 1. Amplification, both at the genetic and RNA level, of miR-17-92 was initially found in several lymphoma cell lines and has subsequently been observed in multiple mouse tumor models [7]. Up-regulation of this particular locus has further been confirmed in miRnome analysis across multiple different tumor types, including lung, breast, stomach, prostate, colon, and pancreatic cancer [8]. The miR-17-92 cluster is directly activated by Myc and modulates a variety of downstream transcription factors important in cell cycle regulation and apoptosis including activation of E2F family and Cyclin-dependent kinase inhibitor (CDKN1A) and downregulation of BCL2L11/BIM and p21 [7]. In addition to promoting cell cycle progression and inhibiting apoptosis, the miR-17-92 cluster also downregulates thrombospondin-1 (Tsp1) and connective tissue growth factor (CTGF), important antiangiogenic proteins [7]. Similarly, microvesicles from colorectal cancer cells contain miR-1246 and TGF-β which are transferred to endothelial cells to silence promyelocytic leukemia protein (PML) and activate Smad 1/5/8 signaling promoting proliferation and migration [9]. Likewise, lung cancer cell line derived microvesicles contain miR-494, in response to hypoxia, which targets PTEN in the endothelial cells promoting angiogenesis through the Akt/eNOS pathway [10]. Lastly, exosomal miR-135b from multiple myeloma cells suppresses the HIF-1/FIH-1 pathway in endothelial cells, increasing angiogenesis [11]. A summary of the studies showing the functions of exosomal miRNAs in shaping the biology of the TME is provided in Table 1.

 

Table 1

Actions of exosomal miRNAs exchanged between cells of the TME

 

Angiogenesis:

 miRNA

Cell of origin

Accepting cell

Pathway/target

Effect on TME

Ref.

 miR-135b

Multiple myeloma

Endothelial cells

HIF-1/FIH-1

↑angiogenesis

[11]

 miR-494

Lung cancer

Endothelial cells

PTEN/AKT/eNOS

↑angiogenesis

[10]

 miR-503

Endothelial cells

Breast cancer

Cyclin D2 and D3

↓Tumor growth and invasion

[22]

 miR-1246

Colorectal cancer

Endothelial Cells

PML/Smad 1/5/8

↑ Growth & migration

[9]

Stromal compartment:

 miR-105

Breast cancer

Endothelial cells

ZO-1

↓Tight junctions

↑Metastatic progression

[68]

 miR-202-3p

CLL

Stromal cells

c-fos/ATM

↑Tumor growth

[53]

Immune system:

 miR-29a

NSCLC

TAM

TLR8/NF-κB

↑Growth & metastasis

[75]

 miR-21

NSCLC

TAM

TLR8/NF-κB

↑Growth & metastasis

[75]

NBL

TAM

TLR8/NF-κB

↑miR-155

[76]

 miR-155

TAM

NBL

TERF1

↑ Drug resistance

[76]

 miR-23a

Hypoxic tumor derived

NK cells

CD107a

↓ NK cell response

[95]

 miR-210

 miR-214

Tumor cells (various)

Regulatory T cells

PTEN

↑Immunosuppression

[96]

 miR-223

TAM

Breast cancer

Mef2c/β-catenin

↑ Invasion

[82]

Abbreviations: TAMs Tumor Associated Macrophages, CLL chronic lymphocytic leukemia, NSCLCnon-small cell lung cancer, NBL Neuroblastoma

The most common target of anti-angiogenic therapy is VEGF, and not unsurprisingly, multiple miRNAs (including miR-9, miR-20b, miR-130, miR-150, and miR-497) promote angiogenesis through the induction of the VEGF pathway. The most studied of these is the up-regulation of miR-9 which has been linked to a poor prognosis in multiple tumor types, including breast cancer, non-small cell lung cancer, and melanoma [12]. The two oncogenes MYC and MYCN activate miR-9 and cause E-cadherin downregulation resulting in the upregulated transcription of VEGF [13]. In addition, miR-9 has been shown to upregulate the JAK-STAT pathway, supporting endothelial cell migration and tumor angiogenesis [13]. Both amplification of miR-20b and miR-130 as well as miR-497 suppression regulate VEGF through hypoxia inducible factor 1α (HIF-1α) supporting increased angiogenesis [14, 15, 16, 17]. …..

The pivotal discovery in 2012 by Mitra et al. laid the ground-work for our current knowledge on the interactions between tumor-derived miRNAs and fibroblasts. In combination, the down-regulation of miR-214 and miR-31 and the up-regulation of miR-155 trigger the reprogramming of quiescent fibroblasts to CAFs [32]. As expected, the reverse regulation of these miRNAs reduced the migration and invasion of co-cultured ovarian cancer cells [32]. While the pathway of miR-155’s involvement in CAF biology is still being elucidated, the pathways of miR-214 and miR-31 have been established. In endometrial cancer, miR-31 was found to target the homeobox gene SATB2, leading to enhanced tumor cell migration and invasion [33]. MiR-214 similarly has an inverse correlation with its chemokine target, C-C motif Ligand 5 (CCL5) [32]. CCL5 secretion has been associated with enhanced motility, invasion, and metastatic potential through NF-κB-mediated MMP9 activation and through generation and differentiation of myeloid-derived suppressor cells (MDSCs) [34, 35, 36]. Furthermore, miR-210 and miR-133b overexpression and miR-149 suppression have been subsequently found to independently trigger the conversion to CAFs, possibly through paracrine stimulation, and to additionally promote EMT in prostate and gastric cancer, respectively [37, 38,39]. MiR-210 additionally enlists monocytes and encourages angiogenesis [37].   …

Another function of CAFs is the destruction of the ECM and its remodeling with a tumor-supportive composition and structure which includes modulation of specific integrins and metalloproteinases as some of the most studied miRNA targets. The 23 matrix metalloproteinases (MMPs) are critical in the ECM degradation, disruption of the growth signal balance, resistance to apoptosis, establishment of a favorable metastatic niche, and promotion of angiogenesis [54]. As expected, miRNAs have been found to regulate the actions of MMPs, together working to promote cancer cell growth, invasiveness, and metastasis. In HCC, MMP2 and 9 expression is up-regulated by miR-21 via PTEN pathway downregulation. Similarly, in cholangiocarcinoma it was observed that reduced levels of miR-138 induced up-regulation of RhoC, leading to increased levels of the same two MMPs [55, 56]. ….

As has been shown throughout this review, miRNAs have an important and varied effect on human carcinogenesis by shaping the biology of the TME towards a more permissive pro-tumoral phenotype. The complex events leading to such an outcome are currently quite universally defined as the “educational” process of cancer cells on the surrounding TME. While the initial focus was on the direction from the cancer cell to the surrounding TME, increasingly interest is centered on the implications of a more dynamic bidirectional exchange of genetic information. MiRNAs represent only part of the cargo of the extracellular vesicles, but an increasing scientific literature points towards their pivotal role in creating the micro-environmental conditions for cancer cell growth and dissemination. The nearby future will have to address several questions still unanswered. First, it is absolutely necessary to clarify which miRNAs and to what extent they are involved in this process. The contradictory results of some studies can be explained by the differences in tumor-types and by different concentrations of miRNAs used for functional studies. Understanding whether different concentrations of the same miRNA elicit different target effects and therefore changes the biology of the TME, will represent a significant consideration in the development of this field. It is certainly very attractive (especially in an attempt to develop new and desperately needed better cancer biomarkers) to think that concentrations of miRNAs within the TME are reflected systemically in the circulating levels of that same miRNA, however this has not yet been irrefutably demonstrated. Moreover, the study of the paracrine interactions among different cell populations of the TME and their reciprocal effects has been limited to two, maximum three cell populations. This is still way too far from describing the complexity of the TME and only the development of new tridimensional models of the TME will be able to cast a more conclusive light on such complexity. Finally, the pharmacokinetics of miRNA-containing vesicles is in its infancy at best, and needs to be further developed if the goal is development of new therapies based on the use of exosomic miRNAs. Therefore, the future of miRNA research, particularly in its role in the TME, holds still a lot of questions that need answering. However, for these exact same reasons, this is an incredibly exciting time for research in this field. We can envision a not too far future in which these concerns will be satisfactorily addressed and our understanding of the role of miRNAs within the TME will allow us to use them as new therapeutic weapons to successfully improve the clinical outcome of cancer patients.

 

 

 

Triggering the protein that programs cancer cells to kill themselves
http://www.kurzweilai.net/triggering-the-protein-that-programs-cancer-cells-to-kill-themselves

May 24, 2016

https://youtu.be/DR80Huxp4y8
WEHI | Apoptosis

Researchers at the Walter and Eliza Hall Institute in Australia have discovered a new way to trigger cell death that could lead to drugs to treat cancer and autoimmune disease.

Programmed cell death (a.k.a. apoptosis) is a natural process that removes unwanted cells from the body. Failure of apoptosis can allow cancer cells to grow unchecked or immune cells to inappropriately attack the body.

The protein known as Bak is central to apoptosis. In healthy cells, Bak sits in an inert state but when a cell receives a signal to die, Bak transforms into a killer protein that destroys the cell.

Triggering the cancer-apoptosis trigger

Institute researchers Sweta Iyer, PhD, Ruth Kluck, PhD, and colleagues unexpectedly discovered that an antibody they had produced to study Bak actually bound to the Bak protein and triggered its activation. They hope to use this discovery to develop drugs that promote cell death.

The researchers used information about Bak’s three-dimensional structure to find out precisely how the antibody activated Bak. “It is well known that Bak can be activated by a class of proteins called ‘BH3-only proteins’ that bind to a groove on Bak. We were surprised to find that despite our antibody binding to a completely different site on Bak, it could still trigger activation,” Kluck said.  “The advantage of our antibody is that it can’t be ‘mopped up’ and neutralized by pro-survival proteins in the cell, potentially reducing the chance of drug resistance occurring.”

Drugs that target this new activation site could be useful in combination with other therapies that promote cell death by mimicking the BH3-only proteins. The researchers are now working with collaborators to develop their antibody into a drug that can access Bak inside cells.

Their findings have just been published in the open-access journal Nature Communications. The research was supported by the National Health and Medical Research Council, the Australian Research Council, the Victorian State Government Operational Infrastructure Support Scheme, and the Victorian Life Science Computation Initiative.

Abstract of Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2–α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1–α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1–α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1–α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

references:

 

Catching metastatic cancer cells before they grow into tumors: a new implant shows promise

https://62e528761d0685343e1c-f3d1b99a743ffa4142d9d7f1978d9686.ssl.cf2.rackcdn.com/files/122764/width926/image-20160516-15899-18cgw3m.jpg

Cure” is a word that’s dominated the rhetoric in the war on cancer for decades. But it’s a word that medical professionals tend to avoid. While the American Cancer Society reports that cancer treatment has improved markedly over the decades and the five-year survival rate is impressively high for many cancers, oncologists still refrain from declaring their cancer-free patients cured. Why?

Patients are declared cancer-free (also called complete remission) when there are no more signs of detectable disease.

However, minuscule clusters of cancer cells below the detection level can remain in a patient’s body after treatment. Moreover, such small clusters of straggler cells may undergo metastasis, where they escape from the initial tumor into the bloodstream and ultimately settle in a distant site, often a vital organ such as the lungs, liver or brain.

Cancer cells can move throughout the body, like these metastatic melanoma cells. NIH Image Gallery/FlickrCC BY

When a colony of these metastatic cells reaches a detectable size, the patient is diagnosed with recurrent metastatic cancer. About one in three breast cancer patients diagnosed with early-stage cancer later develop metastatic disease, usually within five years of initial remission.

By the time metastatic cancer becomes evident, it is much more difficult to treat than when it was originally diagnosed.

What if these metastatic cells could be detected earlier, before they established a “foothold” in a vital organ? Better yet, could these metastatic cancer cells be intercepted, preventing them them from lodging in a vital organ in the first place?

To catch a cancer cell

With these goals in mind, our biomaterials lab joined forces with surgical oncologist Jacqueline Jeruss to create an implantable medical device that acts as a metastatic cancer cell trap.

The implant is a tiny porous polymer disc (basically a miniature sponge, no larger than a pencil eraser) that can be inserted just under a patient’s skin. Implantation triggers the immune system’s “foreign body response,” and the implant starts to soak up immune cells that travel to it. If the implant can catch mobile immune cells, then why not mobile metastatic cancer cells?

The disc can detect cancer cells in mice. Lab mouse via www.shutterstock.com.

We gave implants to mice specially bred to model metastatic breast cancer. When the mice had palpable tumors but no evidence of metastatic disease, the implant was removed and analyzed.

Cancer cells were indeed present in the implant, while the other organs (potential destinations for metastatic cells) still appeared clean. This means that the implant can be used to spot previously undetectable metastatic cancer before it takes hold in an organ.

For patients with cancer in remission, an implant that can detect tumor cells as they move through the body would be a diagnostic breakthrough. But having to remove it to see if it has captured any cancer cells is not the most convenient or pleasant detection method for human patients.

Detecting cancer cells with noninvasive imaging

There could be a way around this, though: a special imaging method under development at Northwestern University called Inverse Spectroscopic Optical Coherence Tomography (ISOCT). ISOCT detects molecular-level differences in the way cells in the body scatter light. And when we scan our implant with ISOCT, the light scatter pattern looks different when it’s full of normal cells than when cancer cells are present. In fact, the difference is apparent when even as few as 15 out of the hundreds of thousands of cells in the implant are cancer cells.

There’s a catch – ISOCT cannot penetrate deep into tissue. That means it is not a suitable imaging technology for finding metastatic cells buried deep in internal organs. However, when the cancer cell detection implant is located just under the skin, it may be possible to detect cancer cells trapped in it using ISOCT. This could offer an early warning sign that metastatic cells are on the move.

This early warning could prompt doctors to monitor their patients more closely or perform additional tests. Conversely, if no cells are detected in the implant, a patient still in remission could be spared from unneeded tests.

The ISOCT results show that noninvasive imaging of the implant is feasible. But it’s a method still under development, and thus it’s not widely available. To make scanning easier and more accessible, we’re working to adapt more ubiquitous imaging technologies like ultrasound to detect tiny quantities of tumor cells in the implant.

Detect and capture. Joseph Xu, Michigan EngineeringCC BY-NC-ND

Not just detecting, but quarantining cancer

Besides providing a way to detect tiny numbers of cancer cells before they can form new tumors in other parts of the body, our implant offers an even more intriguing possibility: diverting metastatic cells away from vital organs, and sequestering them where they cannot cause any damage.

In our mouse studies, we found that metastatic cells got caught in the implant before they were apparent in vital organs. When metastatic cells eventually made their way into the organs, the mice with implants still had significantly fewer tumor cells in their organs than implant-free controls. Thus, the implant appears to provide a therapeutic benefit, most likely by taking the metastatic cells it catches out of the circulation, preventing them from lodging anywhere vital.

Interestingly, we have not seen cancer cells leave the implant once trapped, or form a secondary tumor in the implant. Ongoing work aims to learn why this is. Whether the cells can stay safely immobilized in the implant or if it would need to be removed periodically will be important questions to answer before the implant could be used in human patients.

What the future may hold

For now, our work aims to make the implant more effective at drawing and detecting cancer cells. Since we tested the implant with metastatic breast cancer cells, we also want to see if it will work on other types of cancer. Additionally, we’re studying the cells the implant traps, and learning how the implant interacts with the body as a whole. This basic research should give us insight into the process of metastasis and how to treat it.

In the future (and it might still be far off), we envision a world where recovering cancer patients can receive a detector implant to stand guard for disease recurrence and prevent it from happening. Perhaps the patient could even scan their implant at home with a smartphone and get treatment early, when the disease burden is low and the available therapies may be more effective. Better yet, perhaps the implant could continually divert all the cancer cells away from vital organs on its own, like Iron Man’s electromagnet that deflects shrapnel from his heart.

This solution is still not a “cure.” But it would transform a formidable disease that one out of three cancer survivors would otherwise ultimately die from into a condition with which they could easily live.

 

New PSA Test Examines Protein Structures to Detect Prostate Cancers

5/16/2016  by Cleveland Clinic

A promising new test is detecting prostate cancer more precisely than current tests, by identifying molecular changes in the prostate specific antigen (PSA) protein, according to Cleveland Clinic research presented today at the American Urological Association annual meeting.

The study – part of an ongoing multicenter prospective clinical trial – found that the IsoPSATM test can also differentiate between high-risk and low-risk disease, as well as benign conditions.

Although widely used, the current PSA test relies on detection strategies that have poor specificity for cancer – just 25 percent of men who have a prostate biopsy due to an elevated PSA level actually have prostate cancer, according to the National Cancer Institute – and an inability to determine the aggressiveness of the disease.

The IsoPSA test, however, identifies prostate cancer in a new way. Developed by Cleveland Clinic, in collaboration with Cleveland Diagnostics, Inc., IsoPSA identifies the molecular structural changes in protein biomarkers. It is able to detect cancer by identifying these structural changes, as opposed to current tests that simply measure the protein’s concentration in a patient’s blood.

“While the PSA test has undoubtedly been one of the most successful biomarkers in history, its limitations are well known. Even currently available prostate cancer diagnostic tests rely on biomarkers that can be affected by physiological factors unrelated to cancer,” said Eric Klein, M.D., chair of Cleveland Clinic’s Glickman Urological & Kidney Institute. “These study results show that using structural changes in PSA protein to detect cancer is more effective and can help prevent unneeded biopsies in low-risk patients.”

The clinical trial involves six healthcare institutions and 132 patients, to date. It examined the ability of IsoPSA to distinguish patients with and without biopsy-confirmed evidence of cancer. It also evaluated the test’s precision in differentiating patients with high-grade (Gleason = 7) cancer from those with low-grade (Gleason = 6) disease and benign findings after standard ultrasound-guided biopsy of the prostate.

Substituting the IsoPSA structure-based composite index for the standard PSA resulted in improvement in diagnostic accuracy. Compared with serum PSA testing, IsoPSA performed better in both sensitivity and specificity.

“We took an ‘out of the box’ approach that has shown success in detecting prostate cancer but also has the potential to address other clinically important questions such as clinical surveillance of patients after treatment,” said Mark Stovsky, M.D., staff member, Cleveland Clinic Glickman Urological & Kidney Institute’s Department of Urology. Stovsky has a leadership position (Chief Medical Officer) and investment interest in Cleveland Diagnostics, Inc. “In general, the clinical utility of prostate cancer early detection and screening tests is often limited by the fact that biomarker concentrations may be affected by physiological processes unrelated to cancer, such as inflammation, as well as the relative lack of specificity of these biomarkers to the cancer phenotype. In contrast, clinical research data suggests that the IsoPSA assay can interrogate the entire PSA isoform distribution as a single stand-alone diagnostic tool which can reliably identify structural changes in the PSA protein that correlate with the presence or absence and aggressiveness of prostate cancer.”

 

Point of Care, Highly Accurate Cervical Cancer Screening

5/20/2016 by Avi Rosenzweig, VP of Business Development, Biop Medical
http://www.mdtmag.com/article/2016/05/point-care-highly-accurate-cervical-cancer-screening

Fifty-five million times a year, American women go to their gynecologist for a Pap Smear. After waiting a few weeks for the results, more than 3.5 million of them are called back to the physician for a follow up visualization of the cervix. Beyond the stress related to possibly having cancer, the women are then subjected to a colposcopic exam, and all too often, a painful biopsy. Then more stressful waiting for a final diagnosis from the pathologist.

Cervical cancer develops slowly, allowing for successful treatment, when identified on time. Regions with high screening compliancy have low mortality rates from this cancer. In the US, for instance, where screening rates are close to 90%, only 4,200 women die from cervical cancer, annually, or 2.6 women per 100,000. However, the screening process in the developed world is long, complicated and not optimized.

In developing regions however, cervical cancer is a leading cause of women death. Over 85% of the total deaths from this cancer are in developing countries. Regions suffering from low screening rates include not only Africa, India and China, but many Eastern European countries as well. According to an OECD report from 2014, the cervical cancer screening rates in Romania and Hungary are as low as 14.6% and 36.7% respectively. The mortality rates in these countries are high, 16 in 100,000 women in Romania and 7.7 in 100,000 in Hungary.

The current screening process for cervical cancer detection is long, beginning with a Pap or HPV test. Cytology results take weeks to receive. A positive result requires follow-up testing by colposcopy and often biopsy. In countries where there is little access to medical care, or where screening compliancy is low, the chances of successful detection via this multi-step process are small. Developing regions and non-compliant countries require a point of care diagnostic method, which eliminates the need for return visits.

Additional limitations to cervical cancer screening are the low sensitivity and specificity rates of Pap tests and the high false positive rates of HPV test, leading to unnecessary colposcopies. Both cytology and colposcopy testing are highly dependent on operator proficiency for accurate diagnosis.

Biop has developed a new technology for the optimization of this process, into one, three minute, painless optical scan. The vaginal probe uses advanced optical, imaging and non-imaging technologies to identify and classify epithelium based cancers and pre-cancerous lesions. The probe is inserted into the vaginal canal, and scans the entire cervix. The resulting images and optical signatures created from the light, and captured by the sensors, are analyzed by the proprietary algorithm. The result is two pictures, on the physician’s screen; a high resolution photograph of the patient’s cervix, immediately next to a hot/cold map indicating a precise classification and location of any diseased lesions.

 

Deep learning applied to drug discovery and repurposing

May 27, 2016  http://www.kurzweilai.net/deep-learning-applied-to-drug-discovery-and-repurposing

Deep neural networks for drug discovery (credit: Insilico Medicine, Inc.)

Scientists from Insilico Medicine, Inc. have trained deep neural networks (DNNs) to predict the potential therapeutic uses of 678 drugs, using gene-expression data obtained from high-throughput experiments on human cell lines from Broad Institute’s LINCS databases and NIH MeSH databases.

The supervised deep-learning drug-discovery engine used the properties of small molecules, transcriptional data, and literature to predict efficacy, toxicity, tissue-specificity, and heterogeneity of response.

“We used LINCS data from Broad Institute to determine the effects on cell lines before and after incubation with compounds, co-author and research scientist Polina Mamoshina explained to KurzweilIAI.

“We used gene expression data of total mRNA from cell lines extracted and measured before incubation with compound X and after incubation with compound X to identify the response on a molecular level. The goal is to understand how gene expression (the transcriptome) will change after drug uptake. It is a differential value, so we need a reference (molecular state before incubation) to compare.”

The research is described in a paper in the upcoming issue of the journal Molecular Pharmaceutics.

Helping pharmas accelerate R&D

Alex Zhavoronkov, PhD, Insilico Medicine CEO, who coordinated the study, said the initial goal of their research was to help pharmaceutical companies significantly accelerate their R&D and increase the number of approved drugs. “In the process we came up with more than 800 strong hypotheses in oncology, cardiovascular, metabolic, and CNS spaces and started basic validation,” he said.

The team measured the “differential signaling pathway activation score for a large number of pathways to reduce the dimensionality of the data while retaining biological relevance.” They then used those scores to train the deep neural networks.*

“This study is a proof of concept that DNNs can be used to annotate drugs using transcriptional response signatures, but we took this concept to the next level,” said Alex Aliper, president of research, Insilico Medicine, Inc., lead author of the study.

Via Pharma.AI, a newly formed subsidiary of Insilico Medicine, “we developed a pipeline for in silico drug discovery — which has the potential to substantially accelerate the preclinical stage for almost any therapeutic — and came up with a broad list of predictions, with multiple in silico validation steps that, if validated in vitro and in vivo, can almost double the number of drugs in clinical practice.”

Despite the commercial orientation of the companies, the authors agreed not to file for intellectual property on these methods and to publish the proof of concept.

Deep-learning age biomarkers

According to Mamoshina, earlier this month, Insilico Medicine scientists published the first deep-learned biomarker of human age — aiming to predict the health status of the patient — in a paper titled “Deep biomarkers of human aging: Application of deep neural networks to biomarker development” by Putin et al, in Aging; and an overview of recent advances in deep learning in a paper titled “Applications of Deep Learning in Biomedicine” by Mamoshina et al., also in Molecular Pharmaceutics.

Insilico Medicine is located in the Emerging Technology Centers at Johns Hopkins University in Baltimore, Maryland, in collaboration with Datalytic Solutions and Mind Research Network.

* In this study, scientists used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the Library of Integrated Network-Based Cellular Signatures (LINCS) project developed by the National Institutes of Health (NIH) and linked those to 12 therapeutic use categories derived from MeSH (Medical Subject Headings) developed and maintained by the National Library of Medicine (NLM) of the NIH.

To train the DNN, scientists utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. Cross-validation experiments showed that DNNs achieve 54.6% accuracy in correctly predicting one out of 12 therapeutic classes for each drug.

One peculiar finding of this experiment was that a large number of drugs misclassified by the DNNs had dual use, suggesting possible application of DNN confusion matrices in drug repurposing.
FutureTechnologies Media Group | Video presentation Insilico medicine

Abstract of Deep learning applications for predicting pharmacological properties of drugs and drug repurposing using transcriptomic data

Deep learning is rapidly advancing many areas of science and technology with multiple success stories in image, text, voice and video recognition, robotics and autonomous driving. In this paper we demonstrate how deep neural networks (DNN) trained on large transcriptional response data sets can classify various drugs to therapeutic categories solely based on their transcriptional profiles. We used the perturbation samples of 678 drugs across A549, MCF-7 and PC-3 cell lines from the LINCS project and linked those to 12 therapeutic use categories derived from MeSH. To train the DNN, we utilized both gene level transcriptomic data and transcriptomic data processed using a pathway activation scoring algorithm, for a pooled dataset of samples perturbed with different concentrations of the drug for 6 and 24 hours. When applied to normalized gene expression data for “landmark genes,” DNN showed cross-validation mean F1 scores of 0.397, 0.285 and 0.234 on 3-, 5- and 12-category classification problems, respectively. At the pathway level DNN performed best with cross-validation mean F1 scores of 0.701, 0.596 and 0.546 on the same tasks. In both gene and pathway level classification, DNN convincingly outperformed support vector machine (SVM) model on every multiclass classification problem. For the first time we demonstrate a deep learning neural net trained on transcriptomic data to recognize pharmacological properties of multiple drugs across different biological systems and conditions. We also propose using deep neural net confusion matrices for drug repositioning. This work is a proof of principle for applying deep learning to drug discovery and development.

references:

 

Transistor-based biosensor detects molecules linked to cancer, Alzheimer’s, and Parkinson’s

May 23, 2016  http://www.kurzweilai.net/transistor-based-biosensor-detects-molecules-linked-to-cancer-alzheimers-and-parkinsons

An inexpensive portable biosensor developed by researchers at Brazil’s National Nanotechnology Laboratory (credit: LNNano)  http://www.kurzweilai.net/images/Biosensor-LNNano.jpg

A novel nanoscale organic transistor-based biosensor that can detect molecules associated with neurodegenerative diseases and some types of cancer has been developed by researchers at the National Nanotechnology Laboratory (LNNano) in Brazil.

The transistor, mounted on a glass slide, contains the reduced form of the peptide glutathione (GSH), which reacts in a specific way when it comes into contact with the enzyme glutathione S-transferase (GST), linked to Parkinson’s, Alzheimer’s and breast cancer, among other diseases.

http://www.kurzweilai.net/images/CuPc-transistor.png

Sensitive water-gated copper phthalocyanine (CuPc) thin-film transistor (credit: Rafael Furlan de Oliveira et al./Organic Electronics)

“The device can detect such molecules even when they’re present at very low levels in the examined material, thanks to its nanometric sensitivity,” explained Carlos Cesar Bof Bufon, Head of LNNano’s Functional Devices & Systems Lab (DSF).

Bufon said the system can be adapted to detect other substances by replacing the analytes (detection compounds). The team is working on paper-based biosensors to further lower the cost, improve portability, and facilitate fabrication and disposal.

The research is published in the journal Organic Electronics.

Abstract of Water-gated phthalocyanine transistors: Operation and transduction of the peptide–enzyme interaction

The use of aqueous solutions as the gate medium is an attractive strategy to obtain high charge carrier density (1012 cm−2) and low operational voltages (<1 V) in organic transistors. Additionally, it provides a simple and favorable architecture to couple both ionic and electronic domains in a single device, which is crucial for the development of novel technologies in bioelectronics. Here, we demonstrate the operation of transistors containing copper phthalocyanine (CuPc) thin-films gated with water and discuss the charge dynamics at the CuPc/water interface. Without the need for complex multilayer patterning, or the use of surface treatments, water-gated CuPc transistors exhibited low threshold (100 ± 20 mV) and working voltages (<1 V) compared to conventional CuPc transistors, along with similar charge carrier mobilities (1.2 ± 0.2) x 10−3 cm2 V−1 s−1. Several device characteristics such as moderate switching speeds and hysteresis, associated with high capacitances at low frequencies upon bias application (3.4–12 μF cm−2), indicate the occurrence of interfacial ion doping. Finally, water-gated CuPc OTFTs were employed in the transduction of the biospecific interaction between tripeptide reduced glutathione (GSH) and glutathione S-transferase (GST) enzyme, taking advantage of the device sensitivity and multiparametricity.

references:

 

First Large-Scale Proteogenomic Study of Breast Cancer    

Tues, May 31, 2016     http://www.technologynetworks.com/rnai/news.aspx?ID=191934

The study offers understanding of potential therapeutic targets.

Building on data from The Cancer Genome Atlas (TCGA) project, a multi-institutional team of scientists have completed the first large-scale “proteogenomic” study of breast cancer, linking DNA mutations to protein signaling and helping pinpoint the genes that drive cancer. Conducted by members of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC), including Baylor College of Medicine, Broad Institute of MIT and Harvard, Fred Hutchinson Cancer Research Center, New York University Langone Medical Center, and Washington University School of Medicine, the study takes aim at proteins, the workhorses of the cell, and their modifications to better understand cancer.

Appearing in the Advance Online Publication of Nature, the study illustrates the power of integrating genomic and proteomic data to yield a more complete picture of cancer biology than either analysis could do alone. The effort produced a broad overview of the landscape of the proteome (all the proteins found in a cell) and the phosphoproteome (the sites at which proteins are tagged by phosphorylation, a chemical modification that drives communication in the cell) across a set of 77 breast cancer tumors that had been genomically characterized in the TCGA project. Although the TCGA produced an extensive catalog of somatic mutations found in cancer, the effects of many of those mutations on cellular functions or patients’ outcomes are unknown.

In addition, not all mutated genes are true “drivers” of cancer — some are merely “passenger” mutations that have little functional consequence. And some mutations are found within very large DNA regions that are deleted or present in extra copies, so winnowing the list of candidate genes by studying the activity of their protein products can help identify therapeutic targets. “We don’t fully understand how complex cancer genomes translate into the driving biology that causes relapse and mortality,” said Matthew Ellis, director of the Lester and Sue Smith Breast Center at Baylor College of Medicine and a senior author of the paper.

“These findings show that proteogenomic integration could one day prove to be a powerful clinical tool, allowing us to traverse the large knowledge gap between cancer genomics and clinical action.” In this study, the researchers at the Broad Institute analyzed breast tumors using accurate mass, high-resolution mass spectrometry, a technology that extends the coverage of the proteome far beyond the coverage that can be achieved by traditional antibody-based methods. This allowed them to scale their efforts and quantify more than 12,000 proteins and 33,000 phosphosites, an extremely deep level of coverage.

 

Breakthrough Approach to Breast Cancer Treatment

May 24, 2016    http://www.technologynetworks.com/rnai/news.aspx?ID=191771

Scripps scientists have designed a drug candidate that decreases growth of breast cancer cells.

In a development that could lead to a new generation of drugs to precisely treat a range of diseases, scientists from the Florida campus of The Scripps Research Institute (TSRI) have for the first time designed a drug candidate that decreases the growth of tumor cells in animal models in one of the hardest to treat cancers—triple negative breast cancer.

“This is the first example of taking a genetic sequence and designing a drug candidate that works effectively in an animal model against triple negative breast cancer,” said TSRI Professor Matthew Disney. “The study represents a clear breakthrough in precision medicine, as this molecule only kills the cancer cells that express the cancer-causing gene—not healthy cells. These studies may transform the way the lead drugs are identified—by using the genetic makeup of a disease.”

The study, published by the journal Proceedings of the National Academy of Sciences, demonstrates that the Disney lab’s compound, known as Targaprimir-96, triggers breast cancer cells to kill themselves via programmed cell death by precisely targeting a specific RNA that ignites the cancer.

Short-Cut to Drug Candidates

While the goal of precision medicine is to identify drugs that selectively affect disease-causing biomolecules, the process has typically involved time-consuming and expensive high-throughput screens to test millions of potential drug candidates to identify those few that affect the target of interest. Disney’s approach eliminates these screens.

The new study uses the lab’s computational approach called Inforna, which focuses on developing designer compounds that bind to RNA folds, particularly microRNAs.

MicroRNAs are short molecules that work within all animal and plant cells, typically functioning as a “dimmer switch” for one or more genes, binding to the transcripts of those genes and preventing protein production. Some microRNAs have been associated with diseases. For example, microRNA-96, which was the target of the new study, promotes cancer by discouraging programmed cell death, which can rid the body of cells that grow out of control.

In the new study, the drug candidate was tested in animal models over a 21-day course of treatment. Results showed decreased production of microRNA-96 and increased programmed cell death, significantly reducing tumor growth. Since targaprimir-96 was highly selective in its targeting, healthy cells were unaffected.

In contrast, Disney noted, a typical cancer therapeutic targets and kills cells indiscriminately, often leading to side effects that can make these drugs difficult for patients to tolerate.

Benjamin Zealley and Aubrey D.N.J. de Grey
Commentary on Some Recent Theses Relevant to Combating Aging: June 2015

REJUVENATION RESEARCH 2015; 18(3), 282 – 287   http://dx.doi.org:/10.1089/rej.2015.1728

Cancer Autoantibody Biomarker Discovery and Validation Using Nucleic Acid Programmable Protein Array
Jie Wang, PhD, Arizona State University

Currently in the United States, many patients with cancer do not benefit from population-based screening due to challenges associated with the existing cancer screening scheme. Blood-based diagnostic assays have the potential to detect diseases in a non-invasive way. Proteins released from small early tumors may only be present intermittently and are diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce autoantibody (AAb) responses, which can amplify the signal and persist in the blood even if the antigen is gone. Circulating autoantibodies are a promising class of molecules that have the potential to serve as early detection biomarkers for cancers. This PhD thesis aims to screen for autoantibody biomarkers for the early detection of two deadly cancers, basal-like breast cancer and lung adenocarcinoma. First, a method was developed to display proteins in both native and denatured conformations on a protein array. This method adopted a novel protein tag technology, called a HaloTag, to immobilize proteins covalently on the surface of a glass slide. The covalent attachment allowed these proteins to endure harsh treatment without becoming dissociated from the slide surface, which enabled the profiling of antibody responses against both conformational and linear epitopes. Next, a plasma screening protocol was optimized to increase significantly the signal-to-noise ratio of protein array–based AAb detection. Following this, the AAb responses in basal-like breast cancer were explored using nucleic acid programmable protein arrays (NAPPA) containing 10,000 full-length human proteins in 45 cases and 45 controls. After verification in a large sample set (145 basal-like breast cancer cases, 145 controls, 70 non-basal breast cancer) by enzyme-linked immunosorbent assay (ELISA), a 13-AAb classifier was developed to differentiate patients from controls with a sensitivity of 33% at 98% specificity. A similar approach was also applied to the lung cancer study to identify AAbs that distinguished lung cancer patients from computed tomography–positive benign pulmonary nodules (137 lung cancer cases, 127 smoker controls, 170 benign controls). In this study, two panels of AAbs were discovered that showed promising sensitivity and specificity. Six out of eight AAb targets were also found to have elevated mRNA levels in lung adenocarcinoma patients using TCGA data. These projects as a whole provide novel insights into the association between AAbs and cancer, as well as general B cell antigenicity against self-proteins.

Comment: There are two widely supported models for cancer development and progression—the clonal evolution (CE) model and the cancer stem cell (CSC) model. Briefly, the former claims that most or all cells in a tumor contribute to its maintenance; as newer and more aggressive clones develop by random mutation, they become responsible for driving growth. The range of different mutational profiles generated is assumed to be large enough to account for disease recurrence after therapy (due to rare resistant clones) and metastasis (clones arising with the ability to travel to distant sites). The CSC model instead asserts that a small number of mutated stem cells are the origin of the primary cell mass, drive metastasis through the intermittent release of undifferentiated, highly mobile progeny, and account for recurrence due to a generally quiescent metabolic profile conferring potent resistance to chemotherapy. In either case, the immunological visibility of an early tumor may be highly sporadic. Clones arising early in CE differ little in proteomic terms from healthy host cells; those that do trigger a response are unlikely to have acquired robust resistance to immune attack, so are destroyed quickly in favor of their stealthier brethren. Likewise, CSCs share some of the immune privilege of normal stem cells and, due to their inherent ability to produce differentiated progeny with distinct proteomic signatures, are partially protected from attacks on their descendants. Consequently, such well-hidden cells may remain in the body for years to decades. The autoantibody panel developed in this study for basal-like breast cancer exhibits exceptional specificity despite a comparatively small training set. Given its ease of application, this suggests great promise for a more exhaustively trained classifier as a populationlevel screening tool.

 

Condition-Specific Differential Sub-Network Analysis for Biological Systems
Deepali Jhamb, PhD, Indiana University

Biological systems behave differently under different conditions. Advances in sequencing technology over the last decade have led to the generation of enormous amounts of condition-specific data. However, these measurements often fail to identify low-abundance genes and proteins that can be biologically crucial. In this work, a novel textmining system was first developed to extract condition-specific proteins from the biomedical literature. The literaturederived data was then combined with proteomics data to construct condition-specific protein interaction networks. Furthermore, an innovative condition-specific differential analysis approach was designed to identify key differences, in the form of sub-networks, between any two given biological systems. The framework developed here was implemented to understand the differences between limb regenerationcompetent Ambystoma mexicanum and regeneration-deficient Xenopus laevis. This study provides an exhaustive systems-level analysis to compare regeneration competent and deficient sub-networks to show how different molecular entities inter-connect with each other and are rewired during the formation of an accumulation blastema in regenerating axolotl limbs. This study also demonstrates the importance of literature-derived knowledge, specific to limb regeneration, to augment the systems biology analysis. Our findings show that although the proteins might be common between the two given biological conditions, they can have a high dissimilarity based on their biological and topological properties in the sub-network. The knowledge gained from the distinguishing features of limb regeneration in amphibians can be used in future to induce regeneration chemically in mammalian systems. The approach developed in this dissertation is scalable and adaptable to understanding differential sub-networks between any two biological systems. This methodology will not only facilitate the understanding of biological processes and molecular functions that govern a given system, but will also provide novel intuitions about the pathophysiology of diseases/conditions.

Comment: We have long advocated a principle of directly comparing young and old bodies as a means to identify the classes of physical damage that accumulate in the body during aging. This approach circumvents our ignorance of the full etiology of each particular disease manifestation, a phenomenally difficult question given the ethical issues of experimenting on human subjects, the lengthy ‘‘incubation time’’ of aging-related diseases, and the complex interconnections between their risk factors—innate and environmental. Repairing such damage has the potential to prevent pathology before symptoms appear, an approach now becoming increasingly mainstream.11 However, a naı¨ve comparison faces a number of difficulties, even given a sufficiently large sample set to compensate for inter-individual variation. Most importantly, the causal significance of a given species cannot be reliably determined from its simple prevalence.12 The catalytic nature of cell biology means that those entities whose abundance changes the most profoundly in absolute terms are quite unlikely to be the drivers of that change and may even spontaneously revert to baseline levels in the absence of on-going stimulation. Meanwhile, functionality is often heavily influenced independently of abundance by post-translational modifications that may escape direct detection. Sub-network analysis uses computational means to identify groups of genes and/or proteins that vary in a synchronized way with some parameter, indicating functional connectivity. The application of methods such as those developed here to the comparison of a wide range of younger and older conditions will facilitate the identification of processes—not merely individual factors—that are impaired with age, and thus will help greatly in identifying the optimal points for intervention.

 

Development of a Light Actuated Drug Delivery-on-Demand System
Chase Linsley, PhD, University of California, Los Angeles

The need for temporal–spatial control over the release of biologically active molecules has motivated efforts to engineer novel drug delivery-on-demand strategies actuated via light irradiation. Many systems, however, have been limited to in vitro proof-of-concept due to biocompatibility issues with the photo-responsive moieties or the light wavelength, intensity, and duration. To overcome these limitations, the objective of this dissertation was to design a light-actuated drug delivery-on-demand strategy that uses biocompatible chromophores and safe wavelengths of light, thereby advancing the clinical prospects of light-actuated drug delivery-on-demand systems. This was achieved by: (1) Characterizing the photothermal response of biocompatible visible light and near-infrared-responsive chromophores and demonstrating the feasibility and functionality of the light actuated on-demand drug delivery system in vitro; and (2) designing a modular drug delivery-on-demand system that could control the release of biologically active molecules over an extended period of time. Three biocompatible chromophores—Cardiogreen, Methylene Blue, and riboflavin—were identified and demonstrated significant photothermal response upon exposure to near-infrared and visible light, and the amount of temperature change was dependent upon light intensity, wavelength, as well as chromophore concentration. As a proof-of-concept, pulsatile release of a model protein from a thermally responsive delivery vehicle fabricated from poly(N-isopropylacrylamide) was achieved over 4 days by loading the delivery vehicle with Cardiogreen and irradiating with near-infrared light. To extend the useful lifetime of the light-actuated drug delivery-on-demand system, a modular, reservoir-valve system was designed. Using poly(ethylene glycol) as a reservoir for model small molecule drugs combined with a poly(N-isopropylacrylamide) valve spiked with chromophore-loaded liposomes, pulsatile release was achieved over 7 days upon light irradiation. Ultimately, this drug delivery strategy has potential for clinical applications that require explicit control over the presentation of biologically active molecules. Further research into the design and fabrication of novel biocompatible thermally responsive delivery vehicles will aid in the advancement of the light-actuated drug delivery-on-demand strategy described here. Comment: Our combined comments on this thesis and the next one appear after the next abstract.

 

Light-Inducible Gene Regulation in Mammalian Cells
Lauren Toth, PhD, Duke University

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue-engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible. The recent emergence of the field of optogenetics—the ability to control gene expression using light—has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific 3-bp DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or enhanced green fluorescent protein (eGFP) transgene enabled expression of the transgene in response to blue light. Gene activation was both reversible and tunable on the basis of duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be patterned spatially by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore, we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a lightactivated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the amino-terminal fragment of CIB1 to the amino, carboxyl, or amino and carboxyl terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic ‘‘on’’ switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase–mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system, could be used to express MyoD or the synthetic transcription factor VP64- MyoD in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated up-regulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T1/2 cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased micro-vessel density compared to mice that were not illuminated. Analysis of human vascular endothelial growth factor (VEGF) and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

Comment: Although it is easy to characterize technological progress as following in the wake of scientific discoveries, the reverse is almost equally true; advances in technique open the door to types of experiment previously intractable or impossible. Such is currently the case for the field of optically controlled biotechnology, which has exploded into prominence, particularly over the last half-decade. Light of an appropriate wavelength can penetrate mammalian tissues to a depth of up to a couple of centimeters, rendering much of the living body accessible to optical study and control—still more if the detector/source is integrated into an endoscopic or fiber optic probe. Techniques borrowed from the semiconductor industry allow patterns of illumination to be controlled down to the nanometer scale, ideal for addressing individual cells. The highly controlled time course of such experiments, as compared to traditional means of gene activation, such as the addition of a chemical agent to the medium, eliminates confounding variables, and simplifies data analysis. Furthermore, this level of immediate control opens the door to closed-loop systems where the activity of entities under optical control can be continuously tuned in relation to some parameter(s). In the first of these two illuminating theses, a vehicle is developed that permits light-driven release of a small molecule. Such a system could be employed to target a systemically administered antibiotic or anti-neoplastic agent to a site of infection or cancer while sparing other bodily tissues from toxicity. Because most modern drugs cannot be produced in the body, even given arbitrarily good control of cellular biochemistry, this technique will have lasting value in numerous clinical contexts. In the second thesis, the level of precision achieved is even more profound; the CRISPR/Cas9 system has received much recent attention13 in its own right for its capacity to target arbitrary genetic sequences without an arduous protein-engineering step. The LACE system described stands to permit genetic manipulation with almost arbitrarily good spatial, temporal, and genomic site-specific control, using only means available to a typical university laboratory.

 

Targeting T Cells for the Immune-Modulation of Human Diseases
Regina Lin, PhD, Duke University

Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers, and sentinels of host immunity, T cells represent a compelling target population for immune-modulation. In fact, the antigen-specificity, cytotoxicity, and promise of long-lived of immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell–mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and pre-clinically characterizing their mechanisms of action and in vivo therapeutic efficacy.

CD8+ cytotoxic T cells have potent anti-tumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo– expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA (miRNA)-targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, miR- 23a was up-regulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired anti-tumor potential of patient CD8+ T cells. We determined that tumor-derived transforming growth factor-b (TGF-b) directly suppresses CD8+ T cell immune function by elevating miR-23a and down-regulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR-23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGF-bmediated tumor immune-evasion pathway

Having established that miR-23a-inhibition can enhance the quality and functional resilience of anti-tumor CD8+ T cells, especially within the immune-suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers—such as TGF-b—within the tumor microenvironment. Because TGF-b poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wild-type and miR-23a–deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumorspecific epitope expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wild-type EGFRvIIICAR T cells were vulnerable to functional suppression by TGF-b, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGF-b. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23adeficient EGFRvIII-CAR T cells for GBM immunotherapy.

Last, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune-suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immunesuppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro. Subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro-inflammatory interferon-c (IFN-c) and interleukin-17 (IL-17) production by fully differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases.

Comment: Immunotherapy is among the most promising approaches to cancer treatment, having the specificity and scope to selectively target transformed cells wherever they may reside within the body and the potential to install a permanent defense against disease recurrence. By the time a typical cancer is clinically diagnosed, however, it has already found means to survive a prolonged period of potential immune attack. The mechanisms by which tumors evade immune surveillance are beginning to be elucidated,15,16 and include both direct suppression of effector cells and progressive editing of the host’s immune repertoire to disfavor future attack. It is inherently difficult to interfere with these defenses directly, due to the selection pressures in genetically heterogeneous neoplastic tissue. Much effort is thus being focused on methods for rendering therapeutically delivered immune cells resistant to their effects. The cytokine TGF-b is paradoxically known to function as both a tumor suppressor in healthy tissue and as a tumorderived species associated with multiple cancer-promoting activities, including enhanced immune evasion. This work identifies the pathway by which TGF-b compromises cytotoxic T cell function in the tumor microenvironment, and demonstrates an effective method for blocking this signal. In many clinical cases, however, editing of the patient’s immune repertoire has already removed or rendered anergic those immune cells able to recognize their cancer. Thus, the finding that blocking TGF-b signaling also appears to enhance the effectiveness of CAR-modified T cells— engineered with an antibody fragment targeting them with high affinity to a particular tumor-associated epitope—is a welcome addition to these already promising results.

 

Novel Fibonacci and non-Fibonacci structure in the sunflower: results of a citizen science experiment

Jonathan Swinton, Erinma Ochu, The MSI Turing’s Sunflower Consortium

Published 18 May 2016. DOI http://dx.doi.org:/10.1098/rsos.160091

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

  1. Introduction

Fibonacci structure can be found in hundreds of different species of plants [1]. This has led to a variety of competing conceptual and mathematical models that have been developed to explain this phenomenon. It is not the purpose of this paper to survey these: reviews can be found in [14], with more recent work including [510]. Instead, we focus on providing empirical data useful for differentiating them.

These models are in some ways now very mathematically satisfying in that they can explain high Fibonacci numbers based on a small number of plausible assumptions, though they are not so satisfying to experimental scientists [11]. Despite an increasingly detailed molecular and biophysical understanding of plant organ positioning [1214], the very parsimony and generality of the mathematical explanations make the generation and testing of experimental hypotheses difficult. There remains debate about the appropriate choice of mathematical models, and whether they need to be central to our understanding of the molecular developmental biology of the plant. While sunflowers provide easily the largest Fibonacci numbers in phyllotaxis, and thus, one might expect, some of the stronger constraints on any theory, there is a surprising lack of systematic data to support the debate. There have been only two large empirical studies of spirals in the capitulum, or head, of the sunflower: Weisse [15] and Schoute [16], which together counted 459 heads; Schoute found numbers from the main Fibonacci sequence 82% of the time and Weise 95%. The original motivation of this study was to add a third replication to these two historical studies of a widely discussed phenomenon. Much more recently, a study of a smaller sample of 21 seedheads was carried out by Couder [17], who specifically searched for non-Fibonacci examples, whereas Ryan et al. [18] studied the arrangement of seeds more closely in a small sample of Helianthus annuus and a sample of 33 of the related perennial H. tuberosus.

Neither the occurrence of Fibonacci structure nor the developmental biology leading to it are at all unique to sunflowers. As common in other species, the previous sunflower studies found not only Fibonacci numbers, but also the occasional occurrence of the double Fibonacci numbers, Lucas numbers and F4 numbers defined below [1]. It is worth pointing out the warning of Cooke [19] that numbers from these sequences make up all but three of the first 17 integers. This means that it is particularly valuable to look at specimens with large parastichy numbers, such as the sunflowers, where the prevalence of Fibonacci structure is at its most striking.

Neither Schoute nor Weisse reported their precise technique for assigning parastichy numbers to their samples, and it is noteworthy that neither author reported any observation of non-Fibonacci structure. One of the objectives of this study was to rigorously define Fibonacci structure in advance and to ensure that the assignment method, though inevitably subjective, was carefully documented.

This paper concentrates on the patterning of seeds towards the outer rim of sunflower seedheads. The number of ray florets (the ‘petals’, typically bright yellow) or the green bracts behind them tends to have a looser distribution around a Fibonacci number. In the only mass survey of these, Majumder & Chakravarti [20] counted ray florets on 1002 sunflower heads and found a distribution centred on 21.

This citizen science study evaluates the occurrence of Fibonacci structure in the spirals of sunflower (Helianthus annuus) seedheads. This phenomenon has competing biomathematical explanations, and our core premise is that observation of both Fibonacci and non-Fibonacci structure is informative for challenging such models. We collected data on 657 sunflowers. In our most reliable data subset, we evaluated 768 clockwise or anticlockwise parastichy numbers of which 565 were Fibonacci numbers, and a further 67 had Fibonacci structure of a predefined type. We also found more complex Fibonacci structures not previously reported in sunflowers. This is the third, and largest, study in the literature, although the first with explicit and independently checkable inclusion and analysis criteria and fully accessible data. This study systematically reports for the first time, to the best of our knowledge, seedheads without Fibonacci structure. Some of these are approximately Fibonacci, and we found in particular that parastichy numbers equal to one less than a Fibonacci number were present significantly more often than those one more than a Fibonacci number. An unexpected further result of this study was the existence of quasi-regular heads, in which no parastichy number could be definitively assigned.

Incorporation of irregularity into the mathematical models of phyllotaxis is relatively recent: [17] gave an example of a disordered pattern arising directly from the deterministic model while more recently the authors have begun to consider the effects of stochasticity [10,21]. Differentiating between these models will require data that go beyond capturing the relative prevalence of different types of Fibonacci structure, so this study was also designed to yield the first large-scale sample of disorder in the head of the sunflower.

The Fibonacci sequence is the sequence of integers 1,2,3,5,8,13,21,34,55,89,144… in which each member after the second is the sum of the two preceding. The Lucas sequence is the sequence of integers 1,3,4,7,11,18,29,47,76,123… obeying the same rule but with a different starting condition; the F4 sequence is similarly 1,4,5,9,14,23,37,60,97,…. The double Fibonacci sequence 2,4,6,10,16,26,42,68,110,… is double the Fibonacci sequence. We say that a parastichy number which is any of these numbers has Fibonacci structure. The sequencesF5=1,5,6,11,17,28,45,73,… and F8=1,8,9,17,26,43,69,112… also arise from the same rule, but as they had not been previously observed in sunflowers we did not include these in the pre-planned definition of Fibonacci structure for parsimony. One example of adjacent pairs from each of these sequences was, in fact, observed but both examples are classified as non-Fibonacci below. A parastichy number which is any of 12,20,33,54,88,143 is also not classed as having Fibonacci structure but is distinguished as a Fibonacci number minus one in some of the analyses, and similarly 14,22,35,56,90,145 as Fibonacci plus one.

When looking at a seedhead such as in figure 1 the eye naturally picks out at least one family of parastichies or spirals: in this case, there is a clockwise family highlighted in blue in the image on the right-hand side.

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Distribution and type of parastichy pairs

Figure 5 plots the individual pairs observed. On the reference line, the ratio of the numbers is equal to the golden ratio so departures from the line mark departures from Fibonacci structure, which are less evident in the more reliable photoreviewed dataset. It can be seen from table 3 that Fibonacci pairings dominate the dataset.

 

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Table 3.

Observed pairings of Fibonacci types of clockwise and anticlockwise parastichy numbers. Other means any parastichy number which neither has Fibonacci structure nor is Fibonacci ±1. Of all the Fibonacci ±1/Fibonacci pairs, only sample 191, a (21,20) pair, was not close to an adjacent Fibonacci pair.

One typical example of a Fibonacci pair is shown in figure 6, with a double Fibonacci case infigure 1 and a Lucas one in figure 7. There was no photoreviewed example of an F4 pairing. The sole photoreviewed assignment of a parastichy number to the F4 sequence was the anticlockwise parastichy number 37 in sample 570, which was relatively disordered. The clockwise parastichy number was 55, lending support to the idea this may have been a perturbation of a (34,55) pattern. We also found adjacent members of higher-order Fibonacci series. Figures 8 and 9 each show well-ordered examples with parastichy counts found adjacent in the F5 and F8 series, respectively: neither of these have been previously reported in the sunflower.

Figure 6.

 

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Sunflower 095. An (89,55) example with 89 clockwise parastichies and 55 anticlockwise ones, extending right to the rim of the head. Because these are clear and unambiguous, the other parastichy families which are visible towards the centre are not counted here.

Figure 7.   Sunflower 171. A Lucas series (76,47) example.

Sunflower 667. Anticlockwise parastichies only, showing competing parastichy families which are distinct but in some places overlapping.

Our core results are twofold. First, and unsurprisingly, Fibonacci numbers, and Fibonacci structure more generally, are commonly found in the patterns in the seedheads of sunflowers. Given the extent to which Fibonacci patterns have attracted pseudo-scientific attention [33], this substantial replication of limited previous studies needs no apology. We have also published, for the first time, examples of seedheads related to the F5 and F8 sequences but by themselves they do not add much to the evidence base. Our second core result, though, is a systematic survey of cases where Fibonacci structure, defined strictly or loosely, did not appear. Although not common, such cases do exist and should shed light on the underlying developmental mechanisms. This paper does not attempt to shed that light, but we highlight the observations that any convincing model should explain. First, the prevalence of Lucas numbers is higher than those of double Fibonacci numbers in all three large datasets in the literature, including ours, and there are sporadic appearances of F4, F5 and F8 sequences. Second, counts near to but not exactly equal to Fibonacci structure are also observable: we saw a parastichy count of 54 more often than the most common Lucas count of 47. Sometimes, ambiguity arises in the counting process as to whether an exact Fibonacci-structured number might be obtained instead, but there are sufficiently many unambiguous cases to be confident this is a genuine phenomenon. Third, among these approximately Fibonacci counts, those which are a Fibonacci number minus one are significantly more likely to be seen than a Fibonacci number plus one. Fourth, it is not uncommon for the parastichy families in a seedhead to have strong departures from rotational symmetry: this can have the effect of yielding parastichy numbers which have large departures from Fibonacci structure or which are completely uncountable. This is related to the appearance of competing parastichy families. Fifth, it is common for the parastichy count in one direction to be more orderly and less ambiguous than that in the other. Sixth, seedheads sometimes possess completely disordered regions which make the assignment of parastichy numbers impossible. Some of these observations are unsurprising, some can be challenged by different counting protocols, and some are likely to be easily explained by the mathematical properties of deformed lattices, but taken together they pose a challenge for further research.

It is in the nature of this crowd-sourced experiment with multiple data sources that it is much easier to show variability than it is to find correlates of that variability. We tried a number of cofactor analyses that found no significant effect of geography, growing conditions or seed type but if they do influence Fibonacci structure, they are likely to be much easier to detect in a single-experimenter setting.

We have been forced by our results to extend classifications of seedhead patterns beyond structured Fibonacci to approximate Fibonacci ones. Clearly, the more loose the definition of approximate Fibonacci, the easier it is to explain away departures from model predictions. Couder [17] found one case of a (54,87) pair that he interpreted as a triple Lucas pair 3×(18,29). While mathematically true, in the light of our data, it might be more compellingly be thought of as close to a (55,89) ideal than an exact triple Lucas one. Taken together, this need to accommodate non-exact patterns, the dominance of one less over one more than Fibonacci numbers, and the observation of overlapping parastichy families suggest that models that explicitly represent noisy developmental processes may be both necessary and testable for a full understanding of this fascinating phenomenon. In conclusion, this paper provides a testbed against which a new generation of mathematical models can and should be built.

 

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Immune System in Perspective

Curator: Larry H. Bernstein, MD, FCAP

LPBI

How regulatory T cells work
Vignali DAA, Collison LW & Workman CJ
Nature Reviews Immunology 8, 523-532 (July 2008) |   doi:10.1038/nri2343
http://www.nature.com/nri/journal/v8/n7/full/nri2343.html

Regulatory T (TReg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting antitumour immunity. Given that TReg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we describe the basic mechanisms used by TReg cells to mediate suppression and discuss whether one or many of these mechanisms are likely to be crucial for TReg-cell function. In addition, we propose the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate TReg-cell function.

How regulatory T cells work.

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Basic mechanisms used by Treg cells

This schematic depicts the various regulatory T (Treg)-cell mechanisms arranged into four groups centred around four basic modes of action. ‘Inhibitory cytokines’ include interleukin-10 (IL-10), interleukin-35 (IL-35) and transforming growth factor-β (TGF-β). ‘Cytolysis’ includes granzyme-A- and granzyme-B-dependent and perforin-dependent killing mechanisms. ‘Metabolic disruption’ includes high affinity IL-2 receptor α (CD25)-dependent cytokine-deprivation-mediated apoptosis, cyclic AMP (cAMP)-mediated inhibition, and CD39- and/or CD73-generated, adenosine–purinergic adenosine receptor (A2A)-mediated immunosuppression. ‘Targeting dendritic cells’ includes mechanisms that modulate DC maturation and/or function such as lymphocyte activation gene-3 (LAG3; also known as CD223)–MHC-class-II-mediated suppression of DC maturation, and cytotoxic T lymphocyte antigen-4 (CTLA4)–CD80/CD86-mediated induction of indoleamine 2,3-dioxygenase (IDO), which is an immunosuppressive molecule, by DCs.

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Model for how effector T cells might boost Treg-cell function

This occurs in three stages. (a) Initial regulatory T (Treg)-cell activation induces production of regulatory factors such as interleukin-35 (IL-35). (b) Treg cells ‘sense’ the presence of recently activated effector T cells through a receptor–ligand interaction (cell surface or soluble). (c) This in turn boosts or potentiates Treg-cell function resulting in the enhanced production of regulatory mediators, such as IL-35, and perhaps the induction of new mediators.

 

Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Given that Treg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we discuss the basic mechanisms used by Treg cells to mediate suppression, and discuss whether one or many of these mechanisms are likely to be crucial for Tregcell function. In addition, we present the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate Treg-cell function.

Several sophisticated regulatory mechanisms are used to maintain immune homeostasis, prevent autoimmunity and moderate inflammation induced by pathogens and environmental insults. Chief amongst these are regulatory T (Treg) cells that are now widely regarded as the primary mediators of peripheral tolerance. Although Treg cells play a pivotal role in preventing autoimmune diseases, such as type 1 diabetes1,2, and limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD)3,4, they also block beneficial responses by preventing sterilizing immunity to certain pathogens5,6 and limiting anti-tumour immunity7. A seminal advance in the analysis of Treg cells came with the identification of a key transcription factor, forkhead box P3 (FOXP3), that is required for their development, maintenance and function8,9. Mice and patients that lack FOXP3 develop a profound autoimmune-like lymphoproliferative disease that graphically emphasizes the importance of Treg cells in maintaining peripheral tolerance10-12 (BOX 1). Although FOXP3 has been proposed as the master regulator of Treg cells that controls the expression of multiple genes that mediate their regulatory activity13,14, this has been recently challenged raising the possibility that other transcriptional events may operate upstream of and/or concurrently with FOXP3 to mediate Treg-cell development15.

While Foxp3 has proven to be an invaluable marker for murine Treg cells, its role in human Treg cells is less straightforward (see BOX 2 for a discussion of Treg-cell markers). Humans that lack FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), a severe autoimmune disease that presents early in infancy. Although FOXP3 appears to be required for human Treg-cell development and function, expression of FOXP3 alone is clearly not sufficient as a significant percentage of human activated T cells express FOXP3 and yet do not possess regulatory activity16-20. Furthermore, induction of FOXP3 in human T cells by transforming growth factor-β (TGFβ) does not confer a regulatory phenotype, in contrast to their murine counterparts20. Consequently, FOXP3 is not a good marker for human Treg cells (BOX 2). Whether this distinction is due to intrinsic differences between mouse and human FOXP3 and/or a requirement for an additional cofactor/ transcription factor is an important question that needs to be resolved.

Significant progress has been made over the last few years in delineating the molecules and mechanisms that Treg cells use to mediate suppression21,22. In this Review, we outline our current understanding of the mechanisms used by Treg cells to mediate suppression, and the challenges that lie ahead in defining their mode of action. We also discuss whether Treg cells are likely to depend on one, a few or many of these mechanisms. In addition, we propose that effector T cells may have a significant role in boosting and/or modulating Treg-cell function. Unless stated, we focus here primarily on the mechanisms that are used by thymus-derived natural CD4+CD25+ FOXP3+ Treg cells.

Basic mechanisms of Treg-cell function Defining the mechanisms of Treg-cell function is clearly of crucial importance. Not only would this provide insight into the control processes of peripheral tolerance but it would probably provide a number of potentially important therapeutic targets. Although this quest has been ongoing since interest in Treg cells was reignited in 199523, there has been significant progress in the last few years. From a functional perspective, the various potential suppression mechanisms of Treg cells can be grouped into four basic ‘modes of action’: suppression by inhibitory cytokines, suppression by cytolysis, suppression by metabolic disruption, and suppression by modulation of dendritic-cell (DC) maturation or function (FIG. 1).

Suppression by inhibitory cytokines Inhibitory cytokines, such as interleukin-10 (IL-10) and TGFβ, have been the focus of considerable attention as a mechanism of Treg-cell-mediated suppression. There has also been significant interest in their ability to generate induced (also known as adaptive) Treg-cell populations, either naturally in vivo or experimentally as a potential therapeutic modality (BOX 3). Although the general importance of IL-10 and TGFβ as suppressive mediators is undisputed, their contribution to the function of thymus-derived, natural Treg cells is still a matter of debate24. This is partly due to the general perception that Treg cells function in a contactdependent manner25,26. Indeed, in vitro studies using neutralizing antibodies or T cells that are unable to produce or respond to IL-10 and TGFβ suggested that these cytokines may not be essential for Treg-cell function25-28. However, this contrasts with data from in vivo studies29,30.

In allergy and asthma models, evidence suggests that both natural and antigen-specific Treg cells control disease in a manner that is, in part, dependent on IL-1029 and in some reports dependent on both IL-10 and TGFβ 31. Adoptive transfer of allergen-specific Treg cells induced significant IL-10 production by CD4+ effector T cells in the lung following allergen challenge and this Treg-cell-mediated control of disease was reversed by treatment with an IL-10- receptor-specific antibody32. However, suppression of allergic inflammation and airway hyper-reactivity, and increased production of IL-10 still occurred following transfer of IL-10- deficient Treg cells, suggesting that Treg cells can suppress the Th2-driven response to allergens in vivo through an IL-10-dependent mechanism, but that the production of IL-10 by Treg cells themselves is not required for the suppression observed. This contrasts with a recent study suggesting that the Treg-cell-specific ablation of IL-10 expression resulted in increased lung allergic inflammation and hyperreactivity33.

This scenario might occur in other disease models. For instance, the effects of IL-10 can only be partially attributed to Treg-cell-derived IL-10 in the immune response to hepatitis B virus34 and in the allograft tolerance response elicited by splenocytes exposed to non-inherited maternal antigens35. Recently, it was also shown that IL-10 is crucial for the control of various infections in which Treg cells have been reported to be involved including Mycobacterium tuberculosis36, Toxoplasma gondii37, Leishmania major38, and Trichinella spiralis39. However, Treg cells were not the source of IL-10 in all of these infection models.

By contrast, several studies have shown that IL-10 production by Treg cells is essential for the prevention of colitis in mouse models of IBD40. Moreover, it appears that the tumour microenvironment promotes the generation of FOXP3+ Treg cells that mediate IL-10- dependent, cell-contact independent, suppression41. Similarly, in UV-radiation-induced carcinogenesis, IL-10 production by Treg cells appears to be important for blocking anti-tumour immunity42. IL-10 produced by Treg cells also appears to be crucial for IL-10-mediated tolerance in a model of hepatitis induced by concanavalin A43 and tolerance to bacterial and viral superantigens44. In addition, recent papers suggest new roles for Treg-cell-derived IL-10 in the induction of feto-maternal tolerance45 and B-cell-enhanced recovery from experimental autoimmune encephalomyelitis46. Collectively, the picture that appears to be emerging is that the relative importance of Treg-cell-derived IL-10 is very dependent on the target organism or disease and on the experimental system. Furthermore, the Treg-cell-specific deletion of IL-10 did not result in the development of spontaneous systemic autoimmunity, but did result in enhanced pathology in the colon of older mice and in the lungs of mice with induced airway hypersensitivity, suggesting that the function of Treg-cell-derived IL-10 may be restricted to controlling inflammatory responses induced by pathogens or environmental insults33.

While some early in vitro studies using neutralizing antibodies to TGFβ or Treg cells lacking TGFβ 25,47 indicated that TGFβ was not required for natural Treg-cell function, other studies, both in vitro and in vivo suggested a critical role for Treg-cell surface bound TGFβ 48,49. Therefore, the importance of TGFβ for natural Treg-cell function has also been a controversial topic. Indeed, there has been considerably more focus recently on the importance of TGFβ in the development of induced Treg cells and perhaps in Treg-cell maintenance in general (BOX 3). However, there are studies that suggest that TGFβ produced by Treg cells may directly participate in effector T-cell suppression. For instance, effector T cells that are resistant to TGFβ-mediated suppression cannot be controlled by Treg cells in an IBD model50. In addition, TGFβ produced by Treg cells has been found to be important in the control of the host immune response to M. tuberculosis36, suppression of allergic responses31 and prevention of colitis in an IBD model51. Interestingly, TGFβ produced by Treg cells has also been implicated in limiting anti-tumour immunity in head and neck squamous-cell carcinoma52 and in follicular lymphoma53 by rendering T cells unresponsive to the tumour. TGFβ also appears to limit the anti-tumour activity of cytokine-induced killer cells54.

Membrane-tethered TGFβ can also mediate suppression by Treg cells in a cell-cell contactdependent manner48. Treg cells can control islet infiltration of CD8+ T cells and delay the progress of diabetes through membrane-tethered TGFβ 49. However, experiments using mice deficient in TGFβ-receptor (TGFβR) signalling in effector T cells or using TGFβ or TGFβR blocking reagents failed to show that membrane-tethered TGFβ is required for natural Treg cell development or function47. More recently, however, interest in membrane-tethered TGFβ has re-surfaced with the description of a previously unappreciated role for it in the tumour microenvironment. TGFβ associated with tumour exosome membranes appears to enhance the suppressive function of Treg cells and skew T cells away from their effector functions and towards regulatory functions55. Furthermore, ovalbumin-induced airway inflammation can be attenuated by heme oxygenase-1 through membrane-tethered TGFβ and IL-10 secretion by Treg cells56, a process that activates the Notch1–HES1 (hairy and enhancer of split 1) axis in target cells57. Thus, in light of the most current data, it now appears that soluble and/or membrane-tethered TGFβ may have a previously unappreciated role in natural Treg-cell function.

Recently, a new inhibitory cytokine, IL-35, has been described that is preferentially expressed by Treg cells and is required for their maximal suppressive activity58. IL-35 is a novel member of the IL-12 heterodimeric cytokine family and is formed by the pairing of Epstein–Barr virus induced gene 3 (Ebi3), which normally pairs with p28 to form IL-27, and p35 (also known as  Il12a), which normally pairs with p40 to form IL-12. Both Ebi3 and Il12a are preferentially expressed by murine Foxp3+ Treg cells58,59, but not resting or active effector T cells, and are significantly upregulated in actively suppressing Treg cells58. As predicted for a heterodimeric cytokine, both Ebi3−/− and Il12a−/− Treg cells had significantly reduced regulatory activity in vitro and failed to control homeostatic proliferation and cure IBD in vivo. This precise phenocopy suggested that IL-35 is required for the maximal suppressive activity of Treg cells. Importantly IL-35 was not only required but sufficient, as ectopic expression of IL-35 conferred regulatory activity on naive T cells and recombinant IL-35 suppressed T cell proliferation in vitro58. Although IL-35 is an exciting addition to the Treg-cell portfolio, there is clearly much that remains to be defined about this cytokine and its contribution to Treg-cell function. For instance, it remains to be determined if IL-35 suppresses the development and/or function of other cell types such as DCs and macrophages.

It is now clear that three inhibitory cytokines, IL-10, IL-35 and TGFβ, are key mediators of Treg-cell function. Although they are all inhibitory, the extent to which they are utilized in distinct pathogenic/homeostatic settings differs suggesting a non-overlapping function, which needs further refinement.

……….

How many mechanisms do Treg cells need? Although efforts to define the suppressive mechanisms used by Treg cells continue, an important question looms large. Is it likely that all these molecules and mechanisms will be crucial for Treg-cell function? There are three broad possibilities.

One, a single, overriding suppressive mechanism is required by all Treg cells Until the entire mechanistic panoply of Treg cells is defined, one cannot completely rule out this possibility. However, this possibility would seem unlikely as none of the molecules and/ or mechanisms that have been defined to date, when blocked or deleted, result in the complete absence of regulatory activity — a consequence that one might predict would result in a ‘Scurfy-like’ phenotype (BOX 1). So, although Treg cells that lack a single molecule, for instance IL-10, IL-35 or granzyme B, exhibit significantly reduced suppressor function, a scurfy phenotype does not ensue. Given that none of the current Treg-cell mechanisms can exclusively claim this distinction, it seems unlikely that any ‘unknown’ molecules or mechanisms could do so either.

Two, multiple, non-redundant mechanisms are required for maximal Treg-cell function In the studies conducted to date, Treg cells that lack various suppressive molecules have been shown to be functionally defective. This favours a scenario where there are multiple mechanisms that can be used by Treg cells but they are non-redundant, with each molecule contributing to the mechanistic whole. At present, this possibility would seem plausible. Indeed, this is supported by the recent analysis of mice possessing a Treg-cell-specific ablation of IL-10 expression, in which enhanced pathology was observed following environmental insult33. One would predict that at some point we should be able to generate knockout mice that lack a particular set of genes which results in a complete loss of Treg-cell activity. For this to be truly non-redundant, this list would probably be restricted and small (2–4 genes).

Three, multiple, redundant mechanisms are required for maximal Treg-cell function With the plethora of regulatory mechanisms described to date and the possibility of more yet to be identified, it is conceivable that there are multiple mechanisms that function redundantly. Such a redundant system would help to mitigate against effector T-cell escape from regulatory control. Also, given the very small size of the Treg-cell population, a sizable arsenal may be required at the height of an effector T-cell attack. Of course, it is possible that a semi-redundant scenario exists.

These possibilities have been discussed from the perspective of there being a single homogeneous Treg-cell population. However, as for helper T cell subsets it remains possible that a few or even many different Treg-cell subsets exist24. Each of these may rely on one or multiple regulatory mechanisms. Several recent studies have provided support for both phenotypic and functional heterogeneity amongst Treg cells. For instance, it has recently been shown that a small sub-population of Treg cells express the chemokine receptor CCR6, which is associated with T cells possessing an effector-memory phenotype102. CCR6+ Treg cells appeared to accumulate in the central nervous systems of mice with experimental autoimmune encephalomyelitis (EAE) suggesting that they may have a prevalent role in controlling responses in inflamed tissues. Heterogeneous expression of HLA-DR has also been suggested to mark different subpopulations of functionally distinct human Treg cells103. Indeed, HLADR positive Treg cells were found to be more suppressive than their DR negative counterparts. One might speculate that their enhanced inhibitory activity is due to DR-mediated ligation of the inhibitory molecule LAG3 expressed by activated effector T cells95,96.

So, if multiple suppressor mechanisms exist, how might these be integrated and used productively by Treg cells in vivo? We would propose the following possible models21. First, a ‘hierarchical’ model in which Treg cells possess many mechanisms that could be used but only one or two that are really crucial and consistently important in a variety of regulatory settings. Second, a ‘contextual’ model where different mechanisms become more or less important depending on the background or context in which the Treg cells reside and the type of target cell that they have to repress. For example, some cell types may be inhibited primarily by cytokines, whereas others are most effectively suppressed through lysis by Treg cells. Alternatively, different mechanisms may be more effective in different tissue compartments or in different disease settings. This notion is supported by the recent analysis of mice in which IL-10 expression was specifically ablated in Treg cells33. Whereas Treg-cell-derived IL-10 was not required for the systemic control of autoimmunity, it did seem to be required from the control of inflammatory events at mucosal interfaces such as the lungs and colon. As a clear picture of the available Treg-cell weaponry emerges, an important challenge will be to determine their relative importance and contribution to Treg-cell function in different disease models.

A hypothesis: effector T cells potentiate Treg-cell function? Most cellular interactions within the immune system are bidirectional, with molecular signals moving in both directions even though the interaction has broader unidirectional intentions (for example, CD4+ T-cell help). However, to date the general perception is that Treg cells suppress and effector T cells capitulate. We hypothesize that this is in fact an incomplete picture and that effector T cells have a very active role in their own functional demise. Three recent observations support this view. First, we have recently examined the molecular signature of activated Treg cells in the presence and absence of effector T cells and were surprised to find that it was strikingly different, with hundreds of genes differentially modulated as a consequence of the presence of effector T cells (C.J.W. and D.A.A.V., unpublished observations). Second, we have shown that Ebi3 and Il12a mRNA are markedly upregulated in Treg cells that were co-cultured with effector T cells, supporting the idea that effector T cells may provide signals which boost IL-35 production in trans58. Third, we found that Treg cells were able to mediate suppression of effector T cells across a permeable membrane when placed in direct contact with effector T cells in the upper chamber of a Transwell™ plate (L.W.C. and D.A.A.V., unpublished observations). Interestingly, this suppression was IL-35 dependent, as Ebi3−/− Treg cells were unable to mediate this ‘long-distance’ suppression. Collectively, these data suggest that it is the ‘induction’, rather than the ‘function’, of Treg-cell suppression that is contact-dependent and that effector T cells have an active role in potentiating Treg-cellmediated suppression. Therefore, we hypothesize that receptor–ligand interactions between the co-cultured CD4+ effector T cells and Treg cells initiate a signalling pathway that leads to enhanced IL-35 secretion and regulatory activity (FIG. 2). While the molecule that mediates this enhanced Treg-cell suppression is unknown, it is possible that IL-2 may serve this function104. Given the contrasting genetic profiles of activated Treg cells in the presence and absence of effector T cells, it seems possible that this interaction may boost the expression of other regulatory proteins. It may well be that effector T cells unwittingly perform the ultimate act of altruism.

Concluding remarks Although significant progress has been made over the last few years in defining the mechanisms that Treg cells use to mediate their suppressive function, there is clearly much that remains to be elucidated and many questions persist. First, are there more undiscovered mechanisms and/ or molecules that mediate Treg-cell suppression? What is clear is that the transcriptional landscape of Treg cells is very different from naive or activated effector T cells. There are literally thousands of genes that are upregulated (or downregulated) in Treg cells compared with effector T cells. Although it seems unlikely that all or many of these will be crucial for Treg-cell function, it is quite possible that a few undiscovered genes might be important. It should be noted that although we are discussing mechanisms here, it is clear that some of these molecules may perform key Treg-cell functions, such as Treg-cell homing and homeostasis, which are likely to indirectly influence their suppressive capacity in vivo but don’t directly contribute to their inhibitory activity. It is also possible that some of these unknown molecules may represent more specific markers for the characterization and isolation of Treg cells, a particularly important issue for the analysis and use of human Treg cells (BOX 2).

Second, which mechanisms are most important? An important but potentially complex challenge will be to determine if a few mechanisms are important in many Treg-cell settings or whether different mechanisms are required in different cellular scenarios. At present it is difficult to assess this objectively as these mechanisms have predominantly been elucidated in different labs using distinct experimental systems and thus none have really been compared in side-by-side experiments. Furthermore, only recently have conditional mutant mice been examined that have a regulatory component specifically deleted in Treg cells33.

It almost goes without saying that although defining the Treg-cell mode of action is of great academic importance, it is also essential in order to develop effective approaches for the clinical manipulation of Treg cells. Given the capacity of Treg cells to control inflammation and autoimmunity, and their implication in blocking effective anti-tumour immunity and preventing sterilizing immunity, it seems probable that a clear understanding of how Treg cells work will present definitive opportunities for therapeutic intervention.

Box 1 Scurfy mice: misplaced mechanistic expectations?

Mice that carry a spontaneous loss-of-function mutation (known as Scurfy mice) or a deletion of Foxp3 develop a fatal autoimmune-like disease with hyperresponsive CD4+ T cells9,12. More recently Foxp3:diptheria toxin receptor (DTR) knockin mice have allowed for the selective depletion of Treg cells following DT treatment105. These mice have been invaluable for dissecting the role of Foxp3 in Treg-cell function. Given the profound phenotype in these mice, there is a general expectation that genetic disruption of any key Treg-cell inhibitory molecule or mechanism would probably result in a Scurfy-like phenotype. Of course, it is also possible that deletion of a key Treg-cell gene may be more synonymous with DT-mediated Treg-cell depletion where Foxp3 may still serve to prevent expression of proinflammatory cytokines105. Nonetheless, this has lead to the notion that if mutant mice don’t have a Scurfy-like or a Treg-cell-depleted phenotype, then the disrupted gene probably isn’t important for Treg-cell function. This may not necessarily be correct. Indeed, it is possible that no mouse lacking a Treg-cell inhibitory effector molecule will ever be generated that develops a profound, spontaneous autoimmune disease21. It should be noted that mutant mice that are Helicobacter spp. and/or Citrobacter rodentium positive may have an exacerbated phenotype, as several studies have shown that opportunistic enteric bacteria can significant exacerbate gut pathology4. Ultimately, the occurrence of disease in knockout mice will depend on whether Treg cells rely on a single or multiple suppressive mechanisms. Given the number of genes induced or modulated by FOXP3, it is probable that a programme of intrinsic and extrinsic regulation is induced that involves multiple proteins9,13. Therefore, it would not be surprising if deletion of a single molecule does not provoke the profound Scurfy-like phenotype seen in mice that lack Foxp3.

Box 2. Treg-cell markers

Identifying discriminatory cell surface markers for the characterization and isolation of Treg cells has always been a critical goal. Although excellent markers exist for murine Treg cells, this goal has remained elusive for human Treg cells. Traditionally, murine and human Treg cells have been characterized as CD4+CD25+ (also known as interleukin-2 receptor α (IL-2Rα)). Indeed, murine Treg cells can be effectively isolated based on staining for CD4+CD25+CD45RBlow expression. However, the purity of isolated human Treg cells has always been an issue because T cells up-regulate CD25 upon activation106. Indeed, during the influenza or allergy season a substantial proportion of human CD4+ T cells can express CD25. Although the identification of forkhead box P3 (Foxp3) as a key regulator of Treg-cell development and function has facilitated their identification in the mouse8, many activated (non-regulatory) human T cells express FOXP3, precluding it as a useful marker for human Treg cells16-20. Consequently, the search for Treg-cell-specific cellsurface markers, particularly in humans, has continued in earnest with a growing number of candidates proposed (reviewed by Zhao and colleagues107). For instance, it was shown that the expression of CD127 (also known as IL-7R) is down-regulated on Treg cells and that this could be used to increase the purity of human Treg-cell isolation. Indeed, there is a 90% correlation between CD4+CD25+CD127low T cells and FOXP3 expression108, 109. In addition, it was recently found that Treg cells expressed a higher level of folate receptor 4 (FR4) compared with activated effector T cells110. It is also important to recognize that Treg cells, like their T helper cell counterparts, may be heterogeneous and thus a collection of cell surface markers could facilitate their isolation and functional characterization. Indeed, such heterogeneity has recently been described based on differential expression of HLA-DR or CCR6102,103. However, the general use of both markers remains to be fully established so it is quite probable that the search for better Treg-cell markers will continue for some time.

Box 3 Induced or adaptive Treg cells: development and mode of action

Naturally occurring FOXP3+CD4+CD25+ Treg cells develop in the thymus and display a diverse T-cell receptor (TCR) repertoire that is specific for self-antigens111,112. However, Treg cells can also be ‘induced’, ‘adapted’ or ‘converted’ from effector T cells during inflammatory processes in peripheral tissues, or experimentally generated as a possible therapeutic29,113,114. For instance, T regulatory 1 cells (Tr1) and T helper 3 cells (Th3) can be generated experimentally by, and mediate their suppressive activity through interleukin-10 (IL-10) and transforming growth factor-β (TGFβ), respectively114,115. Typically, these regulatory populations do not express FOXP3. In vivo, it has recently been suggested that stimulation of mouse effector T cells by CD103+ dendritic cells (DCs) in the presence of TGFβ and retinoic acid induces the generation of Foxp3+ T cells in the gutassociated lymphoid tissue (GALT)116-121. Furthermore, Treg cells can be preferentially induced in the periphery by exposure to αVβ8-integrin-expressing DCs122 or suppressor of cytokine signalling 3 (Socs3) −/− DCs123. Interestingly, independent of its role in generating induced Treg cells, TGFβ may also have an important role in helping to maintain Foxp3 expression in natural Treg cells124, a process that can be blocked by IL-4 or interferon-γ (IFNγ) 125. In contrast to mouse T cells, FOXP3 induction by TCR stimulation in the presence of TGFβ in human T cells does not confer a regulatory phenotype20. The mechanism of action of adaptive Treg cells may not necessarily be restricted to suppressive cytokines. Indeed, human adaptive Treg cells (CD4+CD45RA+ T cells stimulated with CD3- and CD46-specific antibodies) have also been shown to express granzyme B and killing target cells in a perforin-dependent manner126. Treg cells often have a restricted specificity for particular cell types, tumours or foreign antigens127. Therefore, induced Treg cells may be ideally suited to respond to infectious agents. This may also be of particular importance in the GALT and in the tumour microenvironment where TGFβ drives the conversion of induced Treg cells118,128. A significant challenge in deciphering data from in vivo experiments is to assess the contribution of natural Treg cells versus induced Treg cells, and to determine whether inhibitory molecules, such as IL-10 or TGFβ, were derived from the former or the latter (or elsewhere).

 

 

Aberrant PD-L1 expression through 3′-UTR disruption in multiple cancers.

Keisuke Kataoka, Yuichi Shiraishi, Yohei Takeda, Seiji Sakata, et al.
Nature may 23,2016; http://dx.doi.org:/10.1038/nature18294

Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development1, 2, 3, 4, 5, 6. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma6, 7, 8, 9, 10. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3′ region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3′-untranslated region (UTR). Disruption of the Pd-l1 3′-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3′-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

 

Viruses are a dominant driver of protein adaptation in mammals.

David Enard, Le Cai, Carina Gwennap and Dmitri A Petrov.
eLife May 16, 2016; 5:e12469. http://dx.doi.org/10.7554/eLife.12469

Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.

 

Purdue scientists use adaptors to advance CAR-T therapy

by Oliver Worsley | May 25, 2016

http://www.fiercebiotech.com/research/purdue-scientists-use-adaptors-to-advance-car-t-therapy

Chimeric antigen receptor (CAR) T cells, developed in the 1990s, are a genetically engineered type of T cell that can target a specific cancer. Now, scientists at Purdue University say they’ve made improvements in this strategy–overcoming the several limitations of traditional CAR-T therapy.

Purdue professor of chemistry Philip Low and his team presented their findings at the American Association for Cancer Research meeting in New Orleans last month.

T cells are a type of immune cell that recognizes and clears the body of invading cells or pathogens, like cancer. They are fine-tuned by the immune system in order to specifically target and kill these foreign invaders–but cancer cells may respond by jumping these safety barriers.

CAR-T therapy was therefore proposed and has been recently used for cancer treatment. It has been hailed for its promising remission rates after early stage clinical trials for acute lymphoblastic leukemia.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said in a university news release. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

They did this by teaming up with Endocyte ($ECYT) scientist Haiyan Chu and designing CAR T cells that require activation by a small molecule adaptor before proceeding. In this way, they can carefully control the amount of active CAR T cells in the circulation.

So far, they have only tried the novel therapy in animal models, but when they tested it in mice they observed antitumor activity only when both the CAR T cells and the correct adaptor molecules were present.

Low believes it will allow clinicians to target multiple cancer subtypes at once. “Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said in the release. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

– here’s the release

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Read More: CAR-T   Cancer

 

Purdue research may expand engineered T-cell cancer treatment

PURDUE UNIVERSITY

http://www.eurekalert.org/multimedia/pub/116141.php

A graphic depicting the activation and inactivation of CAR T cells through a small molecule adaptor is shown. Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery, and graduate student Yong Gu Lee led a team that designed new engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The system has the potential to control the engineered cells to overcome existing limitations in CAR T-cell therapy. CREDIT Purdue University image courtesy of Yong Gu Lee

Purdue University researchers may have figured out a way to call off a cancer cell assassin that sometimes goes rogue and assign it a larger tumor-specific “hit list.”

T cells are the immune system’s natural defense against cancer and other harmful entities in the human body. However, the cells must be activated and taught by the immune system to recognize cancer cells in order to seek out and destroy them. Unfortunately, many types of cancer manage to thwart this process.

 

In the 1990s scientists found a way to genetically engineer T cells to recognize a specific cancer. These engineered T cells, called chimeric antigen receptor, or CAR, T cells, have been recently used as treatment for cancer, said Philip S. Low, Purdue’s Ralph C. Corley Distinguished Professor of Chemistry and director of the Purdue Center for Drug Discovery who led the work.

“The problem is that the traditional engineered T-cell treatment can be too effective, sometimes killing tumor cells too fast and triggering a toxic reaction in a patient, and sometimes not stopping once the tumor has been destroyed and continuing to seek out and destroy healthy cells important to bodily functions,” Low said. “We have found a potential way to control the engineered immune cells to overcome the limitations posed by CAR T-cell therapy.”

Low and Purdue graduate student Yong Gu Lee collaborated with Endocyte Inc. scientist Haiyan Chu to design genetically engineered CAR T cells that must be activated and targeted by a small molecule adaptor before they can kill cancer cells. The technology has been tested in animal models but no human trials have been performed. A poster presentation describing the work was presented Tuesday (April 19, 2016) at the American Association for Cancer Research annual meeting in New Orleans.

“While the traditional CAR T cells could remain and replicate in the human body for many years, the adaptors we have created are expected to be excreted fairly quickly,” Lee said. “By controlling the level of adaptors in the system, we can control the numbers and potencies of active CAR T cells. Those that aren’t stimulated by an adaptor molecule are blind and do not recognize or target any cells. Eventually, if they remain inactive for a while, they should die and be eliminated from the body.”

A study in mice showed the anti-tumor activity was induced only when both the engineered CAR T cell and the correct adaptor molecules were present.

The system also offers the potential to treat multiple cancer subtypes at once, Low said.

“Most tumors are heterogeneous and contain cancer cells that express different characteristics, including having different tumor-specific proteins on their surface,” he said. “The cancer-targeting molecule on the adaptor we designed can be swapped out to target different molecules on other unrelated cancer cell surfaces. The idea is that a mixture of these adaptors can be given to a patient so that a single CAR T cell clone can be targeted to all of the relevant cancer subtypes in a patient.”

The adaptor molecule serves as a bridge between the CAR T-cell and the cancer cell. It is made with a yellow dye called fluorescein isothiocyanate on one end, to which the engineered CAR T cells have been designed to bind, and a cancer-targeting molecule on the other.

Low’s research has focused on the design and synthesis of technologies for targeted delivery of therapeutic and imaging agents to treat cancer, inflammatory and autoimmune diseases, and infectious diseases.

He has developed molecules that target folate-receptors and prostate-specific membrane antigen on the surfaces of cancer cells. Approximately 85 percent of ovarian cancers; 80 percent of endometrial and lung cancers; and 50 percent of breast, kidney and colon cancers express folate receptors on their cellular surfaces. Prostate-specific membrane antigen receptors are found on nearly 90 percent of all prostate cancers. Other tumor-specific ligands developed by Low’s lab can target each of the other major human cancers, he said.

Each CAR T cell has thousands of receptors on its surface to which an adaptor molecule can bind. One CAR T cell could have a variety of adaptor molecules bound to its surface and the cancer cell it targets will depend on which of those adaptors first encounters a targeted cancer cell. Once the CAR T cell binds to a cancer cell, it begins the process of destroying it. When that process is complete, the CAR T cell is released and can bind to a new cancer cell, he said.

“In the past a new CAR T cell had to be designed for each desired cancer target,” Low said. “This system uses the same blind CAR T cell for all treatments. The adaptor molecule is what needs to be changed, and it is far easier to manipulate and swap pieces in and out of it than the T cells.”

In addition to Low, Chu and Lee, members of the research group include Purdue postdoctoral research associates at the time of the study Srinivasarao Tenneti and Ananda Kumar Kanduluru.

Drug discovery is one of the priorities within Purdue Moves, an initiative designed to broaden the university’s global impact and enhance educational opportunities for its students. All of the moves fall into four broad categories: science, technology, engineering and math (STEM) leadership; world-changing research; transformative education; and affordability and accessibility.

The Purdue University Center for Drug Discovery supports more than 100 faculty in six colleges with research focused on several major disease categories: cancer; diabetes, obesity and cardiovascular; immune and infectious disease; and neurological disorders and trauma.

The center and drug discovery initiative builds upon Purdue’s strengths along all points of the drug discovery pipeline, including 14 core units to provide shared resources for analysis, screening, synthesis and testing of potential therapeutic compounds.

With more than 44 Purdue-developed compounds at various stages of preclinical development, and 16 in human clinical trials, Purdue is among the most productive universities in the world of drug discovery.

The center also is aligned with the university’s recently announced $250 million investment in the life sciences.

Endocyte Inc., a Purdue Research Park-based company that develops receptor-targeted therapeutics for the treatment of cancer and autoimmune diseases, funded the study, holds exclusive rights to the technology and assisted Purdue researchers in the development of the technology. Low is a founder and chief science officer of Endocyte Inc. and serves on the Endocyte board of directors.

AACR press release: http://www.aacr.org/Newsroom/Pages/News-Release-Detail.aspx?ItemID=874#.VxZFs2N8V0c

Endocyte press release: http://investor.endocyte.com/releasedetail.cfm?ReleaseID=965753

ABSTRACT

A Universal Remedy for CAR T Cell Limitations

Yong Gu Lee, Haiyan Chu, Srinivasarao Tenneti, Ananda Kumar Kanduluru, Philip S. Low

Chimeric antigen receptor (CAR) T cells show significant potential for treating cancer due to their tumor-specific activation and ability to focus their killing activity on cells that express a tumor antigen. Unfortunately, this promising therapeutic technology is still limited by: (1) an inability to control the rate of cytokine release and tumor lysis; (2) the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is complete; (3) a failure to eliminate tumor cells that do not express the targeted antigen; and (4) a requirement to generate a different CAR T cell for each unique tumor antigen. In order to address these limitations, we have exploited a low molecular weight bi-specific adaptor molecule that must bridge between the CAR T cell and its targeted tumor cell by simultaneously binding to the chimeric antigen receptor on the CAR T cell and the unique antigen on the tumor. Using this bispecific adaptor, one can control CAR T cell cytotoxicity by adjusting the concentration and rate of administration of the adaptor. Because the half life of the adapter is <20 minutes in vivo, termination of CAR T cell killing can be accomplished by cessation of adapter administration. Moreover, when heterogeneous tumors containing cells that express orthogonal antigens must be treated, the same CAR T cell can be targeted to multiple antigens by attachment of the same CAR ligand to the appropriate selection of tumor-specific ligands. Finally, when the targeted tumor antigen is also expressed at low levels on normal cells, tumor specificity can be achieved by adjusting the affinity of the tumor-specific ligand to enable CAR T cell engagement only when a highly multivalent interaction is possible. To experimentally demonstrate the aforementioned benefits of using low molecular weight bispecific adaptors, CAR T cells were constructed by fusing an anti-fluorescein isothiocyanate (FITC) scFv to a CD3 zeta chain containing the intracellular domain of CD137 (i.e. CAR4-1BBZ T cells). Then, to enable their tumor-specific cytotoxicity, a bispecific adaptor molecule comprised of fluorescein linked to a small organic ligand with high affinity and specificity for a tumor-specific antigen (FITC-SMC) was synthesized. For these studies, the tumor-specific ligands were: i) folate for recognition of the folate receptor that is over-expressed on ~1/3 of human cancers, ii) DUPA for binding to prostate specific membrane antigen that is over-expressed on prostate cancers, and iii) NK-1R ligand that is over-expressed on neuroendocrine tumors. The ability of the same clone of CAR4-1BBZ T cells to eliminate tumors expressing each of the above antigens was then demonstrated by administration of the desired FITC-SMC to mice injected with the CAR4-1BBZ T cells. Our data show that anti-tumor activity: i) is only induced when both CAR4-1BBZ T cells and the correct antigen-specific FITC-SMC are present, ii) anti-tumor activity and toxicity can be sensitively controlled by adjusting the dosing of FITC-SMC, and iii) treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired FITC-SMCs. Taken together, these data show that many of the limitations of CAR T cell technology can be addressed by use of a bi-specific adaptor molecule to mediate tumor cell recognition and killing.

 

 

CTLA-4 found in dendritic cells suggests New cancer treatment possibilities

Matthew Halpert, et al. Dendritic Cell Secreted CTLA-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Stem Cells and Development, 2016; http://dx.doi.org:/10.1089/scd.2016.0009

Both dendritic cells and T cells are important in triggering the immune response, whereas antigen presenting dendritic cells act as the “general” leading T cells “soldiers” to chase and eliminate enemies in the battle against cancer. The well-known immune checkpoint break, CTLA-4, is believed to be present only in T cells (and cells of the same lineage). However, a new study published in Stem Cells and Development suggests that CTLA-4 also presents in dendritic cells. It further explores the mechanism on how turning off the dendritic cells in the immune response against tumors.

  Dendritic Cell-Secreted Cytotoxic T-Lymphocyte-Associated Protein-4 Regulates the T-cell Response by Downmodulating Bystander Surface B7.
Halpert MM1, Konduri V1, Liang D1, Chen Y1, Wing JB2, Paust S3,4, Levitt JM1,5, Decker WK1,6.  Stem Cells Dev. 2016 May 15;25(10):774-87. doi: 10.1089/scd.2016.0009. Epub 2016 May 2.

The remarkable functional plasticity of professional antigen-presenting cells (APCs) allows the adaptive immune system to respond specifically to an incredibly diverse array of potential pathogenic insults; nonetheless, the specific molecular effectors and mechanisms that underpin this plasticity remain poorly characterized. Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), the target of the blockbuster cancer immunotherapeutic ipilimumab, is one of the most well-known and well-studied members of the B7 superfamily and negatively regulates T cell responses by a variety of known mechanisms. Although CTLA-4 is thought to be expressed almost exclusively among lymphoid lineage hematopoietic cells, a few reports have indicated that nonlymphoid APCs can also express the CTLA-4 mRNA transcript and that transcript levels can be regulated by external stimuli. In this study, we substantially build upon these critical observations, definitively demonstrating that mature myeloid lineage dendritic cells (DC) express significant levels of intracellular CTLA-4 that they constitutively secrete in microvesicular structures. CTLA-4(+) microvesicles can competitively bind B7 costimulatory molecules on bystander DC, resulting in downregulation of B7 surface expression with significant functional consequences for downstream CD8(+) T-cell responses. Hence, the data indicate a previously unknown role for DC-derived CTLA-4 in immune cell functional plasticity and have significant implication for the design and implementation of immunomodulatory strategies intended to treat cancer and infectious disease.

 

Non-invasive strategy to guide personalized cancer immunotherapy

Cancer immunotherapy is the rising hope to offer ultimate solutions for cancer. Neoantigens, derived from products of mutated genes in tumor cells, are found to be closely related to the efficacy of cancer immunotherapies. A non-invasive approach to identify unique, patient-specific neoantigens has been advanced by Dr. Steven Rosenberg’s group. A recent article published in Nature Medicine reported that a small population of circulating CD8+PD-1+ tumor-reactive T lymphocytes can be used to identify neoantigens, in addition to tumor-infiltrating T cells. The study paves the way for designing personalized cancer immunotherapy with a novel non-invasive approach.

Gros, A. et al.
Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients.
Nat. Med. (2016)   http://dx. doi.org:/10.1038/nm.4501

Detection of lymphocytes that target tumor-specific mutant neoantigens-derived from products encoded by mutated genes in the tumor-is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8+ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8+ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8+lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8+ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8+ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8+PD-1+ compared with CD8+PD-1 TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8+ and the CD8+PD-1+ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8+ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Cancer immunotherapy has experienced major progress in the last decade. Adoptive transfer of ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can cause substantial regression of metastatic melanoma (1, 2). Blockade of the interaction of cytotoxic T lymphocyte antigen 4 (CTLA-4; also known as CD152) or programmed cell death 1 receptor (PD-1; also known as CD279) with their ligands using blocking antibodies alone or in combination have been shown to unleash an otherwise-ineffective immune response against melanoma (37), renal cell carcinoma (3), and non–small cell lung cancer (3). The antitumor responses observed in these clinical trials support the presence of naturally occurring tumor-reactive CD8+ T cells and their immunotherapeutic potential. In the particular case of TIL therapy, persistence of transferred tumor-specific T cell clones is associated with tumor regression (8). Moreover, retrospective clinical studies have shown an association of autologous tumor recognition by TILs and clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. However, the identification of the diverse repertoire of tumor-reactive cells limits the ability to study these cells, enhance clinical efficacy, and extend this therapy to other malignancies.

Melanoma TILs represent a heterogeneous population that can target a variety of antigens, including melanocyte differentiation antigens, cancer germline antigens, self-antigens overexpressed by the tumor, and mutated tumor neoantigens (11). The latter appear to be of critical importance for the antitumor responses observed after transfer of TILs, given the substantial regression of metastatic melanoma in up to 72% of patients in phase 2 clinical trials, in the absence of any autoimmune side effects in the great majority of patients (2). This contrasts with the modest antitumor activity but high prevalence of severe autoimmune manifestations observed after transfer of peripheral blood gene-engineered T cells expressing TCRs targeting shared melanocyte differentiation antigens MART1 and gp100 (12,13). Furthermore, T cells targeting mutated neoepitopes are not subject to negative selection in the thymus and may constitute the predominant naturally occurring tumor-reactive population in cancer patients. In support of this notion, a recent study reported the frequent detection and dominance of T cell populations targeting mutated epitopes in melanoma-derived TILs (14). Conversely, T cells targeting shared melanocyte differentiation antigens and cancer germline antigens in bulk melanoma TILs were represented at a strikingly low frequency (15). These findings have shifted our interest from the more accessible and commonly studied T cells targeting melanocyte differentiation antigens to T cells targeting unique patient-specific mutations. However, the often rare availability of autologous tumor cell lines necessary to study these reactivities, and the hurdles associated with the identification of the unique mutations targeted, have thus far hindered immunobiological studies of these T cell populations in the tumor.

Naturally occurring tumor-reactive cells are exposed to their antigen at the tumor site. Thus, the immunobiological characterization of T cells infiltrating tumors represents a unique opportunity to study their function and to identify the patient-specific repertoire of tumor-reactive cells. TCR stimulation triggers simultaneous upregulation of both costimulatory and coinhibitory receptors, which can either promote or inhibit T cell activation and function. Expression of the inhibitory receptors PD-1, CTLA-4, lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) is regulated in response to activation and throughout differentiation (16, 17). Chronic antigen stimulation has been shown to induce coexpression of inhibitory receptors and is associated with T cell hyporesponsiveness, termed exhaustion (18). Exhaustion in response to persistent exposure to antigen was first delineated in a murine model of chronic lymphocytic choriomeningitis virus (19), but has been observed in multiple human chronic viral infections (2022) as well as in tumor-reactive MART1-specific TILs (23, 24), and has provided the rationale for restoring immune function using immune checkpoint blockade. Conversely, 4-1BB (also known as CD137) is a costimulatory member of the TNF receptor family that has emerged as an important mediator of survival and proliferation, particularly in CD8+ T cells (2527). 4-1BB is transiently expressed upon TCR stimulation, and its expression has been used to enrich for antigen-specific T cells in response to acute antigen stimulation (28). However, expression of this marker has not been extensively explored in CD8+ lymphocytes infiltrating human tumors. In addition to changes in the expression of cosignaling receptors on the surface of T cells, antigen-specific stimulation typically results in clonal expansion. TCR sequence immunoprofiling can be used to monitor T cell responses to a given immune challenge even without a priori knowledge of the specific epitope targeted, through determination of the abundance of specific clonotypes (29, 30). However, there is limited knowledge regarding the TCR repertoire and the frequency of tumor-reactive clonotypes infiltrating human tumors.

We hypothesized that the assessment of unique phenotypic traits expressed by CD8+ TILs and TCR β chain (TCRβ; encoded by TRB) clonotypic immunoprofiling of lymphocytes infiltrating the tumor could provide a powerful platform to study antitumor T cell responses and evaluated their usefulness in identifying the diverse repertoire of tumor-reactive cells. Despite the accepted negative regulatory role of PD-1 in T cells, our findings establish that expression of PD-1 on CD8+ melanoma TILs accurately identifies the repertoire of clonally expanded tumor-reactive, mutation-specific lymphocytes and suggest that cells derived from this population play a critical role in tumor regression after TIL administration.

PD-1 was initially described to be expressed on a T cell hybridoma undergoing cell death (37). Its negative effect on T cell responses was first delineated in PD-1 knockout mice (38, 39). Since then, PD-1 expression and coexpression of other inhibitory receptors such as CTLA-4, TIM-3, BTLA, CD160, LAG-3, and 2B4 have become a hallmark of chronically stimulated T cells during chronic infection or in the tumor microenvironment. This altered phenotype, and the interaction of these receptors with their corresponding ligands on target cells, is associated with impaired proliferation and effector function frequently referred to as exhaustion (18, 24, 40). Expression of PD-1 in patients with chronic viral infections correlates with disease progression (22, 41). Additionally, CD8+ lymphocytes targeting melanoma differentiation antigens in the tumor express PD-1, CTLA-4, TIM-3, and LAG-3 and exhibit impaired IFN-γ and IL-2 secretion (23, 24), supporting a negative regulatory role of PD-1 and inhibitory receptors in naturally occurring T cell responses to cancer and providing a rationale for the treatment of cancer with immune checkpoint inhibitors.

In the present study, we found that expression of PD-1 on CD8+ melanoma TILs captured the diverse repertoire of clonally expanded tumor-reactive lymphocytes. TCRβ sequencing revealed that tumor-reactive and mutation-specific clonotypes were highly expanded in the CD8+ population and preferentially expanded in the PD-1+ population. This is consistent with the TCR stimulation-driven expression of this receptor on T cells (42). The inhibitory receptors TIM-3 and LAG-3 and the costimulatory receptor 4-1BB were also expressed on CD8+PD-1+ TILs and could also be used to enrich for tumor-reactive cells. PD-1 was consistently expressed at a higher frequency and was found to be more comprehensive at identifying the diverse repertoire of tumor-reactive cells infiltrating melanoma tumors, although the less frequent PD-1/TIM-3+ and PD-1/LAG-3+ subpopulations could also represent tumor-reactive cells (Supplemental Figure 4 and Supplemental Table 6). Additionally, previous studies from our laboratory showing coexpression of PD-1 and CTLA-4 (23), and our preliminary data supporting coexpression of PD-1 and ICOS (Supplemental Figure 5), suggest that other receptors may also be used to distinguish tumor-reactive cells. Our present results further support immunotherapeutic intervention using immune checkpoint blockade using PD-1, TIM-3, and LAG-3 blocking antibodies or 4-1BB agonistic antibody to restore the function of tumor-reactive lymphocytes, which is currently being actively pursued in the clinic (3, 4, 6, 7, 43). The potential cooperative mechanisms of inhibition of these receptors when engaged with their ligands (44, 45) suggests that the combined targeting of different inhibitory receptors can further enhance antitumor efficacy, as already shown with the combination of anti–PD-1 and anti–CTLA-4 (5). Our present results demonstrate that PD-1 identifies the clonally expanded CD8+ tumor-reactive population and suggest that expression of PD-1 on CD8+TILs could function as a potential predictive biomarker of antitumor efficacy using immune checkpoint inhibitors.

Naturally occurring tumor-reactive cells play a pivotal role in mediating antitumor responses after TIL transfer. Currently, expansion of TILs for patient treatment involves nonspecific growth of TILs from tumor fragments in IL-2, and the diversity and frequency of antitumor T cells present in the final T cell product used for treatment remains largely uncharacterized. Prospective clinical studies have reported that in vitro recognition of autologous tumor by TILs is associated with a higher probability of clinical response (9, 10), which suggests that enrichment of tumor-reactive cells could enhance clinical efficacy. This is consistent with the idea that both tumor-reactive and non–tumor-reactive cells may compete for cytokines in vivo, especially in the absence of vaccination. However, the isolation of the patient-specific repertoire of tumor-reactive cells is not possible with current technologies (14, 28, 4650). Our findings established that expression of PD-1, TIM-3, LAG-3, and 4-1BB in CD8+ TILs can be used to enrich for tumor-reactive cells, regardless of the specific antigen targeted. One potential concern with isolating T cells expressing inhibitory receptors for therapy is that these cells may be exhausted or functionally impaired (23, 24, 44, 51, 52). However, we found that PD-1+, TIM-3+, and LAG-3+ CD8+ cells expanded in IL-2 were capable of secreting IFN-γ and lyse tumor in vitro. This supports the notion that immune dysfunction associated with coexpression of inhibitory receptors on CD8+ TILs can be reversed (21, 41, 51, 53), and may enable the reproducible enrichment of tumor-reactive cells for patient treatment. Notably, in a preliminary experiment (n= 8 nonresponders; 14 responders), there was no association between the frequency of expression of any of the markers studied in the CD8+ TILs in the fresh tumor and the clinical response to TILs derived from these tumor samples. However, the fresh tumors included in this study belonged to patients treated in several TIL protocols over the course of 10 years, and TILs were generated from these tumors using different methods, which makes these data difficult to interpret. In addition, the frequency of cells initially expressing PD-1 in the tumor may not reflect the frequency of the PD-1 derived cells in the infusion bag. For example, a low frequency of PD-1+ cells may be highly enriched during the process of TIL culture as a result of the presence of tumor cells. Although in vivo antitumor activity of tumor-isolated TILs based on PD-1 expression requires testing in a clinical trial, the observation that the overwhelming majority of tumor-reactive cells were derived from cells expressing PD-1 suggests that cells expressing PD-1 and inhibitory receptors in the tumor play a critical role in tumor regression after TIL administration.

The functional implications of selecting PD-1–, LAG-3–, TIM-3–, or 4-1BB–expressing T cells to enrich for tumor-reactive cells for patient treatment remain unclear. Although previous studies have reported differential expression of PD-1, LAG-3, and TIM-3 throughout differentiation (17), or preferential expression of TIM-3 in IFN-γ–secreting cells (54), our preliminary results have failed to show consistent phenotypic or functional differences between PD-1+, LAG-3+, TIM-3+, and 4-1BB+ selected TILs, including cytokine secretion, proliferation, and susceptibility to apoptosis (data not shown). We found that PD-1 expression was almost completely lost in the PD-1+ derived populations upon in vitro culture in IL-2. Conversely, TIM-3 and LAG-3 expression increased in the TIM-3 and LAG-3 populations after expansion. Overall, there were no differences in the expression of PD-1, TIM-3, or LAG-3 between any the populations after expansion. Thus, in agreement with previous reports (55, 56), we conclude that expansion in IL-2 alters the expression of these markers and compromises the potential use of inhibitory receptors to select for tumor-reactive cells after in vitro expansion. Recent work in animal models suggests that chronic antigen stimulation (5759) or a tolerizing microenvironment (60) may lead to permanent epigenetic changes in T cells, raising the possibility that the restoration of function observed in previously exhausted or tolerized cells in presence of cytokines may only be transient. These results have not yet been corroborated in human tumor-specific cells. However, given that the overwhelming majority of tumor-reactive cells appear to derive from cells expressing PD-1 in the tumor, studying permanent versus transient reversion of exhaustion may have important implications for adoptive cell transfer of TILs.

Tumor-reactive cells can also be found infiltrating other tumor malignancies, such as renal cell carcinoma (61) or ovarian (62), cervical (63), or gastrointestinal tract cancers (64), albeit at lower frequencies. Our findings provide alternatives to enrich and study tumor-reactive CD8+ TILs through selection of cells expressing the cell surface receptors PD-1, LAG-3, TIM-3, and 4-1BB, a hypothesis that we are actively investigating. Additionally, our present findings showed that the frequency of a specific clonotype in the CD8+ and PD-1+ populations can be used to predict its ability to recognize tumor and isolate tumor-specific TCRs, thus providing means to overcome potential irreversible functional impairments of TILs (52).

2 reports with opposing results have generated controversy regarding which may be the optimal marker for the identification of the tumor-reactive repertoire, PD-1 or 4-1BB. In one report studying PD-1 expression in the tumor, the authors showed promising although inconsistent ability to enrich for shared melanoma-reactive cells (55). In a more recent article studying the role of 4-1BB in fresh ovarian TILs, Ye et al. concluded that expression of 4-1BB, but not PD-1, on lymphocytes defines the population of tumor-reactive cells in the tumor (65). The results of Ye et al. appear to contradict our present findings, showing that expression of PD-1 rather than 4-1BB more comprehensively identifies the repertoire of tumor-reactive cells in the tumor. However, these inconsistencies can be explained by different experimental approaches undertaken to study the immunobiology of TILs. First, Ye et al. found that expression of 4-1BB in fresh ovarian TILs and tumor-associated lymphocytes was low, and thus exposed the tumor to IL-7 and IL-15 (65). In the 1 patient sample in which the authors enriched for tumor-reactive cells from fresh ovarian TILs or tumor-associated lymphocytes exposed to IL-7 and IL-15, expression of 4-1BB was dependent on in vitro activation, but no longer represented the natural expression of 4-1BB in the fresh tumor. Second, with the exception of the 1 experiment described above, the enrichment experiments reported were carried out with melanoma or ovarian TIL lines expanded in IL-2 and cocultured with tumor cell lines in vitro. It is well known that IL-2 can change the activation status and also the expression of inhibitory receptors on T cells (data not shown and ref. 56). Thus, the experiment comparing expression of PD-1 and 4-1BB performed by Ye et al. (65) addressed the significance of these receptors after in vitro coculture of a highly activated melanoma TIL line with a tumor cell line, rather than the role of PD-1 and 4-1BB expression in CD8+ lymphocytes in the fresh tumor. Finally, both Inozume et al. and Ye et al. used matched HLA-A2 cell lines to assess tumor reactivity (55, 65). However, the use of HLA-matched tumor cell lines does not enable the assessment of reactivities against unique mutations that are present only in the autologous tumor cell line. In our current study, we used fresh melanoma tumors for all our experiments, and these were rested in the absence of cytokines to preserve the phenotype of TILs. Moreover, we used autologous tumor cell lines to assess tumor recognition. We believe that our experimental approach overcomes the limitations described above, enabling us to conclude that tumor-reactive cells can be detected in both the PD-1+/4-1BB+ and PD-1+/4-1BB CD8+ TIL populations.

In summary, expression of PD-1 in CD8+ TILs in the fresh tumor identified and selected for the diverse patient-specific repertoire of tumor-reactive cells, including mutation-specific cells. In addition, analysis of the CD8+ TIL TCRβ repertoire in 2 melanomas showed that the frequency of a specific TCRβ clonotype in the CD8+ and PD-1+ populations could be used to predict its ability to recognize the autologous tumor. The use of inhibitory receptors and the frequency of individual TCRs to prospectively identify and select the diverse repertoire of tumor-reactive cells holds promise for the personalized treatment of cancer with T cell therapies, but may also facilitate the dissection and understanding of the immune response in human cancer patients.

Anti-PD-1 is poised to be a blockbuster, which other immune-checkpoint targeting drugs are on the horizon?

Clinical studies of anti-immune-checkpoint protein therapeutics have shown not only an improved overall survival, but also a long-term durable response, compared to chemotherapy and genomically-targeted therapy. To expand the success of immune-checkpoint therapeutics into more tumor types and improving efficacy in difficult-to-treat tumors, additional targets involved in checkpoint-blockade need to be explored, as well as testing the synergy between combining approaches.

Currently, CTLA-4 and PD-1/PD-L1 are furthest along in development, and have shown very promising results in metastatic melanoma patients. This is just a fraction of targets involved in the checkpoint-blockade pathway. Several notable targets include:

  • LAG-3 – Furthest along in clinical development with both a fusion protein and antibody approach, antibody apporach being tested in combination with anti-PD-1
  • TIM-3 – Also in clinical development. Pre-clinical studies indicate that it co-expresses with PD-1 on tumor-infiltrating lymphocytes. Combination with anti-PD-improves anti-tumor response
  • VISTA – Antibody targeting VISTA was shown to improve anti-tumor immune response in mice

In addition, there are also co-stimulatory factors that are also being explored as viable therapeutic targets

  • OX40 – Both OX40 and 4-1BB are part of the TNF-receptor superfamily. Phase I data shows acceptable safety profile, and evidence of anti-tumor response in some patients
  • 4-1BB – Phase I/II data on an antibody therapeutic targeting OX40 shows promising clinical response for melanoma, renal cell carcinoma and ovarian cancer.
  • Inducible co-stimulator (ICOS) – Member of the CD28/B7 family. Its expression was found to increase upon T-cell activation. Anti-CTLA-4 therapy increases ICOS-positive effector T-cells, indicating that it may work in synergy with anti-CTLA-4. Clinical trials of anti-ICOS antibody are planned for 2015.

Sharma P and Allison JP.
Immune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative Potential.
Cell. April 2015;161:205-214

 

Targeting single immune-checkpoint proteins has proven to be clinically effective at treating specific tumor types; can targeting two different proteins synergize effects?

Despite the success of targeting immune-checkpoint proteins, such as CTLA-4, PD-1, LAG-3, TIM-3 among others, percentages of patient response vary and rarely exceed 50%. It is highly tempting to speculate a strategy of dual-targeting of these checkpoint proteins. A recent presentation at the Keystone Symposium for Tumor Immunology: Multidisciplinary Science Driving Combination Therapy detailed findings of dual-targeting two immune-checkpoint proteins in mouse tumor models. Their key findings are summarized below:

  • Dual-targeting PD-1 and LAG-3 demonstrates superior efficacy over blocking either target alone
  • In addition to previous reported data on superior dual-targeting efficacy against fibrosarcoma (Sa1N) and colorectal adenosarcoma (MC38) tumor types1, anti-tumor activity against myeloma (SC J558L) and B-cell lymphoma (A20) hematological tumor types were also reported to be effacious.2

These exciting pre-clinical findings may result in further exploration of dual-targeting antibodies in the clinic, either as combination of existing antibody therapies, or as a new bi-specific antibody therapeutic.

Camelid single domain antibodies are a novel bi-specific antibody platform that may be used to develop a new generation of dual-targeting antibodies against multiple immune-checkpoint proteins.

1Woo SR et al.
Immune Inhibitory Molecules Lag-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape.
Cancer Res. Feb 2012. 15(4):917-927.

2Lewis KE et al.
Dual Targeting of PD-1 and LAG-3 demonstrates Superior Efficacy to Blocking Either PD-1 or LAG-3 Alone in Pre-Clinical Solid and Hematological Tumor Models.
Abstract J7 2033. Keystone Symposia: Tumor Immunology: Multidisciplinary Science Driving Combination Therapy. February 8-13, 2015. Banff, Alberta, Canada.

 

New insight behind the success of fighting cancer by targeting immune checkpoint proteins

Immune checkpoint blockade has proven to be highly successful in the clinic at treating aggressive and difficult-to-treat forms of cancer. The mechanism of the blockade, targeting CTLA-4 and PD-1 receptors which act as on/off switches in T cell-mediated tumor rejection, is well understood. However, little is known about the tumor antigen recognition profile of these affected T-cells, once the checkpoint blockade is initiated.

In a recent published study, the authors used genomics and bioinformatics approaches to identify critical epitopes on 3-methylcholanthrene induced sarcoma cell lines, d42m1-T3 and F244. CD8+ T cells in anti-PD-1 treated tumor bearing mice were isolated and fluorescently labeled with tetramers loaded with predicted mutant epitopes. Out of 66 predicted mutants, mLama4 and mAlg8 were among the highest in tetramer-positive infiltrating T-cells. To determine whether targeting these epitopes alone would yield similar results as anti-PD-1 treatment, vaccines against these two epitopes were developed and tested in mice. Prophylactic administration of the combined vaccine against mLama4 and mAlg8 yielded an 88% survival in tumor bearing mice, thus demonstrating that these two epitopes are the major antigenic targets from checkpoint-blockade and therapies against these two targets are similarly efficacious.

In addition to understanding the mechanism, identification of these tumor-specific mutant antigens is the first step in discovering the next wave of cancer immunotherapies via vaccines or antibody therapeutics. Choosing the right antibody platform can speed the discovery of a new therapeutics against these new targets. Single domain antibodies have the advantage of expedited optimization, flexibility of incorporating multiple specificity and functions, superior stability, and low COG over standard antibody approaches.

Gubin MM. et al.
Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.
Nature. Nov 2014. 515:577-584

 

 

Myeloid-derived-suppressor cells as regulators of the immune system
Dmitry I. Gabrilovich and Srinivas Nagaraj  Nat Rev Immunol. 2009 March ; 9(3): 162–174. http://dx.doi.org:/10.1038/nri2506

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expands during cancer, inflammation and infection, and that has a remarkable ability to suppress T-cell responses. These cells constitute a unique component of the immune system that regulates immune responses in healthy individuals and in the context of various diseases. In this Review, we discuss the origin, mechanisms of expansion and suppressive functions of MDSCs, as well as the potential to target these cells for therapeutic benefit.

The first observations of suppressive myeloid cells were described more than 20 years ago in patients with cancer1-3. However, the functional importance of these cells in the immune system has only recently been appreciated due to accumulating evidence that has demonstrated their contribution to the negative regulation of immune responses during cancer and other diseases. It is now becoming increasingly clear that this activity is contained within a population known as myeloid-derived suppressor cells (MDSCs). Features common to all MDSCs are their myeloid origin, immature state and a remarkable ability to suppress T-cell responses (Box 1). In addition to their suppressive effects on adaptive immune responses, MDSCs have also been reported to regulate innate immune responses by modulating the cytokine production of macrophages4. Non-immunological functions of MDSC have also been described, such as the promotion of tumour angiogenesis, tumour-cell invasion and metastasis. However, as a discussion of these aspects of MDSC biology is beyond the scope of this article, the reader is referred to another recent Review on this topic5.

MDSCs represent an intrinsic part of the myeloid-cell lineage and are a heterogeneous population that is comprised of myeloid-cell progenitors and precursors of myeloid cells. In healthy individuals, immature myeloid cells (IMCs) generated in bone marrow quickly differentiate into mature granulocytes, macrophages or dendritic cells (DCs). In pathological conditions such as cancer, various infectious diseases, sepsis, trauma, bone marrow transplantation or some autoimmune disorders, a partial block in the differentiation of IMCs into mature myeloid cells results in an expansion of this population. Importantly, the activation of these cells in a pathological context results in the upregulated expression of immune suppressive factors such as arginase (encoded by ARG1) and inducible nitric oxide synthase (iNOS; also known as NOS2) and an increase in the production of NO (nitric oxide) and reactive oxygen species (ROS). Together, this results in the expansion of an IMC population that has immune suppressive activity; these cells are now collectively known as MDSCs. In this

Origin and subsets of MDSCs It is important to note that MDSCs that are expanded in pathological conditions (see later) are not a defined subset of myeloid cells but rather a heterogeneous population of activated IMCs that have been prevented from fully differentiating into mature cells. MDSCs lack the expression of cell-surface markers that are specific for monocytes, macrophages or DCs and are comprised of a mixture of myeloid cells with granulocytic and monocytic morphology6. Early studies showed that 1–5% of MDSCs are able to form myeloid-cell colonies7-9 and that about one third of this population can differentiate into mature macrophages and DCs in the presence of appropriate cytokines in vitro and in vivo7-9. In mice, MDSCs are characterized by the co-expression of the myeloid lineage differentiation antigen Gr1 (also known as Ly6G) and CD11b (also known as αM-integrin)10. Normal bone marrow contains 20–30% of cells with this phenotype, but these cells make up only a small proportion (2–4%) of spleen cells and are absent from the lymph nodes in mice (Fig. 1). In humans, MDSCs are most commonly defined as CD14-CD11b+ cells or, more narrowly, as cells that express the common myeloid marker CD33 but lack the expression of markers of mature myeloid and lymphoid cells and the MHC-class-II molecule HLA-DR11, 12. MDSCs have also been identified within a CD15+ population in human peripheral blood13. In healthy individuals, immature myeloid cells with described above phenotype comprise ∼0.5% of peripheral blood mononuclear cells.
Recently, the morphological heterogeneity of these cells has been defined more precisely in part based on their expression of Gr1. Notably, Gr1-specific antibodies bind to both Ly6G and Ly6C,  which are encoded by separate genes. However, these epitopes are recognized by different antibodies specific for each individual epitopes: anti-Ly6C and anti-Ly6G. Granulocytic MDSCs have a CD11b+Ly6G+Ly6Clow phenotype, whereas MDSCs with monocytic morphology are CD11b+Ly6G-Ly6Chigh 6,14. Importantly, evidence indicates that these two subpopulations may have different functions in cancer and infectious and autoimmune diseases15-17. During the analysis of ten different experimental tumour models, we found that both of these subsets of MDSCs were expanded. In most cases, however, the expansion of the granulocytic MDSC population was much greater than that of the monocytic subset6 and, interestingly, the two subpopulations used different mechanisms to suppress Tcell function (see later). In addition, the ability to differentiate into mature DCs and macrophages in vitro has been shown to be restricted to monocytic MDSCs6.
In recent years, several other surface molecules have been used to identify additional subsets of suppressive MDSCs, including CD80 (also known as B7.1)18, CD115 (the macrophage colony-stimulating factor receptor)19, 20 and CD124 (the IL-4 receptor α-chain)20. In our own studies, we observed that many MDSCs in tumour-bearing mice co-express CD115 and CD1246; however, direct comparison of MDSCs from tumour-bearing mice and Gr1+CD11b+ cells from naive mice showed that they expressed similar levels of CD115 and CD124. In addition, sorted CD115+ or CD124+ MDSCs from EL-4 tumour-bearing mice had the same ability to suppress T-cell proliferation on a per cell basis as did CD115- or CD124-MDSCs. This suggests that, although these molecules are associated with MDSCs, they might not be involved in the immunosuppressive function of these cells in all tumour models.

Overall, current data suggest that MDSCs are not a defined subset of cells but rather a group of phenotypically heterogeneous myeloid cells that have common biological activity.

MDSCs in pathological conditions MDSCs were first characterized in tumour-bearing mice or in patients with cancer. Inoculation of mice with transplantable tumour cells, or the spontaneous development of tumours in transgenic mice with tissue-restricted oncogene expression, results in a marked systemic expansion of these cells (Fig. 1 and Table 1). In addition, up to a tenfold increase in MDSC numbers was detected in the blood of patients with different types of cancer11, 12, 21, 22. In many mouse tumour models, as many as 20–40% of nucleated splenocytes are represented by MDSCs (in contrast to the 2-4% seen in normal mice). In addition, these cells are found in tumour tissues and in the lymph nodes of tumour-bearing mice.
Although initial observations and most of the current information regarding the role of MDSCs in immune responses has come from studies in the cancer field, accumulating evidence has shown that MDSCs also regulate immune responses in bacterial and parasitic infections, acute and chronic inflammation, traumatic stress, surgical sepsis and transplantation. A systemic expansion of both the granulocytic and monocytic subset of MDSCs was observed in mice primed with Mycobacterium tuberculosis as part of complete Freund’s adjuvant (CFA). Acute Trypanosoma cruzi infection, which induces T-cell activation and increased production of interferon-γ (IFNγ), also leads to the expansion of MDSCs23, 24. A similar expansion of MDSCs has been reported during acute toxoplasmosis25, polymicrobial sepsis26, acute infection with Listeria monocytogenes or chronic infection with Leishmania major27 and infection with helminths28,29, 30, Candida albicans31 or Porphyromonas gingivalis32.

MDSC expansion is also associated with autoimmunity and inflammation. In experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, an increase in CD11b+Ly6ChiLy6G− MDSCs was observed in the spleen and blood and these cells were found to enter the central nervous system during the inflammatory phase of the disease16. A significant increase in the number of MDSCs was also detected in experimental autoimmune uveoretinitis, an animal model of human intraocular inflammatory disease33, in the skin and spleens of mice that were repeatedly treated with a contact sensitizer to induce an inflammatory response34 and in inflammatory bowel diseases35. MDSCs were also found to infiltrate the spleen and suppress T-cell function in a model of traumatic stress36. Finally, a significant transient increase in MDSC numbers was also demonstrated in normal mice following immunization with different antigens such as ovalbumin or peptide together with CFA, a recombinant vaccinia virus expressing interleukin-2 (IL-2) or staphylococcal enterotoxin A 8, 37, 38. Therefore, current information clearly indicates that the expansion of an immunosuppressive MDSC population is frequently observed in many pathological conditions.

Expansion and activation of MDSCs Studies have demonstrated that the MDSC population is influenced by several different factors (Table 1), which can be divided into two main groups. The first group includes factors that are produced mainly by tumour cells and promote the expansion of MDSC through stimulation of myelopoiesis and inhibiting of the differentiation of mature myeloid cells. The second group of factors is produced mainly by activated T cells and tumour stroma, and is involved in directly activating MDSCs.
Mechanisms of MDSC expansion—Factors that induce MDSC expansion can include cyclooxygenase-2 (COX2), prostaglandins 39-41, stem-cell factor (SCF)39, macrophage colony-stimulating factor (M-CSF), IL-642, granulocyte/macrophage colony-stimulating factor (GM-CSF)41 and vascular endothelial growth factor (VEGF) 43 (Table 1). The signalling pathways in MDSCs that are triggered by most of these factors converge on Janus kinase (JAK) protein family members and signal transducer and activator of transcription 3 (STAT3) (Fig. 2), which are signalling molecules that are involved in cell survival, proliferation, differentiation and apoptosis44. STAT3 is arguably the main transcription factor that regulates the expansion of MDSCs. MDSCs from tumour-bearing mice have markedly increased levels of phosphorylated STAT3 compared with IMCs from naive mice45. Exposure of haematopoietic progenitor cells to tumour-cell-conditioned medium resulted in the activation of JAK2 and STAT3 and was associated with an expansion of MDSCs in vitro, whereas inhibition of STAT3 expression in haematopoietic progenitor cells abrogated the effect of tumour-derived factors on MDSC expansion46. Ablation of STAT3 expression in conditional knockout mice or selective STAT3 inhibitors markedly reduced the expansion of MDSCs and increased T-cell responses in tumour-bearing mice45, 47. STAT3 activation is associated with increased survival and proliferation of myeloid progenitor cells, probably through upregulated expression of STAT3 target genes including B-cell lymphoma XL, (BCL-XL), cyclin D1, MYC and survivin. So, abnormal and persistent activation of STAT3 in myeloid progenitors prevents their differentiation into mature myeloid cells and thereby promotes MDSC expansion.

Recent findings suggest that STAT3 also regulates MDSC expansion through inducing the expression of S100A8 and S100A9 proteins. In addition, it has been shown that MDSCs also express receptors for these proteins on their cell surface. S100A8 and S100A9 belong to the family of S100 calcium-binding proteins that have been reported to have an important role in inflammation48. STAT3-dependent upregulation of S100A8 and S100A9 expression by myeloid progenitor cells prevented their differentiation and resulted in the expansion of MDSCs in the spleens of tumor-bearing and naive S100A9-transgenic mice. By contrast, MDSCs did not expand in the peripheral blood and spleens of mice deficient for S100A9 following challenge with tumour cells or CFA49. In a different study, S100A8 and S100A9 proteins were shown to promote MDSC migration to the tumour site through binding to carboxylated N-glycan receptors expressed on the surface of these cells 50. Blocking the binding of S100A8 and S100A9 to their receptors on MDSCs in vivo with a carboxylated glycan-specific antibody reduced MDSC levels in the blood and secondary lymphoid organs of tumour-bearing mice50. In human colon tumour tissue, and in a mouse model of colon cancer, myeloid progenitor cells expressing S100A8 and S100A9 have been shown to infiltrate regions of dysplasia and adenoma. Furthermore, administration of a carboxylated glycan-specific monoclonal antibody (mAbGB3.1) was found to markedly reduced chronic inflammation and tumorigenesis51. Although the mechanisms involved require further study, these studies suggest that S100A9 and/or S100A8 proteins have a crucial role in regulating MDSC expansion, and may provide a link between inflammation and immune suppression in cancer.

Mechanisms of MDSC activation—Recently, it has become clear that the suppressive activity of MDSCs requires not only factors that promote their expansion but those that induce their activation. The expression of these factors, which are produced mainly by activated T cells and tumour stromal cells, is induced by different bacterial or viral products or as a result of tumour cell death 26. These factors, which include IFNγ, ligands for Toll-like receptors (TLRs), IL-13, IL-4 and transforming growth factor-β (TGFβ), activate several different signalling pathways in MDSCs that involve STAT6, STAT1, and nuclear factor-κB (NF-κB) (Fig. 2).

Blockade of IFNγ, which is produced by activated T cells, abolishes MDSC-mediated T-cell suppression17, 52. STAT1 is the major transcription factor activated by IFNγ-mediated signalling and, in the tumour microenvironment, the upregulation of ARG1 and iNOS expression in MDSCs involved a STAT1-dependent mechanism. Indeed, MDSCs from Stat1-/- mice failed to up regulate ARG1 and iNOS expression and therefore did not inhibit Tcell responses53. Consistent with other findings, IFNγ produced by activated T cells and by MDSCs triggered iNOS expression and synergized with IL-4Rα and ARG1 pathways that have been implicated in the suppressive function of MDSCs20.
An important role for the signalling pathway that involves IL-4 receptor α-chain (IL-4Rα) and STAT6 (which is activated by the binding of either IL-4 or IL-13 to IL-4Rα) in MDSC activation has been demonstrated in several studies. It has been shown that ARG1 expression is induced by culturing freshly isolated MDSCs or cloned MDSC lines with IL-454. In addition, IL-4 and IL-13 upregulate arginase activity, which increases the suppressive function of MDSCs55. In line with these observations, other experiments have shown that STAT6 deficiency prevents signalling downstream of the IL-4Rα and thereby blocks the production of ARG1 by MDSCs56. In addition, the IL-4Rα–STAT6 pathway was also found to be involved in IL-13-induced TGFβ1 production by MDSCs in mice with sarcoma, which resulted in decreased tumour immunosurveillance57. This could be regulated by neutralizing both TGFβ and IL-1357. However, in breast tumor model IL-4Rα knockout mice retain high levels of MDSC after surgery56. In a different study that evaluated the separate role of TGFβ (not involving study of IL-4Rα) TGFβ-specific blocking antibody failed to reverse T-cell anergy in B-cell lymphoma in vitro58. It is possible that, the IL4Rα–STAT6 pathway might not be involved in promoting tumour immunosuppression in all tumour models.

TLRs have a central role in the activation of innate immune responses. Polymicrobial sepsis induced by the ligation and puncture of the caecum, which releases microbial products into the peritoneum and systemic circulation, was shown to result in an expansion of the MDSC population in the spleen that was dependent on the TLR adaptor molecule myeloid differentiation primary-response gene 88 (MyD88)26. However, wild-type mice and mice lacking a functional TLR4 protein had comparable expansion of the MDSC during polymicrobial sepsis, which suggests that signalling through TLR4 is not required for MDSC expansion and that MyD88-dependent signalling pathways that are triggered by other TLRs probably contribute to the expansion of MDSCs in sepsis26. This indicates that the activation of MDSCs is a fundamental outcome of the host innate immune response to pathogens that express TLR ligands.

It is important to note that an increase in the production and/or recruitment of IMCs in the context of acute infectious diseases or following vaccination does not necessarily represent an expansion of an immunosuppressive MDSC population. It is likely that under pathological conditions, the expansion of a suppressive MDSC population is regulated by two different groups of factors that have partially overlapping activity: those that induce MDSC expansion and those that induce their activation (which leads to increased levels of ROS, arginase, and/ or NO). This two-tiered system may allow for flexibility in the regulation of these cells under physiological and pathological conditions.
Mechanisms of MDSC suppressive activity Most studies have shown that the immunosuppressive functions of MDSCs require direct cell– cell contact, which suggests that they act either through cell-surface receptors and/or through the release of short-lived soluble mediators. The following sections describe the several mechanisms that have been implicated in MDSC-mediated suppression of T-cell function.

Arginase and iNOS—Historically, the suppressive activity of MDSCs has been associated with the metabolism of L-arginine. L-arginine serves as a substrate for two enzymes: iNOS, which generates NO, and arginase, which converts L-arginine into urea and L-ornithine. MDSCs express high levels of both arginase and iNOS, and a direct role for both of these enzymes in the inhibition of T-cell function is well established; this has been reviewed recently59, 60. Recent data suggest that there is a close correlation between the availability of arginine and the regulation of T-cell proliferation11, 61. The increased activity of arginase in MDSCs leads to enhanced L-arginine catabolism, which depletes this non-essential amino acid from the microenvironment. The shortage of L-arginine inhibits T-cell proliferation through several different mechanisms, including decreasing their CD3ζ expression62 and preventing their upregulation of the expression of the cell cycle regulators cyclin D3 and cyclin-dependent kinase 4 (CDK4)63. NO suppresses T-cell function through a variety of different mechanisms that involve the inhibition of JAK3 and STAT5 in T cells64, the inhibition of MHC class II expression 65 and the induction of T-cell apoptosis66.

ROS—Another important factor that contributes to the suppressive activity of MDSCs is ROS. Increased production of ROS has emerged as one of the main characteristics of MDSCs in both tumour-bearing mice and patients with cancer6, 10, 13, 53, 67-70. Inhibition of ROS production by MDSCs isolated from mice and patients with cancer completely abrogated the suppressive effect of these cells in vitro10, 13, 67. Interestingly, ligation of integrins expressed on the surface of MDSCs was shown to contribute to increased ROS production following the interaction of MDSCs with T cells10. In addition, several known tumour-derived factors, such as TGFβ, IL-10, IL-6, IL-3, platelet-derived growth factor (PDGF) and GM-CSF, can induce the production of ROS by MDSCs (for review see Ref 71).

The involvement of ROS and NO in mechanisms of MDSC suppression are not restricted to neoplastic conditions, as inflammation and microbial products are also known to induce the development of a MDSC population that produces ROS and NO following interactions with activated T cells15. Similar findings were observed in models of EAE16 and acute Toxoplasmosis infection 16. In addition, it has been observed that MDSCs mediated their suppressive function through IFNγ-dependent NO production in an experimental model of Trypanosoma cruzi infection23.

Peroxynitrite—More recently, it has emerged that peroxynitrite (ONOO-) is a crucial mediator of MDSC-mediated suppression of T-cell function. Peroxynitrite is a product of a chemical reaction between NO and superoxide anoion (O2-) and is one of the most powerful oxidants produced in the body. It induces the nitration and nitrosylation of the amino acids cystine, methionine, tryptophan and tyrosine72. Increased levels of peroxynitrite are present at sites of MDSC and inflammatory-cell accumulation, including sites of ongoing immune reactions. In addition, high levels of peroxynitrite are associated with tumour progression in many types of cancer72, 73,74-78, which has been linked with T-cell unresponsiveness. Bronte and colleagues reported that human prostate adenocarcinomas were infiltrated by terminallydifferentiated CD8+ T cells that were in an unresponsive state. High levels of nitrotyrosine were present in the T cells, which suggested the production of peroxynitrites in the tumour environment. Inhibiting the activity of arginase and iNOS, which are expressed in malignant but not in normal prostate tissue and are key enzymes of L-arginine metabolism,, led to decreased tyrosine nitration and restoration of T-cell responsiveness to tumour antigens79. In addition, we have demonstrated that peroxynitrite production by MDSCs during direct contact with T cells results in nitration of the T-cell receptor (TCR) and CD8 molecules, which alters the specific peptide binding of the T cells and renders them unresponsive to antigen-specific stimulation. However, the T cells maintained their responsiveness to nonspecific stimuli80. This phenomenon of MDSC induced antigen-specific T-cell unresponsiveness was also observed in vivo in tumour-bearing mice53.

Subset-specific suppressive mechanisms?—Recent findings indicate that different subsets of MDSC might use different mechanisms by which to suppress T-cell proliferation. As described earlier, two main subsets of MDSCs have been identified: a granulocytic subset and a monocytic subset. The granulocytic subset of MDSC was found to express high levels of ROS and low levels of NO, whereas the monocytic subset expressed low levels of ROS and high levels of NO and both subsets expressed ARG16 (Fig.3). Interestingly, both populations suppressed antigen-specific T-cell proliferation to an equal extent, despite their different mechanisms of action. Consistent with these observations, Movahedi et al. also reported two distinct MDSC subsets in tumour-bearing mice, one that consisted of mononuclear cells that resembled inflammatory monocytes and a second that consisted of polymorphonuclear cells that were similar to immature granulocytes. Again, both populations were found to suppress antigen-specific T-cell responses, although by using distinct effector molecules and signalling pathways. The suppressive activity of the granulocytic subset was ARG1-dependent, in contrast to the STAT1- and iNOS-dependent mechanism of the monocyte fraction17. Finally, the same trend was observed in Trypanosoma cruzii infection. In this case, monocytic MDSCs produced NO and strongly inhibited T-cell proliferation, and granulocytic MDSCs produced low levels of NO and did not inhibit T-cell proliferation, although they did produce superoxide15. The biological significance of such functional dichotomy of these two MDSC subsets remains to be elucidated.
Induction of TReg cells—Recently, the ability of MDSCs to promote the de novo development of FOXP3+ regulatory T (TReg) cells in vivo has been described18, 19. The induction of TReg cells by MDSCs was found to require the activation of tumour-specific Tcells and the presence of IFNγ and IL-10 but was independent of NO19. In mice bearing 1D8 ovarian tumours, the induction of TReg cells by MDSCs required the expression of cytotoxic lymphocyte antigen 4 (CTLA-4; also known as CD152) by MDSCs18. In a mouse model of lymphoma, MDSCs were shown to induce TReg-cell expansion through a mechanism that required arginase and the capture, processing and presentation of tumour-associated antigens by MDSCs, but not TGFβ58. By contrast, Movahedi et al. found that the percentage of TReg cells was invariably high throughout tumour growth and did not relate to the kinetics of expansion of the MDSC population, suggesting that MDSCs were not involved in TReg-cell expansion17. Furthermore, in a rat model of kidney allograft tolerance that was induced with a CD28-specific antibody, MDSCs that were co-expressing CD80 and CD86 were found to have a limited effect on the expansion of the TReg-cell population81. Although further work is required to resolve these discrepancies and to determine the physiological relevance of these studies, it seems possible that MDSCs are involved in TReg-cell differentiation through the production of cytokines or direct cell–cell interactions. Furthermore, MDSCs and TReg cells might be linked in a common immunoregulatory network (see later).
Tissue-specific effects on MDSCs A major unresolved question in this field is whether MDSCs mediate antigen-specific or nonspecific suppression of T-cell responses. Provided that MDSCs and T cells are in close proximity, the factors that mediate MDSC suppressive function (ROS, arginase and NO) can inhibit T-cell proliferation regardless of the antigen specificity of the T cells. Indeed, numerous in vitro studies have demonstrated the antigen nonspecific nature of MDSC-mediated suppression of T cells82 83. However, whether the situation is the same in vivo is not clear, and evidence suggests that MDSC-mediated immunosuppression in peripheral lymphoid organs is mainly antigen-specific. The idea that MDSC-mediated T-cell suppression occurs in an antigen-specific manner is based on findings that antigen-specific interactions between antigen-presenting cells and T cells result in much more stable and more prolonged cell–cell contact than nonspecific interactions82, 84, 85. Such stable contacts are necessary for MDSCderived ROS and peroxynitrite to mediate effects on the molecules on the surface of T cells that render the T cells unresponsive to specific antigen. It should be noted that such modification of cell-surface molecules does not lead to T-cell death nor prevent nonspecific T-cell activation. Other evidence that supports the idea that MDSCs mediate antigen-specific suppression is the finding that that MDSCs can take up soluble antigens, including tumourassociated antigens, and process and present them to T cells17 80; blockade of MDSC–T-cell interactions with a MHC-class-I-specific antibody abrogated MDSC-mediated inhibition of T cell responses in vitro86. The MHC-class-I-restricted nature of MDSC-mediated CD8+ T-cell suppression has also been demonstrated in vivo in tumor models53 and in the model of inflammatory bowel disease 35. This is consistent with the recent observation that large numbers of tumour-induced MDSCs did not inhibit CD8+ T-cell responses specific for unrelated antigens in a model of sporadic cancer87. Notably, it is currently unclear whether similar antigen-specific mechanisms of MDSC-mediated suppression operate on CD4+ T cells, as published studies have only assessed the effects of MDSCs on CD8+ T cells. Addressing this question is complicated by the fact that only a small proportion of MDSCs in many tumour models expresses MHC class II molecules.

The theory that MDSCs suppress T-cell responses in an antigen-specific manner helps to explain the finding that T cells in the peripheral lymphoid organs of tumour-bearing mice and in the peripheral blood of cancer patients can still respond to stimuli other than tumourassociated antigens, including viruses, lectins, co-stimulatory molecules, IL-2 and CD3- and CD28-specific antibodies21, 80, 88-90. Furthermore, even patients with advanced stage cancer do not have systemic immunodeficiency except in cases in which the patient has received high doses of chemotherapy or is at a terminal stage of the disease.

Evidence suggets that the nature of MDSC-mediated suppression at the tumour site is quite different to that which occurs in the periphery. MDSCs actively migrate into the tumour site10, where they upregulate the expression of ARG1 and iNOS, downregulate the production of ROS and/or rapidly differentiate into tumour-associated macrophages (TAMs) 52. The levels of NO and arginase produced by tumour-associated MDSCs and TAMs are much higher than those of MDSCs found in peripheral lymphoid organs of the same animals. In addition, TAMs produce several cytokines (reviewed in REFs91, 92) that suppress T-cell responses in a nonspecific manner (Fig. 4). The mechanisms by which MDSC functions are regulated within the tumour microenvironment, and how they differ from those that operate at peripheral sites, remain unclear. It is possible that tumour stroma, hypoxia and/or the acidophilic environment have a role.
Therapeutic targeting of MDSCs The recognition that immune suppression has a crucial role in promoting tumour progression and contributes to the frequent failure of cancer vaccines to elicit an immune response has resulted in a paradigm shift with respect to approaches for cancer immunotherapy. Indeed, it has become increasingly clear that successful cancer immunotherapy will be possible only with a strategy that involves the elimination of suppressive factors from the body. As MDSCs are one of the main immunosuppressive factors in cancer and other pathological conditions, several different therapeutic strategies that target these cells are currently being explored (Table 2). Although the studies described below were carried out in tumor-bearing hosts, it is likely that the same strategies will be useful in other pathological conditions in which inhibition or elimination of MDSCs is a therapeutic aim.

Promoting myeloid-cell differentiation—One of the most promising approaches by which to target MDSCs for therapy is to promote their differentiation into mature myeloid cells that do not have suppressive abilities. Vitamin A has been identified as a compound that can mediate this effect: vitamin A metabolites such as retinoic acid have been found to stimulate the differentiation of myeloid progenitors into DCs and macrophages 86, 93. Mice that are deficient in vitamin A94 or that have been treated with a pan-retinoic-acid-receptor antagonist95, show an expansion of MDSCs in the bone marrow and spleen. Conversely, therapeutic concentrations of all-trans retinoic acid (ATRA) results in substantial decrease in the presence of MDSCs in cancer patients and tumour-bearing mice. ATRA induced MDSCs to differentiate into DCs and macrophages in vitro and in vivo 12, 86, 96. It is probable that ATRA preferentially induces the differentiation of the monocytic subset of MDSCs, whereas it causes apoptosis of the granulocytic subset. The main mechanism of ATRA-mediated differentiation involved an upregulation of glutathione synthesis and a reduction in ROS levels in MDSCs 97. Decreasing the number of MDSCs in tumour-bearing mice resulted in increased tumour-specific T-cell responses, and the combination of ATRA and two different types of cancer vaccine prolonged the anti-tumour effect of the vaccine treatment in two different tumour models 96. Moreover, administration of ATRA to patients with metastatic renal cell carcinoma resulted in a substantial decrease in the number of MDSCs in the peripheral blood and improved antigen-specific response of T cells 21. Further studies will lead to identification of other agents that have a similar effect. So far, evidence suggests that Vitamin D3 may be another agent with the potential to decrease MDSC numbers in patients with cancer, as it is also known to promote myeloid-cell differentiation98.

Inhibition of MDSC expansion—Because MDSC expansion is known to be regulated by tumour-derived factors (Table 1), several studies have focused on neutralizing the effects of these factors. Recently, SCF has been implicated in causing MDSC expansion in tumourbearing mice39. Inhibition of SCF-mediated signalling by blocking its interaction with its receptor, c-kit, decreased MDSC expansion and tumor angiogenesis39. VEGF, another tumourderived factor that is involved in promoting MDSC expansion, might also be a useful target by which to manipulate MDSC. However, in a clinical trial of 15 patients with refractory solid tumours, treatment with VEGF–trap (a fusion protein that binds all forms of VEGF-A and placental growth factor) showed no effect on MDSC numbers and did not result in increased T-cell responses99. By contrast, treatment of patients with metastatic renal cell cancer with a VEGF-specific blocking antibody (known as avastin) resulted in a decrease in the size of a CD11b+VEGFR1+ population of MDSCs in the peripheral blood 100. However, whether avastatin treatment resulted in an improvement in antitumour responses in these patients has not been determined. Finally, inhibition of matrix metalloproteinase 9 function in tumorbearing mice decreased the number of MDSCs in the spleen and tumour tissues and resulted in a significant delay in the growth of spontaneous NeuT tumours in transgenic BALB/c mice101. However, the mechanism responsible for this outcome remains to be elucidated.

Inhibition of MDSC function—Another approach by which to inhibit MDSCs is to block the signalling pathways that regulate the production of suppressive factors by these cells. One potential target by which this might be achieved is COX2. COX2 is required for the production of prostaglandin E2, which in 3LL tumour cells61 and mammary carcinoma40 has been shown to induce the upregulation of ARG1 expression by MDSCs, thereby inducing their suppressive function. Accordingly, COX2 inhibitors were found to downregulate the expression of ARG1 by MDSCs, which improved antitumour T-cell responses and enhanced the therapeutic efficacy of immunotherapy102, 103. Similarly, phosphodiesterase-5 inhibitors such as sildenafil were found to downregulate the expression of arginase and iNOS expression by MDSCs, thereby inhibiting their suppressive function in growing tumours104. This resulted in the induction of a measurable anti-tumour immune response and a marked delay of tumour progression in several mouse models 104.
ROS inhibitors have also been shown to be effective for decreasing MDSC-mediated immune suppression in tumour-bearing mice. The coupling of a NO-releasing moiety to a conventional non-steroidal anti-inflammatory drug has proven to be an efficient means by which to inhibit the production of ROS. One such drug, nitroaspirin, was found to limit the activity of ARG1 and iNOS in spleen MDSCs105. In combination with vaccination with endogenous retroviral gp70 antigen, nitroaspirin inhibited MDSCs function and increased the number and function of tumour-antigen-specific T cells105.

Elimination of MDSCs—MDSCs can be directly eliminated in pathological settings by using some chemotherapeutic drugs. Administration of one such drug, gemcitabine, to mice that were bearing large tumours resulted in a dramatic reduction in the number of MDSCs in the spleen and resulted in a marked improvement in the anti-tumour response induced by immunotherapy106, 107. This effect was specific to MDSCs, as a significant decrease in the number of T or B cells was not observed in these animals. Furthermore, in a study of 17 patients with early-stage breast cancer that were treated with doxorubicin–cyclophosphamide chemotherapy, a decrease in the level of MDSCs in the peripheral blood was observed22.

Evidence suggests that there is a broad range of methods that will be effective for targeting of the number and/or function of MDSCs in vivo. These strategies will undoubtedly help to further investigate the biology of these cells as well as expedite clinical applications to treat cancer and other pathological conditions.

MDSCs as regulatory myeloid cells? The wealth of information that has accumulated in recent years regarding the biology of MDSCs suggests that these cells might have evolved as a regulatory component of the immune system. These cells are absent under physiological conditions, as IMCs in naive mice are an intrinsic part of normal haematopoiesis that are not immunosuppressive in an unactivated state. In conditions of acute stress, infection or immunization, there is a transient expansion of this IMC population, which then quickly differentiates into mature myeloid cells. This transient IMC population can mediate the suppressive functions that are characteristic of MDSCs but, because the acute conditions are short-lived, the suppressive functions of this transient population have a minimal impact on the overall immune response. However, these cells probably function as important ‘gatekeepers’ that prevent pathological immune-mediated damage.

The role of the MDSC population in settings of chronic infections and cancer is very different. In these pathological conditions, the prolonged and marked expansion of IMCs and their subsequent activation leads to the expansion of a large population of MDSCs with immunosuppressive abilities. MDSCs accumulate in peripheral lymphoid organs and migrate to tumour sites, where they contribute to immunosuppression. Furthermore, some evidence suggests that MDSCs can also induce expansion of regulatory T cells. Future studies will reveal whether MDSCs can be considered part of a natural immune regulatory network.

Concluding remarks The field of MDSC research has more outstanding questions than answers. The roles of specific MDSC subsets in mediating T-cell suppression, and the molecular mechanisms responsible for inhibition of myeloid-cell differentiation, need to be elucidated. The issue of whether Tcell suppression occurs in an antigen-specific manner remains to be clarified, as do the mechanisms that cause MDSC migration to peripheral lymphoid organs. Some of the main priorities in this field should include a better characterization of human MDSCs and a clear understanding of whether targeting these cells in patients with various pathological conditions will be of clinical significance. Conversely, adoptive cellular therapy with MDSCs may be an attractive opportunity by which to inhibit immune responses in the setting of autoimmune disease or transplantation. The challenge for these approaches will be to devise methods by which to generate these cells ex vivo in clinical-grade conditions such that they are suitable for administration to patients. If the past 5–6 years are an indication of the potential for progress in this area, it is safe to estimate that there will soon be significantly more discoveries that further our understanding about the biology and clinical utility of MDSCs.

Box 1. Definition of myeloid-derived suppressor cells (MDSCs)

• a heterogeneous population of cells of myeloid origin that consist of myeloid progenitors and immature macrophages, immature granulocytes and immature dendritic cells

• present in activated state that is characterized by the increased production of reactive oxygen and nitrogen species, and of arginase

• potent suppressors of various T-cell functions • in mice, their phenotype is CD11b+Gr1+, although functionally distinct subsets within this population have been identified (see main text)

• in humans, their phenotype is Lin-HLA-DR-CD33+ or CD11b+CD14-CD33+.

Human cells do not express a marker homologous to mouse Gr1. MDSC have also been identified within a CD15+ population in human peripheral blood.

• in the steady state, immature myeloid cells lack suppressive activity and are present in the bone marrow, but not in secondary lymphoid organs

• accumulation of MDSCs in lymphoid organs and in tumours in response to various growth factors and cytokines is associated with various pathological conditions (most notably cancer)

• in tumour tissues, MDSCs can be differentiated from tumour-associated macrophages (TAMs) by their high expression of Gr1 (not expressed by TAMs) by their low expression of F4/80 (expressed by TAMs), by the fact that a large proportion of MDSCs have a granulocytic morphology and based the upregulated expression of both arginase and inducible nitric oxide synthase by MDSCs but not TAMs.

References

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Aurelian Udristioiu commented on your update
“The proto-oncogenic transcription factor Myc is known to promote transcription of genes for the cell cycle as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role to induce the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor HIF1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions. “

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