Latest Articles Include:
- An unhealthy disregard
- Nat Med 16(6):609 (2010)
Nature Medicine | Editorial An unhealthy disregard Journal name:Nature MedicineVolume:16,Page:609Year published:(2010)DOI:doi:10.1038/nm0610-609 Major pharmaceutical firms are regularly slapped with huge fines for illegally promoting off-label drug use. But as long as the sales of these drugs dwarf the size of the fines, companies will continue to skirt the law. View full text Additional data - In vision trial, some researchers would rather see double
- Nat Med 16(6):611 (2010)
A government-sponsored clinical study of vision loss has come under fire for investigating a proprietary medicine without comparing it to a similar drug that costs a fraction of the price.In late April, first year results from a two-year clinical study of about 700 individuals with a condition known as diabetic macular edema were reported by the Diabetic Retinopathy Clinical Research Network (DRCRnet), a multicenter collaborative group funded by the US National Eye Institute (NEI). - Four years on, clinical partnerships program proves worth
- Nat Med 16(6):612-613 (2010)
Last fall, as the H1N1 'swine flu' pandemic reached full swing, Gordon Bernard of Vanderbilt University Medical Center in Nashville, Tennessee started gearing up to test his idea that statins, which can dampen inflammation, might combat the disease. Whereas clinical trials often take years to get up and running, Bernard's study was approved in record time—two months. - Tool kit for translational research
- Nat Med 16(6):612-613 (2010)
Nature Medicine | News Tool kit for translational research * Charlotte Schubert1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:612–613Year published:(2010)DOI:doi:10.1038/nm0610-612b The CTSA program has sped up the pace of research by promoting shared informatics tools, such as the four featured here. Not all of these tools are funded fully by the CTSA—but the program has enabled individual institutions to support these projects and fostered their dissemination. View full text Additional data Affiliations * Washington, DC * Charlotte Schubert - Avandia outcome may signal change in epidemiologists' sway
- Nat Med 16(6):614 (2010)
Next month, the Obama-era US Food and Drug Administration (FDA) will confront what is likely to be the most defining moment in its short history. In a public meeting scheduled for mid-July, a committee of external experts will advise agency leaders—who may or may not accept their advice—on whether to ban a controversial diabetes drug implicated in causing heart attacks, when an equally effective competitor is also on the market, minus the heart attack risks. - Diabetes drug woes spell trouble for the entire drug family
- Nat Med 16(6):614 (2010)
Although the final fate of the blockbuster diabetes drug Avandia (rosiglitazone) remains to be determined later this year by the US Food and Drug Administration, the damage to the family of 'PPAR-gamma ligand' drugs to which it belongs has already largely been done.Development of such drugs is losing support: "what we have heard back from drug companies is that, if it has the word 'PPAR' in it, it's dead," says Bruce Spiegelman, a cell biologist at the Dana-Farber Cancer Institute in Boston. - Antivenoms needed, say officials, but companies won't bite
- Nat Med 16(6):615 (2010)
"When a doctor in a developing nation sees a patient with an infection, it's an easy choice to prescribe an antibiotic," says Ana Padilla, a technical advisor at the World Health Organization (WHO) in Geneva. "But when a patient shows up with a snake bite, most doctors won't know which antivenom to give or how to administer it—and even if they did, they probably don't have it or know where to get it. - Cancer vaccine approval could open floodgates
- Nat Med 16(6):615 (2010)
The US Food and Drug Administration approved the first-ever vaccine to treat cancer on 29 April. After a three-year battle with the regulatory agency and three phase 3 trials, the treatment—called Provenge, by Seattle-based Dendreon—extended median survival time in men with advanced prostate cancer by more than four months. - When natural disasters strike, tragedy can unfold in the lab
- Nat Med 16(6):616-617 (2010)
When Laura Levy returned to her laboratory in the aftermath of Hurricane Katrina, there was nothing to suggest that a violent storm had pummeled the city. On the fifth floor of the Tulane University School of Medicine, where Levy's lab sits, most things were in their proper place. - Data handling errors spur debate over clinical trial
- Nat Med 16(6):618 (2010)
On 6 May, the US stock market experienced a peculiar 'minicrash' when what seems to be a mishandled trading order temporary sent stocks plummeting. The dramatic episode on Wall Street underscores how small errors can substantially upset data-heavy systems, and deciphering the error afterward can be a seemingly impossible task. - Health reform unhealthy for pharma
- Nat Med 16(6):618 (2010)
The healthcare reform passed in the US in March will expand medical coverage and pharmacy benefits to millions of Americans. This increase in the number of insured people should translate into a windfall for the pharmaceutical industry—but only once that piece of the legislation goes into effect, starting in 2014. - Industry looks to buck bias in emerging 'adaptive' designs
- Nat Med 16(6):619 (2010)
Adaptive clinical trials, in which aspects such as medical endpoints and sample sizes can be modified midtrial, are catching on in the pharmaceutical industry to make drug development both faster and cheaper. But as the industry stakes out shortcuts, many researchers are concerned that they might also shortcut the integrity of clinical trials. - Stem cell decision could rewrite rules of patentability
- Nat Med 16(6):619 (2010)
For the past decade, many researchers have complained bitterly that a trio of hotly contested patents has thwarted potentially life-saving research involving embryonic stem cells. Now, a decision to overturn one of these claims may radically change the notion of what's patentable in the life sciences. - Committee planned to weigh misconduct in Australia
- Nat Med 16(6):620 (2010)
People who have no plans for conducting scientific studies will be the final arbiters of alleged breaches of research ethics when the Australian Research Integrity Committee is fully established in early 2011.Expressions of interest in appointment to the committee, which closed in late May, required applicants to have knowledge of tribunal processes as well as legal or research governance experience, and no current or future involvement in research-related activities. - Lind guidelines offer a checklist for research priorities
- Nat Med 16(6):620 (2010)
When it comes to medical treatment, the research community's agenda doesn't always match up with patients' desires. For instance, when people suffering from knee osteoarthritis were asked in 1999 what research was needed, they said they wanted more information on interventions such as braces, exercise regimens and pain management strategies. - Correction: 'Universal' immunizations get a boost in India
- Nat Med 16(6):620 (2010)
The article "Universal' immunizations get a boost in India' (Nat. Med.16, 497, 2010 - Beyond high school, 'colleges' teach medical marijuana
- Nat Med 16(6):621 (2010)
Amidst the remnants of the failing automobile industry in Michigan rises a new business: the production of medical marijuana.Capitalizing on the fledgling but rapidly growing medical marijuana market is Med Grow Cannabis College, an institution that teaches cannabis growers the how-tos of cultivating the controversial plant. - The state of clinical research in America
- Nat Med 16(6):621 (2010)
The most populous three states in the US—California, Texas and New York—were the only states last year to launch more than 1,000 human clinical trials each. That's not surprising, given the millions of people inhabiting each of these states. - News in brief
- Nat Med 16(6):622-623 (2010)
Apr 20Flights in and out of the UK finally resumed after the cloud of ash from Eyjafjallajökull delayed European travel for more than a week. But, according to reports, the volcano also delayed a polio vaccine campaign for 85 million children in central and western Africa, along with several transplants and medical procedures. - Straight talk with...George Daley
- Nat Med 16(6):624 (2010)
Nature Medicine | News Straight talk with...George Daley * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:624Year published:(2010)DOI:doi:10.1038/nm0610-624 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In 2008, the method of taking skin cells from people suffering from disease and transforming them into embryonic-like stem cells was heralded as the 'breakthrough of the year' by publications such as Time magazine. Two years on, so-called induced pluripotent stem (iPS) cells are just beginning to shed new light on disease biology. From day one of this burgeoning area of study, stem cell pioneer George Daley of Children's Hospital in Boston, who developed the first library of disease-specific iPS cells lines, has remained involved in this fast-paced field. Ahead of the June annual meeting of the International Society for Stem Cell Research (ISSCR) in San Francisco, spoke to Daley about when and how reprogrammed stem cells will deliver. View full text Additional data - When powerhouses fail
- Nat Med 16(6):625-627 (2010)
Nature Medicine | News When powerhouses fail * Erica Westly1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:625–627Year published:(2010)DOI:doi:10.1038/nm0610-625 Each cell in the body possesses hundreds to thousands of mitochondria, known as 'powerhouses' for the energy they provide. But gene mutations can cause these important organelles to fail, often resulting in devastating disease. reports on the patient advocates—and politicians—pushing for new treatments for mitochondrial disease. View full text Additional data Affiliations * Erica Westly is a freelance science writer based in Brooklyn, New York. - Up against the wall
- Nat Med 16(6):628-631 (2010)
Nature Medicine | News Up against the wall * Christian Torres1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:628–631Year published:(2010)DOI:doi:10.1038/nm0610-628 The notoriously drug-resistant bug MRSA has made headlines for years, but a whole other class of bacteria may prove even more troublesome. These microbes, Gram-negative bacteria, are increasingly a threat—and yet not a single late-stage drug in development specifically targets them. follows one man's quest to get the antibacterial pipeline flowing once again. View full text Additional data Affiliations * Christian Torres is a former intern at Nature Medicine who lives in San Diego. - US cancer trials may go the way of the Oldsmobile
- Nat Med 16(6):632 (2010)
Nature Medicine | News US cancer trials may go the way of the Oldsmobile * David Dilts1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:632Year published:(2010)DOI:doi:10.1038/nm0610-632 Cancer clinical trials in the US are at a major crossroads. Current government-sponsored research is some of the best in the world, but the field shares a worrying number of similarities with the American auto industry in its heyday. For clinical research to survive, the field must transform itself now to prevent a similar decline. View full text Additional data Affiliations * David Dilts is the director of the Center for Management Research in Healthcare at the Oregon Health & Science University's Knight Cancer Institute in Portland. A management scientist by training, Dilts was a member of the IOM's committee on cancer cooperative groups and the NCI's Operational Efficiency Working Group. The views expressed are his own and do not reflect the position of any affiliate institutions. - A shameful system of research
- Nat Med 16(6):633 (2010)
In 1958, Eugene Saenger, a cancer researcher at the University of Cincinnati School of Medicine, submitted a proposal to the Department of Defense (DOD) for a study on the effects of total body radiation (TBI) on patients with cancer. Two years later, Saenger began one of the most notorious human radiation experiments of the postwar era. - Activated basophils give lupus a booster shot
- Nat Med 16(6):635-636 (2010)
In systemic lupus erythematosus (SLE), self-reactive antibodies can target the kidney (lupus nephritis), leading to functional failure and possible mortality. We report that activation of basophils by autoreactive IgE causes their homing to lymph nodes, promoting T helper type 2 (TH2) cell differentiation and enhancing the production of self-reactive antibodies that cause lupus-like nephritis in mice lacking the Src family protein tyrosine kinase Lyn (Lyn−/− mice). Individuals with SLE also have elevated serum IgE, self-reactive IgEs and activated basophils that express CD62 ligand (CD62L) and the major histocompatibility complex (MHC) class II molecule human leukocyte antigen-DR (HLA-DR), parameters that are associated with increased disease activity and active lupus nephritis. Basophils were also present in the lymph nodes and spleen of subjects with SLE. Thus, in Lyn−/− mice, basophils and IgE autoantibodies amplify autoantibody production that leads to lupu! s nephritis, and in individuals with SLE IgE autoantibodies and activated basophils are factors associated with disease activity and nephritis. - Behind the paper: muzzling muscle spasticity
Dolgin E - Nat Med 16(6):637 (2010)
In September 2008, Kendall Winter was driving to a friend's house along Highway 20 in central Alberta when her silver, compact Chevrolet slipped onto the shoulder of the road. Her car rolled six times, and she found herself trapped in the mangled wreckage, but still conscious. - Interleukin-33 safeguards neutrophils in sepsis
- Nat Med 16(6):638-639 (2010)
Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options1, 2. Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33–treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (TH1) to TH2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection3. Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration4. We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverse! s the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein–coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors5, 6. Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis. - Microflora in colorectal cancer: a friend to fear
- Nat Med 16(6):639-641 (2010)
Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apcmin/+ mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal–related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apcmin/+/Myd88−/− mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apcmin/+ mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apcmin/+/Myd88−/− mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apcmin/+/Myd88−/− and Apcmin/+ mice and increa! sed their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host. - Shifting HIFs in osteoarthritis
- Nat Med 16(6):641-644 (2010)
Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2α (HIF-2α, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2α enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2α, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2α expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis developme! nt, and a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-κB (NF-κB) family member, as a potent inducer of HIF-2α expression. Hence, HIF-2α is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis. - A mother's gift, minus mitochondria
- Nat Med 16(6):645 (2010)
At least one in 10,000 people in the UK are affected by diseases caused by mutations in their mitochondrial DNA (mtDNA). A new study in human embryos provides a potential route to prevent transmission of such diseases to the offspring of affected women, via nuclear transfer techniques. - Sudden Cardio Arrest: When normal ECG variants turn lethal
- Nat Med 16(6):646-647 (2010)
Nature Medicine | Between Bedside and Bench Sudden Cardio Arrest: When normal ECG variants turn lethal * Stanley Nattel1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:646–647Year published:(2010)DOI:doi:10.1038/nm0610-646 Numerous factors can contribute to sudden cardiac death, from underlying disease after myocardial infarction to genetic variants that can claim young lives. In 'Bedside to Bench', Stanley Nattel examines recent clinical studies suggesting that a particular type of readout on an electrocardiogram (ECG) may increase the risk of the condition. This ECG 'variant' is relatively common and was previously thought to be benign. In 'Bench to Bedside', Gordon Tomaselli and Andreas Barth take a look at studies at the bench examining how oxidative stress may promote sudden cardiac death. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Stanley Nattel is in the Department of Medicine and Research Center, Montreal Heart Institute and Université de Montréal, and the Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Stanley Nattel (Stanley.nattel@icm-mhi.org) Additional data - Sudden Cardio Arrest: Oxidative stress irritates the heart
- Nat Med 16(6):648-649 (2010)
Sudden cardiac death (SCD) and cardiac arrhythmias remain a daunting public health problem. It is estimated that there are between 250,000 and 400,000 SCDs in the United States each year1, most of which occur in the setting of heart failure or as a complication of ischemic heart disease, such as after myocardial infarction. - Research Highlights
- Nat Med 16(6):650-651 (2010)
- Missing pieces in the Parkinson's disease puzzle
- Nat Med 16(6):653-661 (2010)
Nature Medicine | Review Missing pieces in the Parkinson's disease puzzle * Jose A Obeso1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria C Rodriguez-Oroz1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher G Goetz3 Search for this author in: * NPG journals * PubMed * Google Scholar * Concepcion Marin2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey H Kordower3 Search for this author in: * NPG journals * PubMed * Google Scholar * Manuel Rodriguez2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Etienne C Hirsch6 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Farrer7 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony H V Schapira8 Search for this author in: * NPG journals * PubMed * Google Scholar * Glenda Halliday9 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:653–661Year published:(2010)DOI:doi:10.1038/nm.2165Published online23 May 2010 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Parkinson's disease is a neurodegenerative process characterized by numerous motor and nonmotor clinical manifestations for which effective, mechanism-based treatments remain elusive. Here we discuss a series of critical issues that we think researchers need to address to stand a better chance of solving the different challenges posed by this pathology. View full text Figures at a glance * Figure 1: Schematic summary of established etiopathogenic mechanisms and interactions in the dopaminergic cells of the substantia nigra in Parkinson's disease. Cell death may be caused by α-synuclein aggregation, proteosomal and lysosomal (not shown) system dysfunction, and reduced mitochondrial activity. Gene mutations are associated with impairment of one or several of these mechanisms. In addition, secondary changes (not shown) such as excitotoxicity and inflammation are likely to play a relevant role in progressive neuronal degeneration. α-Sp22, a 22-kilodalton glycosylated form of α-synuclein; PAELR, parkin-associated endothelin receptor-like receptor; UbCH7, ubiquitin-conjugating enzyme 7; UbCH8, ubiquitin-conjugating enzyme 8; UCHL1, ubiquitin carboxy-terminal hydrolase L1. * Figure 2: Striatal dopamine innervation assessed by 18F-dopa positron emission tomography. () Mean control values for eight control subjects shows high uptake (highest value in white) in the striatum. () Subject with Parkinson's disease (right) featuring slowness and rigidity on the right limbs but minimal signs on the left limbs. Uptake is markedly reduced (70% below normal) in the left posterior putamen and reduced to a minor extent in the anterior putamen and caudate of the left hemisphere. () SPM2-based analysis (yellow represents the largest statistical difference and red the smallest one), showing the difference in uptake between and to highlight the caudorostral pattern of denervation. The statistical map is rendered over the MRI for anatomical localization. * Figure 3: Distribution of Lewy bodies in Parkinson's disease. Diagrammatic representation of pathological data from longitudinally studied cases showing the severity of midbrain dopamine cell loss and Lewy body infiltration over time in an average individual who develops symptoms around 55 years of age versus one who develops symptoms after the age of 70. The severity of dopamine cell loss is related to the duration of symptoms, with those with longer durations having greater cell loss (represented as progressively darker color). The infiltration of Lewy bodies appears more marked in late-onset disease, and in many instances, individuals with late-onset disease have additional age-related pathologies (represented as cortical plaques). Dementia, as indicated in the lower bar, occurs earlier in the disease in older-onset individuals, consistent with the greater pathology observed. Author information * Abstract * Author information Affiliations * Department of Neurology, Clínica Universitaria and Medical School of Navarra, Neuroscience Centre, Center for Applied Medical Research, Pamplona, Spain. * Jose A Obeso & * Maria C Rodriguez-Oroz * Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Carlos III, Ministerio de Investigación y Ciencias, Madrid, Spain. * Jose A Obeso, * Maria C Rodriguez-Oroz, * Concepcion Marin & * Manuel Rodriguez * Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA. * Christopher G Goetz & * Jeffrey H Kordower * Laboratori de Neurologia Experimental, Àrea de Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona and CIBERNED, Barcelona, Spain. * Concepcion Marin * Departamento de Fisiologia, Facultad de Medicina, Universidad de La Laguna and CIBERNED, Tenerife, Spain. * Manuel Rodriguez * Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de Moelle Epiniére, Paris, France. * Etienne C Hirsch * Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, USA. * Matthew Farrer * University Department of Clinical Neuroscience, Institute of Neurology, London, UK. * Anthony H V Schapira * Prince of Wales Medical Research Institute and the University of New South Wales, Sydney, Australia. * Glenda Halliday Competing financial interests J.A.O. has served on the Advisory Board of GlaxoSmithKline (UK) and received honoraria for lecturing at meetings organized by GlaxoSmithKline (Spain), Lundbeck-Teva and UCB. M.C.R.-O. participates in the Advisory Board of UCB Spain and receives payment for lectures and travel accommodation payments for scientific meetings from GlaxoSmithKline, UCB, Lundbeck and Medtronic and for teaching courses from Medtronic. C.G.G., as of the 12 months ending September 2009, is a consultant to or member of Advisory Boards, with associated honoraria, for Asubio, Boehringer-Ingelheim, Impax Pharmaceuticals, i3 Research, Ingenix, Juvantia Pharmaceuticals, Neurim Pharmaceuticals, Novartis Pharmaceuticals, Osmotica Pharmaceutical, Oxford Biomedica, Santhera Pharmaceuticals, Solvay Pharmaceuticals, Teva Pharmaceuticals, United Biosource Corporation and UCB. J.K. is a founding scientist and Scientific Advisory Board member of Ceregene Inc. A.S. has received honoraria from Lundbeck-Teva, Boehring! er-Ingelheim, GlaxoSmithKline and Orion-Novartis for advice on Parkinson's disease drug research and development and for educational symposia. E.H., G.H., M.R. and C.M. report no conflicts of interest. Corresponding author Correspondence to: * Jose A Obeso (jobeso@unav.es) Additional data - Epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype 6
- Nat Med 16(6):662-664 (2010)
Nature Medicine | Brief Communication Epidermal growth factor receptor is a co-receptor for adeno-associated virus serotype 6 * Melodie L Weller1 Search for this author in: * NPG journals * PubMed * Google Scholar * Panomwat Amornphimoltham2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Schmidt1 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul A Wilson3 Search for this author in: * NPG journals * PubMed * Google Scholar * J Silvio Gutkind2 Search for this author in: * NPG journals * PubMed * Google Scholar * John A Chiorini1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:662–664Year published:(2010)DOI:doi:10.1038/nm.2145Received30 October 2009Accepted07 April 2010Published online09 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A key step in gene therapy is the efficient transfer of genes in a cell type– and tissue-specific manner. To better understand the mechanism of adeno-associated virus serotype 6 (AAV6) transduction, we used comparative gene analysis (CGA) combined with pathway visualization software to identify a positive correlation between AAV6 transduction and epidermal growth factor receptor (EGFR) expression. Subsequent experiments suggested that EGFR is necessary for vector internalization and probably functions as a co-receptor for AAV6. View full text Figures at a glance * Figure 1: AAV6 transduction corresponds with EGFR expression and function. () A stable EGFR 32D clone (32D-EGFR) was used to evaluate the specific impact of EGFR expression on AAV transduction. Wild-type 32D (32Dwt) and 32D-EGFR cells were transduced with AAV1, AAV2, AAV5 or AAV6 containing a vector composed of a CMV promoter driving EGFP expression. Scale bar, 50 μm. () Quantification of FACS analysis of 32D-EGFR cells 96 h after transduction with AAV2, AAV5, AAV1 or AAV6-CMV-EGFP. ***P < 0.0001, n = 3. () HEK293T and HN13 cells were transfected with siRNA against EGFR and EGFR expression levels were quantified by western blotting. Cells were transduced by AAV2 or AAV6-CMV-eGFP (***P < 0.0001, n = 3). () FACS analysis of HEK293T cells preincubated with one of the EGFR-specific inhibitors, AG1478 and gefitinib and subsequently incubated with AAV6-CMV-eGFP to evaluate the impact of EGFR function on AAV6 mediated transduction. AAV2 transduction was not significantly influenced by EGFR inhibition. ***P < 0.0001, n = 3. () Internalization of AAV6 in 3! 2D-EGFR cells was measured in the presence or absence of gefitinib to evaluate the impact of function EGFR on AAV6 internalization. *P < 0.01, n = 3. () Immunoprecipitation of AAV after incubating AAV2, AAV5, or AAV6 with protein A–sepharose beads alone or beads precoated with rhEGFR-Fc or rhFGFR-Fc. ***P < 0.0001, n = 3. * Figure 2: AAV6 mediated transduction of EGFR expressing tumors and delivery of cytotoxic transgene, HSVtk, followed by ganciclovir treatment results in a significant reduction in tumor growth. () Head and neck tumor cell lines, HN12 and HEp-2, were injected subcutaneously into the right and left flank of female nude mice. After tumors were established, AAV6-CMV-luciferase was introduced by direct intratumoral injection to the right flank tumors, with the vehicle control injected into the left flank tumors. Ten days after AAV administration, in vivo luciferase activity was measured by bioluminescence after intraperitoneal injection of luciferin (representative images, n = 5). () Percentage growth of HN12 tumors injected with AAV6-CMV-HSVtk followed by ganciclovir (GCV) treatment and HN12 tumors treated with GCV alone. The HN12 xenograft tumors received intratumoral injections of AAV6-CMV-HSVtk. One week after AAV6 transduction, mice were started on daily GCV injections. Arrow indicates day GCV treatment was started. *P < 0.05, **P < 0.001, n = 9. Mice were housed in a pathogen-free facility, and all procedures involving mice were performed in compliance with the NI! H Guidelines on Use of Animals in Research and approved by the NIDCR Animal Care and Use Committee. Author information * Author information * Supplementary information Affiliations * Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, US National Institutes of Health (NIH), Bethesda, Maryland, USA. * Melodie L Weller, * Michael Schmidt & * John A Chiorini * Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland USA. * Panomwat Amornphimoltham & * J Silvio Gutkind * Custom Applications Branch, Division of Enterprise and Custom Applications Center for Information Technology, NIH, Bethesda, Maryland, USA. * Paul A Wilson Contributions M.L.W. performed experiments, analyzed data and wrote the paper; P.A. performed experiments, analyzed data and wrote the paper; M.S. performed experiments and analyzed data; P.A.W. developed software used in CGA; J.S.G. contributed tumor model and head-and-neck tumor cell lines and analyzed data; J.A.C. analyzed data and wrote the paper. All authors were involved in the review and editing of the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * John A Chiorini (jchiorini@dir.nidcr.nih.gov) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figs. 1–3 and Supplementary Methods Additional data - ERK activation drives intestinal tumorigenesis in Apcmin/+ mice
- Nat Med 16(6):665-670 (2010)
Nature Medicine | Article ERK activation drives intestinal tumorigenesis in Apcmin/+ mice * Sung Hee Lee1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Li-Li Hu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jose Gonzalez-Navajas1 Search for this author in: * NPG journals * PubMed * Google Scholar * Geom Seog Seo1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Shen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan Brick1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Herdman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nissi Varki2 Search for this author in: * NPG journals * PubMed * Google Scholar * Maripat Corr1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jongdae Lee1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eyal Raz1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:665–670Year published:(2010)DOI:doi:10.1038/nm.2143Received18 October 2009Accepted01 April 2010Published online09 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apcmin/+ mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal–related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apcmin/+/Myd88−/− mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apcmin/+ mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apcmin/+/Myd88−/− mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apcmin/+/Myd88−/− and Apcmin/+ mice and increased th! eir survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host. View full text Figures at a glance * Figure 1: Genetic disruption of Myd88 in Apcmin/+ mice suppresses proliferation and enhances apoptosis of IECs. () IHC and BrdU incorporation in IECs (DSI) after intraperitoneal (i.p.) BrdU injection (scale bars, 20 μm; magnification, ×200). BrdU-positive cells, per time point, were enumerated for each indicated position in a crypt (10 crypt-villi units per time point), with position 0 being the base of the crypt39. () Apoptotic IECs (DSI) were determined by TUNEL assay (scale bars, 40 μm; magnification, ×100). () Cleaved product of poly(ADP-ribose) polymerase (PARP) in IECs (DSI) harvested from the indicated mice (n = 2 per group). Error bars represent s.d. * Figure 2: Myd88 signaling in hematopoietic cells is not required for tumorigenesis in Apcmin/+ mice. () Polyp count in bone marrow chimeras in the DSI and colon (n = 7–9 mice per group); n.s., nonsignificant. () Polyp count in the small intestine in Apcmin/+/Il1r1−/− and Apcmin/+/Casp1−/− mice at 20 weeks of age (n = 7 per group). () Polyp count in anakinra-treated Apcmin/+ mice (DSI) (n = 7 per group). P < 0.05. * Figure 3: MyD88 regulates c-Myc expression levels. () IHC analysis of c-Myc protein in IECs from the DSI and colon from 20-week-old mice (scale bars, 10 μm; magnification, ×200). () Immunoblot analysis of the indicated proteins in IECs (DSI) of 20-week-old mice (n = 2). () Transcript abundance of Myc in IECs (DSI) (nonsignificant; n = 3 per group). Error bars represent s.d. () RKO cells transfected with either control or Myd88 siRNA, stimulated with Wnt3a (100 ng ml−1) and subjected to immunoblot analysis. * Figure 4: TLR signaling via MyD88 stabilizes c-Myc protein in IECs through activation of ERK. () RKO cells stimulated with P3C (2 μg ml−1), lysed and analyzed by immunoblotting (IB; top) and transcript levels (qPCR) after TLR2 stimulation (bottom). () Abundance of protein (IB; top) and transcript (qPCR; bottom) in MG-132 treated (10 μM) RKO cells. c-Myc was immunoprecipitated (IP) and then subjected to IB with anti-ubiquitin (Ub) antibody. () RKO cells were treated with P3C (2 μg ml−1), and the abundance of ubiquitinated c-Myc was measured by IP followed by IB. () p-ERK and c-Myc abundance (IB) in U0126- or PD0325901-treated RKO cells. * Figure 5: Activation of ERK restores the Min phenotype in Apcmin/+/Myd88−/− mice. () PD reduces the number of polyps in EGF-treated Apcmin/+/Myd88−/− mice (DSI) (n = 8 per group). (,) Blood hemoglobin concentrations () and body weight () of these mice. () Top, H&E of DSI in control, EGF-treated and EGF + PD–treated mice. The arrows indicate intestinal polyps. Middle, c-Myc expression in IEC. Bottom, p-ERK levels in IECs. Error bars represent s.d. * Figure 6: Activation of ERK is essential for the Min phenotype in Apcmin/+ mice. (–) Polyp count (), hemoglobin level () and body weight () in PD-treated Apcmin/+ mice (n = 6 for vehicle, n = 9 for PD group). () Top, H&E of DSI in control and PD-treated Apcmin/+ mice. Arrows indicate intestinal polyps. Bottom, c-Myc expression in IECs. () Immunoblot analysis of c-Myc and p-ERK levels in IECs (DSI) of these mice. () Survival in PD-treated or vehicle-treated Apcmin/+ mice for 17 weeks (n = 8). () The PD-treated Apcmin/+ mice in were split to PD- and vehicle-treated groups (n = 4 per group). Polyp count (DSI) was performed 15 weeks later. () Microflora induces tumorigenesis in Apcmin/+ mice by triggering the TLR-ERK pathway in IECs. This stabilizes c-Myc and inhibits its proteasomal degradation. Increased c-Myc levels induce the Min phenotype. Additional signals such as growth factors use the MEK-ERK pathway and, like TLR ligands, can enhance c-Myc expression. However, sterile food and water contain TLR ligands (for example, lipopolysaccharides) that are ! capable of stimulating IECs. This may account for the Min phenotype observed in Apcmin/+ mice housed under germ-free conditions40. Error bars represent s.d. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Medicine, University of California, San Diego, La Jolla, California, USA. * Sung Hee Lee, * Li-Li Hu, * Jose Gonzalez-Navajas, * Geom Seog Seo, * Carol Shen, * Jonathan Brick, * Scott Herdman, * Maripat Corr, * Jongdae Lee & * Eyal Raz * Department of Pathology, University of California, San Diego, La Jolla, California, USA. * Nissi Varki * Current addresses: College of Pharmacy, Wonkwang University, Iksan, Chonbuk, Republic of Korea (S.H.L.); Wonkwang University School of Medicine, Iksan, Chonbuk, Republic of Korea (G.S.S.). * Sung Hee Lee & * Geom Seog Seo Contributions E.R. designed the study; S.H.L. and J.L. performed the signaling experiments; C.S., L.-L.H., S.H. and G.S.S. performed the in vivo studies; M.C. generated the bone marrow chimeras; J.B., J.L. and J.G.-N. performed immunohistochemistry and flow cytometry; S.H.L., M.C., N.V., J.L. and E.R. analyzed the data; and S.H.L., J.L. and E.R. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eyal Raz (eraz@ucsd.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–4 Additional data - A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic implications
- Nat Med 16(6):671-677 (2010)
Nature Medicine | Article A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic implications * Zhi Sheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Li Li2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lihua J Zhu3 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas W Smith4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Demers5 Search for this author in: * NPG journals * PubMed * Google Scholar * Alonzo H Ross2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard P Moser5 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Green1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:671–677Year published:(2010)DOI:doi:10.1038/nm.2158Received25 September 2009Accepted23 April 2010Published online23 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS–mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element–binding protein-3–like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results! demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma. View full text Figures at a glance * Figure 1: A genome-wide RNAi screen reveals a signaling pathway required for ATF5 expression. () Schematic summary of the genome-wide RNAi screen. DT, diphtheria toxin. () qRT-PCR analysis of endogenous Atf5 expression in GL261 cells transiently transfected with siRNA to luciferase (Luc), FRS2, PAK1 or CREB3L2. () qRT-PCR analysis of endogenous Atf5 expression in GL261 cells treated with sorafenib (10 μM), U0126 (20 μM), FR180204 (40 μM), LY294002 (20 μM), JNK inhibitor I (20 μM), SB203580 (10 μM) or, as a control, DMSO. () ChIP analysis monitoring binding of CREB3L2 to two regions of the Atf5 promoter in GL261 cells treated with or without sorafenib (10 μM). A consensus CRE motif is present at −632 to −639 bp upstream of the transcription start site (bottom). Enrichment of CREB3L2 is expressed relative to a no antibody control. () Luciferase reporter assay in GL261 cells transiently transfected with an Atf5 promoter reporter construct carrying a wild-type (WT) or mutant (Mut) CRE. () Caspase-3/7 activity assay in GL261 cells stably expressing Flag-ATF5 or! , as a control, Flag, and transiently transfected with a nonsilencing (NS) shRNA or shRNA to FRS2, PAK1 or CREB3L2. () Caspase-3/7 activity assay in GL261 cells expressing Flag or Flag-ATF5 and treated with either DMSO, sorafenib (20 μM), JNK inhibitor I (20 μM) or SB203580 (10 μM). () Mouse tumorigenensis assays. Sorafenib (Sor) treatment decreased tumor volume significantly in the absence of ATF5 (P = 0.0045) but not in the presence of ATF5 (P = 0.62). All experiments with statistical analyses were performed at least three times, and error bars depict means ± s.d.; *P < 0.05. * Figure 2: ATF5 promotes survival through upregulation of MCL1. () qRT-PCR analysis of Mcl1 expression in GL261 cells treated with DMSO, sorafenib (10 μM), U0126 (20 μM), FR180204 (40 μM), JNK inhibitor I (20 μM) or SB203580 (10 μM). () Immunoblot analysis monitoring ATF5, MCL1, BCL2, BCL2L1 or BIRC3 abundance in GL261 cells transiently transfected with a Luc- or ATF5-specific siRNA. β-actin (ACTB) was monitored as a loading control. () qRT-PCR analysis of Mcl1 expression in GL261 cells transiently transfected with a Luc- or ATF5-specific siRNA. () ChIP analysis monitoring binding of ATF5 to three regions of the Mcl1 promoter, as indicated, in the presence or absence of sorafenib (10 μM). () Immunoblot analysis monitoring cleaved caspase-3 (c-CASP3) amounts in GL261 cells stably expressing either Flag or Flag-MCL1 and transiently transfected with a Luc- or ATF5-specific siRNA. () A model depicting the ATF5-mediated survival pathway in solid tumors. Dashed lines represent potential alternate pathways. () Immunoblot analysis monitor! ing ATF5, MCL1, c-CASP3 and ACTB amounts in U87MG, DU145, UACC62, A549 and OVCAR3 cells treated with a Luc- or ATF5-specific siRNA. All experiments with statistical analyses were performed at least three times, and error bars depict means ± s.d.; *P < 0.05. * Figure 3: ATF5 expression correlates with poor prognosis in human malignant glioma. () Immunohistochemical analysis of phosphorylated ERK (pERK), CREB3L2, ATF5 and MCL1 in brain tissue sections from a normal individual and a subject with glioblastoma. Samples were also stained with H&E. () Kaplan-Meier analysis assessing the correlation between survival of 23 individuals with malignant glioma and the expression of ATF5 (left) or pERK (right). 'Positive' and 'negative' refer to the presence and absence, respectively, of ATF5 or pERK expression in glioblastomas. The P value of both ATF5 and pERK expression in subject survival was 0.03. The time in months refers to the time of death after the malignant glioma was first diagnosed. * Figure 4: The ATF5-mediated survival pathway is essential for viability of human malignant glioma cells. () Immunoblot analysis of pERK, ERK, CREB3L2, ATF5, MCL1, GFAP, nestin (NES) and tubulin in GS9-6 GSCs treated in the presence or absence of serum. () qRT-PCR analysis monitoring CREB3L2, ATF5, MCL1, CD133, NES and GFAP expression in D456MG and GS9-6 GSCs treated in the presence of serum, relative to the expression in the absence of serum. () Immunoblot analysis of CREB3L2, ATF5, MCL1, c-CASP3 and ACTB in GS9-6 GSCs treated in the absence or presence of 40 μM sorafenib. () qRT-PCR analysis of CREB3L2, ATF5 and MCL1 in GS9-6 GSCs treated in the absence or presence of 40 μM sorafenib. () Cell viability, monitored by method of transcriptional and translational (MTT) assay, of GS9-6 GSCs incubated with or without serum and then treated with sorafenib for 96 h. Cell viability was significantly affected by sorafenib treatment (P = 3.53 × 10−7). All experiments with statistical analyses were performed at least three times, and error bars depict means ± s.d.; *P < 0.05. * Figure 5: Inhibition of MAPK signaling suppresses development of malignant glioma in mouse xenografts. () Orthotopic mouse experiments. Tumor formation was monitored by MRI (left); the position of the tumor is indicated by the arrowhead. Brain sections were stained with H&E (middle) or an antibody to GFAP (right) to verify the glial origin of the tumor. Expression of CREB3L2, ATF5 and MCL1 in these sections is shown in Supplementary Figure 5. () Quantification of tumor size, on the basis of MRI images. Sorafenib significantly inhibited tumor formation (P = 0.0344). () c-CASP3 staining in brain sections from vehicle- or sorafenib-treated mice; arrowheads indicate c-CASP3–positive (apoptotic) cells. () c-CASP3–positive cells were counted in three different fields under the microscope, and the average value is shown. All experiments with statistical analyses were performed at least three times, and error bars depict means ± s.d.; *P < 0.05. * Figure 6: Sorafenib synergizes with temozolomide to inhibit tumor cell growth. () Cell viability of U87MG cells treated with combinations of sorafenib and temozolomide (TMZ), as measured by MTT assay. The combination of sorafenib and TMZ significantly decreased cell viability (P = 1.468 × 10−5). () Quantification of tumor size in mouse subcutaneous xenograft experiments. The combination of sorafenib and TMZ treatment significantly decreased tumor volume compared to sorafenib (P = 4.4 × 10−5) or TMZ (P = 5.6 × 10−6) alone. All error bars depict means ± s.d. × 10−5. Author information * Abstract * Author information * Supplementary information Affiliations * Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Zhi Sheng & * Michael R Green * Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Li Li & * Alonzo H Ross * Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Lihua J Zhu * Department of Pathology and Neurology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Thomas W Smith * Department of Surgery, Division of Neurosurgery, University of Massachusetts Medical School, Massachusetts Center for Translational Research in Neurosurgical Oncology, Worcester, Massachusetts, USA. * Andrea Demers & * Richard P Moser Contributions Z.S. and M.R.G. designed all experiments. Z.S. performed all experiments. Z.S. and M.R.G. prepared the manuscript. L.L. and A.H.R. assisted with intracranial injections. T.W.S., A.D. and R.P.M. helped analyze human malignant gliomas. L.J.Z. performed all statistical analyses. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael R Green (michael.green@umassmed.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 and Supplementary Tables 1–3 Additional data - Transcriptional regulation of endochondral ossification by HIF-2α during skeletal growth and osteoarthritis development
- Nat Med 16(6):678-686 (2010)
Nature Medicine | Article Transcriptional regulation of endochondral ossification by HIF-2α during skeletal growth and osteoarthritis development * Taku Saito1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Atsushi Fukai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Akihiko Mabuchi3 Search for this author in: * NPG journals * PubMed * Google Scholar * Toshiyuki Ikeda2 Search for this author in: * NPG journals * PubMed * Google Scholar * Fumiko Yano4 Search for this author in: * NPG journals * PubMed * Google Scholar * Shinsuke Ohba4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nao Nishida3 Search for this author in: * NPG journals * PubMed * Google Scholar * Toru Akune5 Search for this author in: * NPG journals * PubMed * Google Scholar * Noriko Yoshimura5 Search for this author in: * NPG journals * PubMed * Google Scholar * Takumi Nakagawa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kozo Nakamura1 Search for this author in: * NPG journals * PubMed * Google Scholar * Katsushi Tokunaga3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ung-il Chung4 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroshi Kawaguchi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:678–686Year published:(2010)DOI:doi:10.1038/nm.2146Received10 February 2010Accepted08 March 2010Published online23 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2α (HIF-2α, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2α enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2α, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2α expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis development, an! d a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-κB (NF-κB) family member, as a potent inducer of HIF-2α expression. Hence, HIF-2α is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis. View full text Figures at a glance * Figure 1: Transcriptional regulation of COL10A1 by HIF-2α. () Luciferase assay for screening transcription factors that activate the COL10A1 promoter by the transfections of candidate genes into ATDC5 and HeLa cells with a reporter construct containing a fragment (−1,028 to +127 bp) of the COL10A1 gene. Data are shown as means ± s.d. () Immunoprecipitation and immunoblotting analysis by co-transfections of Flag-tagged HIF-2α or the control empty vector (EV) and hemagglutinin (HA)-tagged β-subunit members or the EV in ATDC5 cells. () Mammalian two-hybrid assay by transfections of vectors expressing GAL4–HIF-2α and VP16–β-subunit fusion proteins with the luciferase reporter vector with GAL4 binding sites into HeLa cells. Data are shown as means ± s.d. of relative fold increase in luciferase activity as compared to EV + EV (which is arbitrarily set to 1). () Site-directed mutagenesis analyses of the luciferase assay; one in HRE1 and three in HRE2 (+87 and +88 for mut1, +91 and +92 for mut2, and both for mut3), in the two ce! ll lines transfected with GFP, HIF-2α, ARNTL or both HIF-2α and ARNTL. Data are shown as means ± s.d. () EMSA for specific binding (arrowhead) of the wild-type (WT) oligonucleotide probe containing HRE2 or the mutated probes described in (m1, m2 and m3) with in vitro–translated HIF-2α, ARNT or both. Supershift by an antibody to HIF-2α (anti–HIF-2α) and cold competition with a 50-fold excess of unlabeled WT or the mutated probe are presented. () ChIP assay with cell lysates of human chondrogenic SW1353 cells that were amplified by a primer set spanning the HRE2 (+, +32 to +249 bp) or not spanning the HRE2 (−, −2,131 to −1,900 bp) before (input) and after immunoprecipitation with anti-HIF-2α or nonimmune IgG (anti-IgG). * Figure 2: In vitro and in vivo expression patterns of the HIF α- and β-subunit members and Col10a1, Mmp-13 and Vegf during chondrocyte differentiation. () Time course of mRNA levels of the indicated genes during differentiation of mouse chondrogenic ATDC5 cells cultured with ITS (insulin, transferrin and sodium selenite) for 3 weeks and for 2 d more with inorganic phosphate (Pi). Data are expressed as means ± s.d. () H&E staining and immunofluorescence with antibodies to the indicated proteins, as well as a nonimmune control, in the proximal tibias of mouse embryos (embryonic day 18.5 (E18.5)). Scale bars, 100 μm. Red and blue bars to the left of each row indicate layers of proliferative and hypertrophic zones, respectively. * Figure 3: Skeletal abnormality in Epas1-deficient mice. () Wild-type (WT), heterozygous-deficient (Epas1+/−) and homozygous-deficient (Epas1−/−) littermate embryos (E17.5). All Epas1−/− embryos died at mid-gestation. Scale bars, 1 mm. () Double staining with Alizarin red and Alcian blue of the whole skeleton of WT and Epas1+/− littermate embryos (E17.5). Scale bars, 1 mm. () Length of long bones and vertebra (first to fifth lumbar spines) of WT and Epas1+/− littermate embryos. Data are expressed as means ± s.d. *P < 0.05 versus WT. () H&E staining of whole tibias of the WT and Epas1+/− littermate embryos. Inset boxes indicate the regions of the bottom three rows representing proliferative zone, hypertrophic zone and bone area, shown by red, blue and green bars, respectively. Scale bars, 100 μm. () Percentage of the length of proliferative zone (red), hypertrophic zone (blue) and bone area (green) over the total tibial length of the WT and Epas1+/− littermate embryos. () Immunofluorescence with antibodies to Hi! f-2α, Col10a1, Mmp-13 and Vegf, as well as bromodeoxyuridine (BrdU) labeling and von Kossa staining of the proximal tibias of WT and Epas1+/− littermate embryos (E17.5). Color bars indicate layers as indicated in . Scale bars, 200 μm. () The number of BrdU-positive cells in 1 × 104 μm2 of the proximal tibia of WT and Epas1+/− littermate embryos. Data are expressed as means ± s.d. * Figure 4: Effects of gain and loss of function of HIF-2α on endochondral ossification parameters in cultures of chondrogenic cells. () mRNA levels of Col10a1, Mmp13 and Vegfa, alkaline phosphatase (ALP) activity (relative to control) and Alizarin red staining in stable lines of ATDC5 cells retrovirally transfected with GFP, HIF-2α, ARNTL or both HIF-2α and ARNTL and in nontransfected parental cells (−) after culture for 3 weeks with ITS and 2 d with Pi. HIF-2α and ARNTL levels were confirmed by western blotting, with the actin level as the internal control. () Analyses of the parameters in in stable ATDC5 lines transfected with GFP or HIF-2α mutants at the oxygen-dependent hydroxylation residues causing enhancement (N847A and P531A) and abrogation (P849A) of HIF-2α transactivation activity under the culture conditions used in . Gene expression was confirmed by RT-PCR with the EPAS1 primer set inside the coding sequence, with the Gapdh level as the internal control. () Analyses of the same read-outs in in stable ATDC5 lines transfected with GFP or HA-tagged dominant-negative HIF-2α (HA–dnHIF-2α! ) (left) or siRNA specific for GFP or Epas1 mRNA (right) under the cultures conditions used in . HA–dnHIF-2α and Hif-2α amounts were confirmed by western blotting. () mRNA levels of Col10a1, Mmp13 and Vegfa and Epas1 in the pellet cultures of primary chondrocytes derived from wild-type (WT) and Epas1+/− littermate embryos for 2 weeks. For the rescue experiment, adenoviral transfection with HIF-2α (Ax–HIF-2α) or the control GFP (Ax-GFP) was performed before the pellet formation. All data are expressed as means ± s.d. *P < 0.05 versus GFP unless otherwise indicated. * Figure 5: Contribution of HIF-2α to osteoarthritis development in mice and humans. () Cartilage degradation assessed by safranin O staining and expression of Hif-2α, Col10a1, Mmp-13 and Vegf by immunostaining (brown) and immunofluorescence (green) in mouse knee joints 0 and 8 weeks after creating a surgical osteoarthritis model in 8-week-old wild-type (WT) and Epas1+/− littermates. Boxed areas in each safranin O–stained or each immunostained image indicate the regions shown in the enlarged safranin O–stained or immunofluorescent image immediately below. Scale bars, 100 μm. () Quantification of osteoarthritis development by our (two left graphs) and OARSI (two right graphs) grading systems. Data are expressed as means ± s.d. #P < 0.05, *P < 0.01 versus WT. () Safranin O staining, H&E staining and immunofluorescence with an antibody to HIF-2α in human tibial cartilages of various degradation stages, subchondral bone (beneath the cartilage with Mankin score = 8) and synovium (around the cartilage with Mankin score = 8), obtained as surgical specimen! s of total knee arthroplasty. Scale bars, 100 μm. () Top, the identified SNP, rs17039192, and primers used for genotyping (red lines) in the human EPAS1 gene. CDS, coding sequence. Bottom, association of the rs17039192 (+18C/T) SNP with knee osteoarthritis (OA) diagnosed on radiographs using the Kellgren/Lawrence grade in a Japanese population. The odds ratio of the susceptibility allele was 1.44 (95% confidence interval: 1.08–1.92). *P = 0.05, **P = 0.013. () Luciferase activities in chondrogenic SW1353, OUMS27 and ATDC5 cells and nonchondrogenic HeLa cells transfected with a luciferase reporter gene construct ligated to a fragment (−1,000 bp to +488 bp) containing +18C or +18T. Data are shown as means ± s.d. *P < 0.05 versus 18C. * Figure 6: Upstream mechanism that regulates HIF-2α. () Luciferase activities after transfections of putative chondrocyte-related transcription factors into chondrogenic SW1353, OUMS27 and ATDC5 cells and nonchondrogenic HeLa cells with a reporter construct containing a fragment (−1,000 bp to +488 bp) of the EPAS1 gene. OSX, osterix; AP2, transcription factor AP-2α; Notch1-ICD, intercellular domain of Notch1; HES1, hairy and enhancer of split 1. Data are shown as means ± s.d. () Top, depiction of the NF-κB motif (−983 to −973) in the human EPAS1 gene. Bottom, site-directed mutagenesis analyses of the luciferase assay in the three chondrogenic cell lines transfected with GFP or RELA. Luciferase activities were compared with or without mutation in the NF-κB motif and with +18C or +18T of the rs17039192 SNP. Data are shown as means ± s.d. *P < 0.05 versus wild-type NF-κB and 18C with RELA. () mRNA levels of EPAS1 in the three chondrogenic cells cultured with or without TNF-α or IL-1β (each 1 ng ml−1) for 2 d. Data! are expressed as means ± s.d. *P < 0.05 versus control. () Time course of degradation in mouse knee joint cartilage, as shown by Safranin O staining and expression of Rela and Hif-2α by immunostaining and immunofluorescence, respectively, in a surgical osteoarthritis model in 8-week-old mice. Boxed areas in each of the top images are enlarged in the bottom images directly beneath. Scale bar, 100 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Sensory & Motor System Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Taku Saito, * Atsushi Fukai, * Takumi Nakagawa, * Kozo Nakamura & * Hiroshi Kawaguchi * Bone and Cartilage Regenerative Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Taku Saito & * Toshiyuki Ikeda * Human Genetics, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Akihiko Mabuchi, * Nao Nishida & * Katsushi Tokunaga * Center for Disease Biology and Integrative Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Fumiko Yano, * Shinsuke Ohba & * Ung-il Chung * 22nd Century Medical and Research Center, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan. * Toru Akune & * Noriko Yoshimura Contributions T.S., T.I. and H.K. performed project planning; T.S., A.F., A.M., F.Y. and S.O. performed the experiments; T.S., A.M., N.N., T.A., N.Y., T.N., K.N., K.T., U.-i.C. and H.K. conducted data analysis; T.S. and H.K. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hiroshi Kawaguchi (kawaguchi-ort@h.u-tokyo.ac.jp) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–7 and Supplementary Table 1 Additional data - Hypoxia-inducible factor-2α is a catabolic regulator of osteoarthritic cartilage destruction
- Nat Med 16(6):687-693 (2010)
Nature Medicine | Article Hypoxia-inducible factor-2α is a catabolic regulator of osteoarthritic cartilage destruction * Siyoung Yang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonghwan Kim1 Search for this author in: * NPG journals * PubMed * Google Scholar * Je-Hwang Ryu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hwanhee Oh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Churl-Hong Chun2 Search for this author in: * NPG journals * PubMed * Google Scholar * Byoung Ju Kim3 Search for this author in: * NPG journals * PubMed * Google Scholar * Byoung Hyun Min3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jang-Soo Chun1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:687–693Year published:(2010)DOI:doi:10.1038/nm.2153Received22 February 2010Accepted13 April 2010Published online23 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Osteoarthritic cartilage destruction is caused by an imbalance between anabolic and catabolic factors. Here, we show that hypoxia-inducible factor-2α (HIF-2α, encoded by EPAS1) is a catabolic transcription factor in the osteoarthritic process. HIF-2α directly induces the expression in chondrocytes of genes encoding catabolic factors, including matrix metalloproteinases (MMP1, MMP3, MMP9, MMP12 and MMP13), aggrecanase-1 (ADAMTS4), nitric oxide synthase-2 (NOS2) and prostaglandin-endoperoxide synthase-2 (PTGS2). HIF-2α expression was markedly increased in human and mouse osteoarthritic cartilage, and its ectopic expression triggered articular cartilage destruction in mice and rabbits. Moreover, mice transgenic for Epas1 only in chondrocytes showed spontaneous cartilage destruction, whereas heterozygous genetic deletion of Epas1 in mice suppressed cartilage destruction caused by destabilization of the medial meniscus (DMM) or collagenase injection, with concomitant modulati! on of catabolic factors. Our results collectively demonstrate that HIF-2α causes cartilage destruction by regulating crucial catabolic genes. View full text Figures at a glance * Figure 1: Regulatory mechanisms of Epas1 expression in articular chondrocytes. (,) RT-PCR and western blot () and qRT-PCR () analyses of Epas1 in mouse articular chondrocytes treated with pro- and anti-inflammatory cytokines. Values in are means ± s.e.m. (n ≥ 6). *P < 0.005 as compared to untreated control (0 h). Erk and glyceladehyde-3-phosphate dehydrogenase (Gapdh) serve as loading controls. () Western blot analysis of inhibitor of κB (IκB) and MAP kinase subtypes in mouse articular chondrocytes treated with IL-1β (5 ng ml−1) for the indicated time periods. (,) Western blot and RT-PCR () and qRT-PCR () analyses in mouse articular chondrocytes treated with IL-1β (5 ng ml−1) for 36 h in the presence of the indicated inhibitors (μM): Bay (BAY 11-7085), an NF-κB inhibitor; PD (PD98059), an Erk inhibitor; SB (SB203580), a p38 MAP kinase inhibitor; SP (SP600125), a Jnk inhibitor. Values are means ± s.e.m. (n ≥ 6). *P < 0.005. () Immunohistochemical staining of phosphorylated IκB (pIκB) and JNK (pJNK) in both normal cartilage and arthriti! s-affected damaged regions of human osteoarthritic (OA) cartilage. Scale bars, 25 μm. * Figure 2: Hif-2α regulates expression of MMP1, MMP3, MMP9, MMP12, MMP13, ADAMTS4, PTGS2 and NOS2 in chondrocytes as direct target genes. (,) RT-PCR analysis in rabbit articular chondrocytes () and western blot analysis in mouse articular chondrocytes () from the cells either untreated or infected with 800 multiplicity of infection (MOI) of mock adenovirus or the indicated amounts (in MOI) of Ad-Epas1 (left). Chondrocytes were either untreated or transfected with control (C) siRNA (100 nM) or the indicated amounts (in nM) of siRNA specific for rabbit () or mouse () Epas1 and exposed to IL-1β (5 ng ml−1) for 36 h (right). Lamin B and Gapdh are loading controls. () Reporter gene activity in rabbit articular chondrocytes transfected with empty vector (EV, 1.0 μg), Epas1 vector (0.5, 1.0, or 1.5 μg) or Epas1 (1.0 μg) with dominant negative (Δ) Epas1 vector (0.5, 1.0 or 1.5 μg). Values are means ± s.e.m. (n ≥ 6). () Reporter gene activity in cells transfected with empty vector (EV) or Epas1 vector. The cells were co-transfected with wild-type (WT) or single- or double-mutant (Δ) reporter genes (see Supp! lementary Fig. 5a). Values are means ± s.e.m. (n ≥ 6). () NO and PGE2 production in mouse articular chondrocytes treated with IL-1β (5 ng ml−1), infected with Ad-Epas1 (MOI), or transfected with control (C) or Epas1 siRNA (nM), followed by treatment with IL-1β. Data represent means ± s.e.m. (n = 5). *P < 0.05, **P < 0.005. * Figure 3: HIF-2α is overexpressed in OA cartilage of humans and STR/ort mice. () qRT-PCR analyses of EPAS1 and COL2A1 and ICRS grade from both undamaged regions (UD) and arthritis-affected damaged (D) human osteoarthritic cartilage regions. Values are means ± s.e.m. (n ≥ 20). () In situ hybridization of EPAS1 (left) and immunohistochemical staining of HIF-2α protein (right) in damaged and undamaged regions of human osteoarthritic cartilage. Scale bars, 30 μm. () RT-PCR (left) and qRT-PCR (right) analysis from normal (N), undamaged (UD) and damaged (D) human osteoarthritic cartilage. Values are means ± s.e.m. (n ≥ 20). () qRT-PCR analysis of Epas1 and Col2a1 and Mankin score of STR/ort (STR) and control CBA/CaCrl (CBA) mice. Values are means ± s.e.m. (n = 6). () Safranin O staining (left) and immunohistochemical staining of Hif-2α (right) from knee joint cartilage of STR/ort and CBA/CaCrl mice. Scale bars, 150 μm (left) and 30 μm (right). () Results of RT-PCR (left) and qRT-PCR (right) analysis from knee joint cartilage of CBA mice (C) and ! STR/ort mice (S). Values represent means ± s.e.m. (n = 5). *P < 0.01, **P < 0.005. * Figure 4: Overexpression of Hif-2α triggers cartilage destruction. () Immunohistochemical staining of Hif-2α in mouse cartilage injected with mock virus or Ad-Epas1 virus (1 × 109 PFU). Scale bars, 30 μm. () Safranin O staining of cartilage sections (left, middle) and Mankin score (right) in mice injected with mock virus or Ad-Epas1 virus (1 × 109 PFU). The middle images are enlarged versions of the marked regions in the left images. Scale bars, 150 μm. Values are means ± s.e.m. (n = 9). () Results of RT-PCR (left) and qRT-PCR (right) analyses of the indicated genes. Values represent means ± s.e.m. (n = 8). *P < 0.01, **P < 0.005. * Figure 5: Chondrocyte-specific Epas1-transgenic (TG) mice show spontaneous cartilage destruction. () Immunohistochemical staining of Hif-2α in the cartilage sections of WT and Epas1-TG mice. Scale bars, 30 μm. () Safranin O staining of cartilage sections (left) and Mankin score (right) in WT and Epas1-TG mice (12-week-old and 45-week-old). Scale bars, 150 μm. Values represent means ± s.e.m. (n = 8). () Results of RT-PCR (left) and qRT-PCR (right) analyses in WT and Epas1-TG mice cartilage. Data are means ± s.e.m. (n = 6). *P < 0.01, **P < 0.005. * Figure 6: Genetic deletion of one allele of Epas1 inhibits OA cartilage destruction. (,) Safranin O staining of cartilage sections (left) and Mankin score (right) () and RT-PCR analysis of the indicated molecules () in WT (+/+) and Epas1+/− mice injected with collagenase (Coll) or PBS. Scale bars, 150 μm. Values represent means ± s.e.m. (n = 9). (,) Safranin O staining of cartilage sections (left) and Mankin score (right) () and RT-PCR analysis of the indicated molecules () in WT and Epas1+/− mice. Cartilage destruction was caused by DMM. Sham operation was used as a control (Ctrl). Scale bars, 150 μm. Values represent means ± s.e.m. (n = 9). *P < 0.001. Author information * Abstract * Author information * Supplementary information Affiliations * Cell Dynamics Research Center, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Korea. * Siyoung Yang, * Jonghwan Kim, * Je-Hwang Ryu, * Hwanhee Oh & * Jang-Soo Chun * Department of Orthopedic Surgery, Wonkwang University School of Medicine, Iksan, Korea. * Churl-Hong Chun * Department of Molecular Science & Technology and Department of Orthopedic Surgery, Ajou University, Suwon, Korea. * Byoung Ju Kim & * Byoung Hyun Min Contributions S.Y. contributed to the writing of the manuscript and performed most of the experiments, except for the NO and PGE2 assays (performed by J.K.), the cartilage explants and synovitis experiments (performed by J.-H.R.) and immunofluorescence microscopy and subchondral bone assays (performed by H.O.). C.-H.C. provided and evaluated human cartilage samples. B.J.K. and B.H.M. performed microsurgery to induce DMM. J.-S.C. initiated and supervised the project and contributed to the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jang-Soo Chun (jschun@gist.ac.kr) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–11, Supplementary Tables 1–3 and Supplementary Methods Additional data - Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors
Murray KC Nakae A Stephens MJ Rank M D'Amico J Harvey PJ Li X Harris RL Ballou EW Anelli R Heckman CJ Mashimo T Vavrek R Sanelli L Gorassini MA Bennett DJ Fouad K - Nat Med 16(6):694-700 (2010)
Nature Medicine | Article Recovery of motoneuron and locomotor function after spinal cord injury depends on constitutive activity in 5-HT2C receptors * Katherine C Murray1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aya Nakae2 Search for this author in: * NPG journals * PubMed * Google Scholar * Marilee J Stephens1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle Rank1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica D'Amico3 Search for this author in: * NPG journals * PubMed * Google Scholar * Philip J Harvey1 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaole Li1 Search for this author in: * NPG journals * PubMed * Google Scholar * R Luke W Harris1 Search for this author in: * NPG journals * PubMed * Google Scholar * Edward W Ballou1 Search for this author in: * NPG journals * PubMed * Google Scholar * Roberta Anelli3 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles J Heckman4 Search for this author in: * NPG journals * PubMed * Google Scholar * Takashi Mashimo2 Search for this author in: * NPG journals * PubMed * Google Scholar * Romana Vavrek1 Search for this author in: * NPG journals * PubMed * Google Scholar * Leo Sanelli1 Search for this author in: * NPG journals * PubMed * Google Scholar * Monica A Gorassini3 Search for this author in: * NPG journals * PubMed * Google Scholar * David J Bennett1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Karim Fouad1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:694–700Year published:(2010)DOI:doi:10.1038/nm.2160Received04 September 2009Accepted08 April 2010Published online30 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Muscle paralysis after spinal cord injury is partly caused by a loss of brainstem-derived serotonin (5-HT), which normally maintains motoneuron excitability by regulating crucial persistent calcium currents. Here we examine how over time motoneurons compensate for lost 5-HT to regain excitability. We find that, months after a spinal transection in rats, changes in post-transcriptional editing of 5-HT2C receptor mRNA lead to increased expression of 5-HT2C receptor isoforms that are spontaneously active (constitutively active) without 5-HT. Such constitutive receptor activity restores large persistent calcium currents in motoneurons in the absence of 5-HT. We show that this helps motoneurons recover their ability to produce sustained muscle contractions and ultimately enables recovery of motor functions such as locomotion. However, without regulation from the brain, these sustained contractions can also cause debilitating muscle spasms. Accordingly, blocking constitutively act! ive 5-HT2C receptors with SB206553 or cyproheptadine, in both rats and humans, largely eliminates these calcium currents and muscle spasms, providing a new rationale for antispastic drug therapy. View full text Figures at a glance * Figure 1: Constitutive 5-HT2 receptor activity, but not residual 5-HT, causes spasms. () Schematic of tail spasm in an awake chronic spinal rat with S2 sacral transection. () Representative immunofluorescence images of 5-HT fibers (beaded) in the S4 ventral horn of normal rats (top; mn, motoneuron, n = 5 rats) and chronic spinal rats (bottom; the arrow indicates a residual fiber, n = 5; scale bar, 50 μm). (,) Spasms in chronic spinal rat evoked by cutaneous electrical stimulation of the tail (pulse three times the threshold (3×T)) and recorded with EMG (quantified during the length of time indicated by the bar, LLR) before and after blocking effects of residual 5-HT with i.t. injection of the neutral antagonist SB242084 (3 mM in 30 μl saline). () Lack of spasm (LLR) after blocking constitutive receptor activity with the inverse agonist SB206553 (i.t., 3 mM in 30 μl saline). () Tail flexion angle during spasms before and after SB206553 injection, quantified during the length of time indicated by the bar. () Group means of spasms (normalized to predrug cont! rol) with SB242084 (abbreviated SB242; LLR), SB206553 (SB206 for LLR EMG recording; and SB206+ for tail-angle spasms) and cyproheptadine (cypro; LLR; 10 mg per kg body weight, orally), and after depletion of residual 5-HT with para-chlorophenylalanine-methyl-ester (pCPA) (two 300 mg per kg body weight intraperitoneal injections over 48 h; tail-angle), with n = 5 rats per drug. (,) Normalized group means of SLR and background EMG with SB242084 and SB206553. **P < 0.01 relative to predrug control, 100%. Error bars indicate s.e.m. * Figure 2: Constitutive 5-HT2 receptor activity contributes to LLRs in the isolated spinal cord in vitro. () Whole sacrocaudal spinal cord below chronic S2 transection maintained in vitro. () Long-lasting reflex triggered by dorsal root stimulation (single pulse, 3×T) and recorded from the ventral roots (LLR, quantified during the length of time indicated by the horizontal bar; counterpart of spasms in Figure 1) before and after blocking effects of residual 5-HT with the neutral 5-HT2 receptor antagonist SB242084 (3–5 μM). () Elimination of LLR, but not SLR, after blocking constitutive 5-HT2 receptor activity with the inverse agonist SB206553 (3–5 μM). Inset, SLR (expanded time scale). () Group means of LLRs (normalized to predrug LLRs) with SB242084 (abbreviated SB242, n = 11), methysergide (Methys, 10 μM, neutral antagonist, n = 12), SB206553 (SB206, n = 24), cyproheptadine (Cypro, 20 μM; n = 6), and SB206553 after prior application of methysergide (30 μM; white bar; Methy+SB206; n = 8). (,) Normalized group means of the SLR and background ventral root activity with ! SB206553 and SB242084. *P < 0.05, **P < 0.01 relative to control, 100%. Error bars indicate s.e.m. * Figure 3: Constitutively active 5-HT2 receptors on motoneurons contribute to Ca2+ PICs underlying spasms. (,) Intracellular recording from motoneuron (mn) in whole spinal cord, in vitro. () Top, Ca2+ PIC in motoneuron of chronic spinal rat, activated by slowly increasing the membrane potential under voltage-clamp in presence of 2 μM tetrodotoxin (TTX) and quantified at its initial peak, where it produced a downward deflection in the recorded current (thick black plot, at arrow, Ca2+ PIC) relative to the leak current (thin line). Bottom plot, small Ca2+ PIC after SB206553 application (5 μM). () Ca2+ PIC in another motoneuron (arrow), which is unaffected by SB242084 application (5 μM). () Top, PIC-mediated plateau and sustained firing (LLR) evoked by dorsal root stimulation (3×T; without TTX) in a motoneuron at rest (without injected current; top). Bottom, with a hyperpolarizing bias current to prevent PIC activation, the same stimulation only evoked a polysynaptic EPSP (lower plot). () Response of same motoneuron as in to dorsal root stimulation after application of SB206553 ! (5 μM), at rest (top) and with a hyperpolarizing bias current (bottom). () Group means of Ca2+ PIC (normalized to predrug Ca2+ PIC in chronic spinal rats, control), with SB206553 (SB206; n = 7), cyproheptadine (cypro, 20 μM; n = 16) and SB242084 (SB242; n = 5) in chronic spinal rats and in acute spinal rats (white bar, no drugs, n = 7). () Normalized group means of EPSP amplitude (middle bar; control mean 4.4 mV) and duration (right bar, control 480 ms) with inverse agonists cyproheptadine or SB206553 (chronic). **P < 0.01 relative to control, 100%. Error bars represent s.e.m. * Figure 4: A highly constitutively active 5-HT2C receptor isoform is upregulated with injury. () Schematic showing 5-HT2C receptor with various isoforms produced by changing three amino acids on its intracellular loop (green; isoforms named by amino acid triplet). These three amino acids (underlined) are changed by post-transcriptional editing of RNA at five sites (A–E; adenosine editing), leading to various native receptor isoforms, of which the unedited isoform (INI) is most highly constitutively active. () Total 5-HT2C receptor mRNA (normalized to an internal control, 18S rRNA) in chronic spinal rats (n = 6) and normal uninjured rats (n = 6). () Proportion of 5-HT2C receptor mRNA with editing at sites A, B and D (editing efficiency) in chronic spinal and normal rats (C and E site editing efficiency < 30% and not changed, data not shown). () Distribution of 5-HT2C receptor isoform mRNA in the spinal cord of normal and chronic spinal rats (15 isoforms detected; the five most prevalent are shown). () Comparison of change in INI isoform expression (top) and Ca2+ PIC! (bottom, recorded in vitro) after chronic spinal injury. *P < 0.05, **P < 0.01, significant change with injury. Error bars indicate s.e.m. * Figure 5: 5-HT2 receptor inverse agonist blocks spasms in spinal cord injured humans. () Leg spasm triggered by brief electrical stimulation of the medial arch of the foot (3–5×T). TA, tibialis anterior. () Spasm recorded with tibialis anterior muscle surface EMG and quantified over the time windows indicated (LLR and SLR), before and 2 h after blocking constitutively active 5-HT2 receptors with cyproheptadine (8 mg administered orally). The inset on a different scale shows SLR. () Gradual reduction in the spasms (LLRs), but not SLRs, over time after inverse agonist application. (,) Normalized group means for LLRs () and SLRs () with cyproheptadine (n = 7 subjects). **P < 0.01 relative to control, 100%. Error bars represent s.e.m. * Figure 6: Spontaneous recovery of locomotion in staggered-hemisected rats depends on constitutively active 5-HT2 receptors. () Schematic of staggered-hemisection SCI, which transects all descending axons from the brain, including 5-HT neurons (white circles), but leaves local propriospinal neurons (black) that transverse the injury and help relay descending signals for initiation of locomotion (gray)41. () Rat walking with good weight support and toe clearance three weeks after the staggered-hemisection (after second hemisection). () Same rat with little hindlimb weight support (just foot paddling motions), while the forelimbs dragged the hindquarters during walking after blocking constitutively active 5-HT2 receptors with SB206553 (3 mM in 30 μl saline, i.t.; same dose as in Fig. 1). Scale bar, 2 cm. () Group means of BBB locomotor scores before and after SB206553 injection (n = 8) and control SB242084 injection (3 mM in 30 μl saline, i.t.; n = 8 rats). **P < 0.01 relative to preinjection. Error bars represent s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * David J Bennett & * Karim Fouad Affiliations * Centre for Neuroscience, Faculty of Rehabilitation Medicine, University of Alberta, Edmonton, Alberta, Canada. * Katherine C Murray, * Marilee J Stephens, * Michelle Rank, * Philip J Harvey, * Xiaole Li, * R Luke W Harris, * Edward W Ballou, * Romana Vavrek, * Leo Sanelli, * David J Bennett & * Karim Fouad * Department of Anesthesiology & Intensive Care, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan. * Aya Nakae & * Takashi Mashimo * Centre for Neuroscience, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada. * Jessica D'Amico, * Roberta Anelli & * Monica A Gorassini * Department of Physiology, Northwestern University, Chicago, Illinois, USA. * Charles J Heckman Contributions K.C.M. performed the in vitro rat experiments, contributed to all other rat studies and co-wrote the paper. M.R., P.J.H., R.L., W.H., L.S., M.J.S., R.V., X.L. and K.F. contributed to the in vivo rat experiments. K.F., R.V., E.W.B., R.A. and C.J.H. contributed to immunolabeling experiments. K.F. co-wrote the paper and shared equally with D.J.B. in senior authorship (last author). A.N. and T.M. conducted mRNA analysis. J.D. and M.A.G. conducted the human experiments. D.J.B. performed in vitro and in vivo rat experiments, supervised or co-supervised all of the experiments and co-wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David J Bennett (bennettd@ualberta.ca) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (704K) Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Methods Additional data - Basophils and the T helper 2 environment can promote the development of lupus nephritis
Charles N Hardwick D Daugas E Illei GG Rivera J - Nat Med 16(6):701-707 (2010)
Nature Medicine | Article Basophils and the T helper 2 environment can promote the development of lupus nephritis * Nicolas Charles1 Search for this author in: * NPG journals * PubMed * Google Scholar * Donna Hardwick2 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Daugas3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gabor G Illei4 Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Rivera1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:701–707Year published:(2010)DOI:doi:10.1038/nm.2159Received16 October 2009Accepted23 April 2010Published online30 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In systemic lupus erythematosus (SLE), self-reactive antibodies can target the kidney (lupus nephritis), leading to functional failure and possible mortality. We report that activation of basophils by autoreactive IgE causes their homing to lymph nodes, promoting T helper type 2 (TH2) cell differentiation and enhancing the production of self-reactive antibodies that cause lupus-like nephritis in mice lacking the Src family protein tyrosine kinase Lyn (Lyn−/− mice). Individuals with SLE also have elevated serum IgE, self-reactive IgEs and activated basophils that express CD62 ligand (CD62L) and the major histocompatibility complex (MHC) class II molecule human leukocyte antigen-DR (HLA-DR), parameters that are associated with increased disease activity and active lupus nephritis. Basophils were also present in the lymph nodes and spleen of subjects with SLE. Thus, in Lyn−/− mice, basophils and IgE autoantibodies amplify autoantibody production that leads to lupus neph! ritis, and in individuals with SLE IgE autoantibodies and activated basophils are factors associated with disease activity and nephritis. View full text Figures at a glance * Figure 1: The lupus-like nephritis in Lyn−/− mice is IL-4 and IgE dependent. () Glomerulonephritis scores obtained from H&E-stained histological kidney sections from aged mice (over 40 weeks) of the indicated genotypes. Data shown as means ± s.e.m. (for WT and Lyn−/−: n = 8; for WT and Igh-7−/−;Lyn−/−: n = 6; for WT and Il4−/−;Lyn−/−: n = 4 and n = 5; for KitW-sh/W-sh and KitW-sh/W-sh;Lyn−/−: n = 11). Statistical analysis was by a two-tailed unpaired Student's t test; ***P < 0.001; NS, not significant. () Representative glomeruli in H&E-stained histological kidney sections of aged mice (40-weeks-old) of the indicated genotypes. Scale bar, 50 μm. () Immunofluorescent detection of glomerular IgG deposits in aged mice (40 weeks) of the indicated genotypes after staining with fluorescein-conjugated antibody to mouse IgG. Scale bar, 50 μm. () ACR measured in the urine of a minimum of 15 aged mice (40 weeks) of the indicated genotype per group. Data are means ± s.e.m. Statistical analysis was by a two-tailed unpaired Student's t! test; ***P < 0.001. * Figure 2: IgE, basophils and IL-4 regulate autoantibody production in Lyn−/− mice, and basophils alter the kidney cytokine environment. () Quantification of dsDNA-specific IgG in the serum of aged mice (40 weeks) of the indicated genotype. Data are means ± s.e.m. (at least 15 mice per group). () Quantification of ANA-specific IgG in the mice studied in . () Quantification of ANA-specific IgG autoantibodies in the serum of aged mice (32 weeks) of the indicated genotypes before (D0) and six days after (D6) injection of the basophil-depleting antibody MAR-1 (−) or isotype control (+). Data are means ± s.e.m. (WT: n = 3; Lyn−/− (+): n = 4; Lyn−/− (−): n = 5). () Same quantification as in for the serum of mice (20-weeks-old) of the indicated genotypes. Data are means ± s.e.m. (for each group, n = 3). () Proportion of splenic CD138+CD19+ plasma cells determined by flow cytometry in mice 6 d after basophil depletion (−) or isotype injection (+). () Quantification of IL-4 (left) and IFN-γ (right) in kidney homogenates from 40-week-old WT and Lyn−/− mice 6 d after basophil depletion (−) or iso! type injection (+). Cytokine amounts were normalized to the total protein content of the respective homogenates. In and , data are means ± s.e.m. (WT and Lyn−/−, at least n = 4 per group). Statistical analysis was by a two-tailed unpaired (,,,) or paired (,) Student's t test; *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 3: Autoreactive IgEs and IgE-CICs are present in the sera of aged Lyn−/− mice. () Quantification of dsDNA-specific IgE in the sera of aged mice (40 weeks) of the indicated genotypes, as determined by semiquantitative ELISA. Data are means ± s.e.m. (more than ten mice per group) normalized to the respective WT control and expressed as arbitrary units. Statistical analysis was by a two-tailed unpaired Student's t test; *P < 0.05; ***P < 0.001. () IgE-CICs and IgG-CICs from serum samples of Igh7−/−;Lyn−/− and Il4−/−;Lyn−/− aged mice (>30-weeks-old) are reduced. Western blots were probed with antibody to mouse IgE or antibody to mouse IgG. One representative of at least ten mice per genotype is shown. () Serum levels of CICs (IgA, IgM and IgG), as determined by semiquantitative ELISA from at least ten aged mice per genotype on complement factor 1q (C1q)-coated plates. Data are means ± s.e.m. normalized to levels in WT mice and reported as arbitrary units. Statistical analysis was by a two-tailed unpaired Student's t test; *P < 0.05; **P 4) (n = 15), as measured by ELISA. Data are means ± s.e.m. Statistical analysis was by a two-tailed unpaired Student's t test; *P < 0.05; **P < 0.01; ***P < 0.001. () dsDNA-specific IgE levels, as determined by semiquantitative ELISA. dsDNA-coated plates were incubated with sera from healthy controls and subjects with SLE (the same populations as in ). Data are means ± s.e.m. (same n as in ) normalized to healthy controls. Statistical analysis was by a two-tailed unpaired Student's t test; *P < 0.05; ***P < 0.001. () IgE-specific IgG levels, as determined by incubating sera from healthy controls and individuals with SLE on human IgE-coated plates. IgE-specific IgG was detected with antibody to human IgG (Fcγ specific). Data are means ± s.e.m. (same n as in! ) normalized to healthy controls. Statistical analysis was by a two-tailed unpaired Student's t test; **P < 0.01. () dsDNA-specific IgE in sera of subjects with SLE classified on the basis of active nephritis (yes, n = 8) or not (no, n = 34) (see Supplementary Methods). Data are means ± s.e.m. Statistical analysis was by a two-tailed unpaired Student's t test. * Figure 6: Basophils in individuals with SLE are active, upregulate CD62L and HLA-DR and home to secondary lymphoid organs. () Flow cytometric analysis of the levels of activated blood basophils (CD203c expression) relative to disease intensity from subjects with inactive, mild or active SLE ((n = 13, n = 15 and n = 15, respectively) as defined in the legend for Figure 5a compared to controls (n = 41). Data are the ratio of CD203c mean fluorescence intensity (MFI) normalized to controls and expressed in arbitrary units. () Flow cytometric analysis of CD62L expression (MFI) on blood basophils in subject groups as in . Data are normalized as in and are expressed as means ± s.e.m. in arbitrary units (AU) (healthy controls: n = 14; inactive SLE: n = 4; moderate SLE: n = 6; active SLE: n = 6). () Flow cytometric analysis of relative HLA-DR levels on HLA-DR+ blood basophils (MFI) from subjects with SLE (P) compared to healthy controls (). Data are normalized as in and expressed as means ± s.e.m. in AU (healthy controls: n = 13; SLE patients: inactive/mild/active n = 4/6/6). () Absolute number of bloo! d basophils (healthy controls: n = 41; inactive SLE: n = 13; moderate SLE: n = 15; active SLE: n = 15) as determined by flow cytometry. Data are means ± s.e.m. In –, statistical analysis was by a two-tailed unpaired Student's t test; *P < 0.05; **P < 0.01; ***P < 0.001. (,) Immunohistochemistry (with the 2D7 monoclonal antibody) of basophils in the lymph nodes () or spleen () of healthy (normal) controls or subjects with SLE (n = 2). Basophils were found in the B cell zone of lymph node germinal centers in individuals with SLE only (). A spleen biopsy from healthy (normal) controls or from an individual with SLE shows the localization of basophils in the germinal centers of subjects with SLE but not normal controls (). Similar results were obtained with a second basophil-specific antibody (BB1) (data not shown). Original magnification, ×20. Scale bar, 200 μm. Insets show the boxed area of the larger images. Original magnification, ×40. Scale bars, 25 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, Maryland, USA. * Nicolas Charles & * Juan Rivera * Office of the Clinical Director, NIAMS, NIH, Bethesda, Maryland, USA. * Donna Hardwick * Institut National de la Santé et de la Recherche Médicale U699, Department of Nephrology, Assistance Publique–Hôpitaux de Paris, Université Paris Diderot, Hôpital Bichat, Paris, France. * Eric Daugas * Sjogren's Syndrome Clinic, Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA. * Gabor G Illei Contributions N.C. and J.R. conceived and directed the project, designed experiments and wrote the manuscript. N.C. conducted experiments. D.H., E.D. and G.G.I. provided SLE patient history, samples and analysis. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Juan Rivera (juan_rivera@nih.gov) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–15, Supplementary Tables 1 and 2 and Supplementary Methods Additional data - Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection
- Nat Med 16(6):708-712 (2010)
Nature Medicine | Letter Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection * Jose C Alves-Filho1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Fabiane Sônego2 Search for this author in: * NPG journals * PubMed * Google Scholar * Fabricio O Souto2 Search for this author in: * NPG journals * PubMed * Google Scholar * Andressa Freitas2 Search for this author in: * NPG journals * PubMed * Google Scholar * Waldiceu A Verri Jr2 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Auxiliadora-Martins3 Search for this author in: * NPG journals * PubMed * Google Scholar * Anibal Basile-Filho3 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew N McKenzie4 Search for this author in: * NPG journals * PubMed * Google Scholar * Damo Xu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Fernando Q Cunha2 Search for this author in: * NPG journals * PubMed * Google Scholar * Foo Y Liew1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:708–712Year published:(2010)DOI:doi:10.1038/nm.2156Received04 March 2010Accepted16 April 2010Published online16 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options1, 2. Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33–treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (TH1) to TH2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection3. Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration4. We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the ! TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein–coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors5, 6. Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis. View full text Figures at a glance * Figure 1: IL-33 attenuates sepsis and increases neutrophil influx to the site of infection and bacteria clearance. (,) Clinical signs () and mortality () after IL-33 (1 μg per mouse per injection) was injected i.v. 24 and 1 h before CLP on naive BALB/c mice. Data are pooled from three experiments, n = 6 mice per group per experiment. () Time course of IL-33 treatment. IL-33 (1 μg) was injected i.v. as a single dose 1, 3 or 6 h after CLP in BALB/c mice. () Survival rate of untreated Il1rl1−/− and WT mice given a milder form of CLP. () sST2 and IL-33 concentration in the peritoneal lavage fluid of WT and Il1rl1−/− CLP mice, as determined by ELISA. Similar results were obtained in the serum of the CLP mice (data not shown). () Survival rate of Il1rl1−/− and WT CLP mice treated with IL-33 as in . () Number of neutrophils in the peritoneum of CLP or sham-operated mice treated with IL-33 or PBS. (,) Bacterial loads in the peritoneum () and in the blood () of CLP mice treated with IL-33 or PBS. Data are means ± s.e.m., n = 5–10 mice per group and are representative of three exp! eriments (,). *P < 0.05. * Figure 2: IL-33 treatment reduces systemic proinflammatory cytokine, chemokine and lung myeloperoixdase (MPO) activity but increases CXCR2 expression on, and chemotaxis of, neutrophils. Naive BALB/c mice were treated with IL-33 as in Figure 1a and then given CLP or sham operated. Experiments were terminated 6 h after CLP, and blood and lungs were collected for analysis. (,) Serum () and peritoneal lavage () TNF-α, IL-6 and CXCL2 concentrations, as determined by ELISA. () Lung MPO, IL-6 and CXCL2 amounts, as determined by MPO assay and ELISA. ND, not detected. (,) Blood neutrophils were analyzed 4 h after CLP for cell surface CXCR2 expression by FACS () and chemotaxis toward CXCL2 (). Data are means ± s.e.m., n = 5 mice per group, and are representative of three experiments. *P < 0.05 compared to PBS-treated group. * Figure 3: IL-33 blocks the downregulation of CXCR2 and chemotaxis mediated by LPS in vitro. () FACS analysis of naive BALB/c bone marrow neutrophils cultured with IL-33 or medium (RPMI) overnight and stained for ST2. (–) Neutrophils were purified from the bone marrow of naive mice and cultured for 1 h with LPS (1 μg ml−1), LTA (1 μg ml−1), IL-33 (10–50 ng ml−1), CXCL2 (30 ng ml−1) or a combination of these reagents, as indicated. The expression of CXCR2 (,) and chemotaxis to CXCL2 (,,,) were determined. In some experiments (), neutrophils were pretreated with CXCL2 or IL-33 before assaying for chemotaxis toward CXCL2. Data are means ± s.e.m., n = 5 replicates per group. *P < 0.05 versus cultures without IL-33 (,), untreated neutrophils (), or cells not pretreated with CXCL2 (). () Survival of CLP mice treated with IL-33 with or without the CXCR2 inhibitor SB225002 (10 mg per kg body weight). Data are representative of two experiments, n = 10 mice per group. * Figure 4: IL-33 blocks the induction of GRK2 by LPS. () Immunofluorescence staining of GRK2 expression (red) in fixed neutrophils purified from the bone marrow of naive BALB/c mice and cultured for 1 h with LPS (1 μg ml−1), IL-33 (50 ng ml−1) or a combination of LPS and IL-33. Nuclei were counterstained with DAPI (blue). Data show representative individual slide staining of three experiments. Results are shown as the means ± s.d. of the mean fluorescence intensity of each field subtracted from the mean intensity of the area measured as background for each. *P < 0.05 compared to all other groups. Scale bar, 10 μm. () Flow cytometric analysis of GRK2 expression in neutrophils from sham-operated or CLP mice treated with IL-33 or PBS 4 h after CLP (n = 5 mice per group). () Neutrophil chemotaxis (toward CXCL2) in the presence of LPS or LTA in the presence or absence of a GRK2 inhibitor (150 μM). n = 5 replicates per group, *P < 0.05 versus respective cultures without inhibitor. () Chemotaxis (toward CXCL8) of purified peri! pheral blood neutrophils from healthy donors in the presence of LPS and graded concentrations of IL-33. n = 4 donors, *P < 0.05 versus cultures without IL-33. For –, data are means ± s.e.m. () CXCR2 expression of peripheral blood neutrophils from individuals with sepsis and healthy donors, as analyzed by FACS. The cells were also examined for chemotaxis (toward CXCL8), n = 5–10 donors per group, *P < 0.05 compared to healthy donors. () sST2 and IL-33 concentrations in sera collected from individuals with sepsis and healthy donors, as analyzed by ELISA. () Schematic representation of the mechanism by which IL-33 facilitates neutrophil migration during sepsis. Author information * Author information * Supplementary information Affiliations * Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK. * Jose C Alves-Filho, * Damo Xu & * Foo Y Liew * Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. * Jose C Alves-Filho, * Fabiane Sônego, * Fabricio O Souto, * Andressa Freitas, * Waldiceu A Verri Jr & * Fernando Q Cunha * Division of Intensive Care, Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil. * Maria Auxiliadora-Martins & * Anibal Basile-Filho * Medical Research Council Laboratory of Molecular Medicine, Cambridge, UK. * Andrew N McKenzie Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Foo Y Liew (f.y.liew@clinmed.gla.ac.uk) or * Fernando Q Cunha (fdqcunha@fmrp.usp.br) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (852K) Supplementary Figures 1–7 Additional data - The Toll-like receptor 4 ligands Mrp8 and Mrp14 are crucial in the development of autoreactive CD8+ T cells
- Nat Med 16(6):713-717 (2010)
Nature Medicine | Letter The Toll-like receptor 4 ligands Mrp8 and Mrp14 are crucial in the development of autoreactive CD8+ T cells * Karin Loser1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Vogl2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Maik Voskort1 Search for this author in: * NPG journals * PubMed * Google Scholar * Aloys Lueken3 Search for this author in: * NPG journals * PubMed * Google Scholar * Verena Kupas1 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Nacken4 Search for this author in: * NPG journals * PubMed * Google Scholar * Lars Klenner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Annegret Kuhn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Foell3 Search for this author in: * NPG journals * PubMed * Google Scholar * Lydia Sorokin5 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas A Luger1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes Roth2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan Beissert1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:713–717Year published:(2010)DOI:doi:10.1038/nm.2150Received23 April 2009Accepted12 April 2010Published online09 May 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mechanisms linking innate immunity and autoimmune responses are poorly understood1. Myeloid-related protein-8 (Mrp8) and Mrp14 are damage-associated molecular pattern molecules (DAMPs) highly upregulated in various autoimmune disorders. We show in a mouse autoimmune model that local Mrp8 and Mrp14 production is essential for the induction of autoreactive CD8+ T cells and the development of systemic autoimmunity. This effect is mediated via Toll-like receptor 4 (TLR4) signaling leading to increased interleukin-17 (IL-17) expression. Notably, expression of Mrp8 and Mrp14 was upregulated in cutaneous lupus erythematosus, and stimulation of CD8+ T cells from individuals with lupus erythematosus with MRP proteins resulted in an upregulation of IL-17, suggesting a key role for MRP8 and MRP14 for the development of autoreactive lymphocytes during human autoimmunity as well. These results demonstrate a link between local expression of DAMP molecules and the development of systemic a! utoimmunity. View full text Figures at a glance * Figure 1: Impaired development of CD40L-induced systemic autoimmunity in Cd40lg × S100A9−/− mice. () Immunofluorescence staining of ear skin and kidneys from wild-type (WT) and Cd40lg-transgenic (Cd40lg) mice before and after onset of autoimmune disease with antibodies specific for Mrp8 and Mrp14. Scale bar, 25 μm. () Mrp8 and Mrp14 (Mrp8/Mrp14) concentrations in the sera of WT mice and Cd40lg-transgenic mice before and after onset of autoimmunity, as quantified by ELISA (n = 5 mice per group), *P < 0.05 versus Cd40lg-transgenic mice before onset of autoimmunity. () Autoimmune dermatitis score in Cd40lg × S100A9−/− mice compared to Cd40lg-transgenic mice (n = 15 mice per group), *P < 0.05 versus Cd40lg-transgenic mice (means ± s.d.). () Autoantibodies in the serum of Cd40lg × S100A9−/− mice, Cd40lg-transgenic mice and WT controls, as visualized by indirect immunofluorescence staining of HEp-2 cells. Scale bar, 25 μm. () Immunofluorescence staining of renal immunoglobulin depositions in Cd40lg-transgenic mice, Cd40lg × S100A9−/− mice and WT controls. Sca! le bar, 25 μm. () FACS analysis of expression of the activation markers CD44, CD43, and CD69 as well as IL-17 in CD8+ T cells from Cd40lg × S100A9−/− mice compared to CD8+ T cells from Cd40lg-transgenic mice (n = 5 mice per group). Cells are gated for CD8, and IL-17 staining was performed after cell permeabilization. () Ear skin biopsies from Cd40lg-transgenic and Cd40lg × S100A9−/− mice as well as WT controls were stained with antibodies to CD8 and IL-17. Merged images are shown, and cells expressing both markers are indicated by arrows. Scale bar, 25 μm. * Figure 2: Mrp8 and Mrp14 proteins are required for the induction of autoreactivity in CD8+ T cells. () Typical skin pathologies of WT mice 4 weeks after adoptive transfer of CD8+ T cells from WT, Cd40lg-transgenic or Cd40lg × S100A9−/− mice (n = 5 mice per group). () Autoantibodies in the serum of WT recipients of CD8+ T cells from Cd40lg × S100A9−/− mice, Cd40lg-transgenic mice or WT controls, as visualized by indirect immunofluorescence staining of HEp-2 cells. Scale bar, 25 μm. () Immunofluorescence staining of immunoglobulin depositions in the kidneys of WT recipients injected with CD8+ T cells from Cd40lg × S100A9−/− donors, Cd40lg-transgenic donors or WT donors. Scale bar, 25 μm. () FACS analysis of IL-17–expressing CD8+ T cells in cervical lymph nodes of WT recipients injected with CD8+ T cells from Cd40lg × S100A9−/− donors compared to WT recipients of Cd40lg-transgenic CD8+ T cells (n = 5 mice per group). Cells are gated for CD8, and FACS staining was performed after cell permeabilization. () Mrp8 and Mrp14 (Mrp8/Mrp14) serum concentrations! in WT recipients of CD8+ T cells from Cd40lg × S100A9−/− donors, Cd40lg-transgenic donors or WT donors, as quantified by ELISA (n = 5 mice per group). *P < 0.05 versus Cd40lg-transgenic donors. () IL-17 secretion in Mrp8- and Mrp14-stimulated CD8+ T cells from WT mice, Cd40lg × S100A9−/− mice or Cd40lg-transgenic mice before and after onset of autoimmunity (n = 5 mice per group). *P < 0.05 versus PBS-treated CD8+ T cells. Data in and are means ± s.d. () Typical skin pathology (top) and renal IgG depositions, as detected by immunofluorescence staining of kidney tissues (bottom) in WT recipients 4 weeks after adoptive transfer of Mrp8- or Mrp14-stimulated CD8+ T cells (n = 4 mice per group). Scale bar, 25 μm. * Figure 3: MRP8 and MRP14 abundance is increased in subjects with lupus erythematosus, and they upregulate IL-17 expression in CD8+ T cells. () Immunohistochemistry showing MRP8 and MRP14 expression in the skin from healthy donors or subjects with lupus erythematosus (LE) and kidneys from individuals with minimal change nephritis (MCN; which can be considered as healthy) or individuals with LE. Scale bar, 25 μm. () MRP8 and MRP14 concentrations in the sera of subjects with lupus erythematosus and healthy donors, as quantified by ELISA (n = 5 individuals per group). *P < 0.05 versus healthy donors (means ± s.d.). () Immunofluorescence staining of lesional skin from individuals with psoriasis, lichen planus, acute atopic dermatitis, chronic urticaria or granuloma annulare with antibodies directed against MRP8 and MRP14 (MRP8/14). Scale bar, 25 μm. () IL-17 expression in CD8+ T cells from subjects with lupus erythematosus and healthy donors (n = 5 per group) after stimulation with MRP8 or MRP14 proteins, as analyzed by flow cytometry. Cells were permeabilized before FACS staining and are gated for CD8. One repres! entative histogram overlay is shown. * Figure 4: Essential role of TLR4 for Mrp8-mediated effects on CD8+ T cells in the induction of autoreactivity. () Flow cytometry and real-time PCR analyses of TLR4 and RAGE expression in CD8+ T cells from WT and autoimmune-prone Cd40lg-transgenic mice (n = 5 mice per group) or healthy donors and subjects with lupus erythematosus (n = 5 individuals per group). () IL-17 expression in CD8+ T cells from Tlr4−/− and Ager−/− mice after stimulation with Mrp8 in the presence of Cd40lg-transgenic LCs (n = 3 mice per group). Data are presented as percentage IL-17 secretion relative to WT controls, *P < 0.05 versus CD8+ T cells from Tlr4+/+ mice. (,) Relative mRNA expression of IL-17, RORC and RUNX1 in mouse () or human () CD8+ T cells after stimulation with MRP8 and/or TLR4 blockade, as quantified by real-time PCR. CD8+ T cells from WT mice () or subjects with lupus erythematosus () were cultured with LCs from autoimmune-prone Cd40lg-transgenic mice () or antigen-presenting cells from the same individual () in the presence or absence of Mrp8 () or MRP8 () and antibodies to TLR4 (n = 10! mice or individuals per group). *P < 0.05 versus without TLR4 blockade. Data are means ± s.d. Author information * Author information * Supplementary information Affiliations * Department of Dermatology, University of Münster, Münster, Germany. * Karin Loser, * Maik Voskort, * Verena Kupas, * Lars Klenner, * Annegret Kuhn, * Thomas A Luger & * Stefan Beissert * Interdisciplinary Center of Clinical Research, University of Münster, Münster, Germany. * Karin Loser, * Thomas Vogl, * Thomas A Luger, * Johannes Roth & * Stefan Beissert * Institute of Immunology, University of Münster, Münster, Germany. * Thomas Vogl, * Aloys Lueken, * Dirk Foell & * Johannes Roth * Institute of Molecular Virology, University of Münster, Münster, Germany. * Wolfgang Nacken * Institute of Pathobiochemistry, University of Münster, Münster, Germany. * Lydia Sorokin Contributions K.L. conceived of the study, performed the experiments, analyzed data, prepared the figures and wrote the paper. T.V. provided recombinant Mrp8 and Mrp14 proteins as well as Mrp8- and Mrp14-specific antibodies, performed Mrp-specific ELISAs and helped to design some of the experiments. M.V., V.K. and L.K. assisted K.L. with some experiments; A.L. and D.F. provided Ager−/− mice; W.N. bred S100A9−/− mice; A.K. collected a few skin biopsies from subjects with lupus erythematosus; L.S. provided human kidney biopsies; T.A.L. provided most of the human skin biopsies and contributed advice in parts of the study; J.R. provided expertise and helped to design experiments and to write the paper; S.B. contributed expertise and helped to write the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Karin Loser (loserk@uni-muenster.de) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (668K) Supplementary Data, Supplementary Methods and Supplementary Figures 1 and 2 Additional data - In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response
- Nat Med 16(6):718-722 (2010)
Nature Medicine | Technical Report In vivo tracking of 'color-coded' effector, natural and induced regulatory T cells in the allograft response * Zhigang Fan1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Joel A Spencer2, 3, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Lu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Costas M Pitsillides2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Gurbakhshish Singh1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pilhan Kim5 Search for this author in: * NPG journals * PubMed * Google Scholar * Seok H Yun5 Search for this author in: * NPG journals * PubMed * Google Scholar * Vasilis Toxavidis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Terry B Strom1 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles P Lin2 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Koulmanda1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:718–722Year published:(2010)DOI:doi:10.1038/nm.2155Received29 May 2009Accepted01 January 2010Published online23 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we present methods to longitudinally track islet allograft–infiltrating T cells in live mice by endoscopic confocal microscopy and to analyze circulating T cells by in vivo flow cytometry. We developed a new reporter mouse whose T cell subsets express distinct, 'color-coded' proteins enabling in vivo detection and identification of effector T cells (Teff cells) and discrimination between natural and induced regulatory T cells (nTreg and iTreg cells). Using these tools, we observed marked differences in the T cell response in recipients receiving tolerance-inducing therapy (CD154-specific monoclonal antibody plus rapamycin) compared to untreated controls. These results establish real-time cell tracking as a powerful means to probe the dynamic cellular interplay mediating immunologic rejection or transplant tolerance. View full text Figures at a glance * Figure 1: In vivo imaging of color-coded T cells. () FACS sorting of DsRed+CD4+GFP− red Teff cells from DsRed–knock-in mice and CD4+GFP+ green nTreg cells from the original knock-in mice. () Graft survival curves of mice treated with CD154-specific mAb plus rapamycin and untreated rejecting controls. The difference in the survival curves is significant, as calculated by either log-rank (Mantel-Cox) (P = 0.0004) or Gehan-Breslow-Wilcoxon (P = 0.0012) tests. () Representative image of allograft-infiltrating nTreg (green), Teff (red) and iTreg cells (yellow) acquired by intravital microscopy. Scale bar, 50 μm. * Figure 2: Analysis of infiltrating T cells within islet allografts. () Representative intravital microscopy images showing T cell infiltration within islet allografts in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin on week 1 and week 2 after transplantation. Scale bar, 100 μm. (–) Summary of cell density of islet allograft–infiltrating nTreg (), iTreg () and Teff () cells, as detected by intravital imaging. (,) Summary of the ratios of islet allograft–infiltrating nTreg to Teff () and iTreg to Teff () cells, as detected by intravital imaging. Error bars represent means ± s.d. * Figure 3: Serial endomicroscopy of infiltrating T cells within islet allografts. Representative endomicroscopy images within islet allografts on days 3, 5, 7, 10, 12 and 14 after transplantation in untreated hosts and hosts treated with CD154-specific mAb plus rapamycin. Each row of images is from the same mouse at the given time points. Infiltrating nTreg (green), Teff (red) and iTreg cells (green + red) accumulate in the allograft over time. Scale bar, 50 μm. * Figure 4: Detection of nTreg, Teff and iTreg cells by in vivo flow cytometry in the peripheral blood. () A representative in vivo flow cytometry trace showing the identification of single positive nTreg (green box), Teff (red boxes) and double-positive iTreg (yellow box) cells. The second peak in the DsRed channel occurred about 45 ms before the second peak in the GFP channel. As this time difference was greater than the uncertainty of the measurements, these two peaks were distinguished as separate cells and not a double-positive iTreg cell. () In vivo flow cytometry showing Teff (red), nTreg (green) and iTreg cells (yellow) in the peripheral blood. There is a ten-fold difference in scale between mice treated with CD154-specific mAb plus rapamycin and untreated mice. Each curve represents serial analysis of the same blood vessel of the same animal. () Summary of the ratio of circulating Treg to Teff cells, as detected by in vivo flow cytometry. Error bars represent means ± s.d. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zhigang Fan & * Joel A Spencer Affiliations * Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA. * Zhigang Fan, * Yan Lu, * Gurbakhshish Singh, * Vasilis Toxavidis, * Terry B Strom & * Maria Koulmanda * Advanced Microscopy Program, Center for Systems Biology and Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Joel A Spencer, * Costas M Pitsillides & * Charles P Lin * Department of Biomedical Engineering, Science and Technology Center, Tufts University, Medford, Massachusetts, USA. * Joel A Spencer * Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA. * Costas M Pitsillides * Advanced Microscopy Program, Wellman Center for Photomedicine, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Pilhan Kim & * Seok H Yun Contributions Z.F. and J.A.S. designed the experiments, conducted research, collected and analyzed data and wrote the manuscript; Y.L., C.M.P., G.S. and V.T. helped conduct research and collected and analyzed data; P.K. and S.H.Y. developed and performed endoscopic microscopy; T.B.S., C.P.L. and M.K. designed the experiments, sponsored the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Terry B Strom (tstrom@bidmc.harvard.edu) or * Charles P Lin (lin@helix.mgh.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (10M) Z-stack reconstructed movie showing yellow iTreg (green + red), green nTreg and red Teff cells (red) in a tolerized islet allograft at week 2. Z axis step size is 2 μm. PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–5 and Supplementary Table 1 Additional data
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