Hot off the presses! Oct 01 Nat Med

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Latest Articles Include:

• Drug targets slip-sliding away
  - Nat Med 17(10):1155 (2011)
  Nature Medicine | Editorial Drug targets slip-sliding away Journal name:Nature MedicineVolume: 17,Page:1155Year published:(2011)DOI:doi:10.1038/nm.2530Published online11 October 2011 The starting point for many drug discovery programs is a published report on a new drug target. Assessing the reliability of such papers requires a nuanced view of the process of scientific discovery and publication. View full text Additional data
• Cancer drugs find a companion with new diagnostic tests
  - Nat Med 17(10):1157 (2011)
  Article preview View full access options Nature Medicine | News Cancer drugs find a companion with new diagnostic tests * Charlotte SchubertJournal name:Nature MedicineVolume: 17,Page:1157Year published:(2011)DOI:doi:10.1038/nm1011-1157Published online11 October 2011 When Stephen Little co-founded a molecular diagnostics company in 2001, he gave numerous presentations to pharmaceutical companies touting the benefits of tests that can tell which patients are likely to respond to a particular therapy or experience side effects. But most drugmakers were focused on blockbusters, and, since these so-called 'companion diagnostics' divide patient populations into smaller groups, they threatened to contract, not expand, their markets. As such, Little recalls, "there was not a great deal of enthusiasm." Ten years later, the tide has changed for Little and the field of companion diagnostics. In 2009, Little's company, Manchester, UK–based DxS, was bought by the Dutch technology company Qiagen, where Little now serves as vice president of personalized health care. Qiagen currently has partnerships with more than 15 pharmaceutical companies, including, most recently, an agreement announced last month with Eli Lilly to develop a PCR-based test to detect mutations in the gene encoding Janus kinase 2 and thereby identify individuals with blood cancer who are likely to respond to a drug in early-stage development at the Indiana-based company. This deal follows less than a month after the US Food and Drug Administration (FDA) approved two cancer drugs—Zelboraf (vemurafenib) for BRAF-mutation–positive metastatic melanoma on 17 August, and, a week later, Xalkori (crizotinib) for people with non–small-cell lung cancer driven by an ALK (anaplastic lymphoma kinase) fusion gene—together with their companion diagnostic tests. Notably, these two drug-diagnostic combined approvals represent divergent development strategies on the part of big pharma. The test accompanying Roche's Zelboraf was developed in house at the Swiss drug giant's diagnostics arm, Roche Molecular Diagnostics in Pleasanton, California. In contrast, New York–based Pfizer's Xalkori comes with a diagnostic developed with Abbott Molecular of Des Plaines, Illinois. istockphoto Drugmakers develop companion diagnostic tests alongside novel therapeutics. Regardless of the business approach, experts agree that it's best to start developing companion tests alongside therapeutics well before compounds even enter their first clinical trials. "The sooner we can partner, the better," says Little. Walter Koch, head of global research at Roche Molecular Diagnostics, credits the parallel development of a diagnostic with accelerating research on Zelboraf, which first entered phase 3 testing less than 20 months before FDA approval. The drug specifically inhibits a mutant BRAF protein found in about half of all patients with melanoma and can produce dramatic responses in these individuals. Knowing who is likely to respond to the drug up front, notes Koch, helped the company design better clinical trials along the way, and, ultimately, created a better medicine for patients. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Charlotte Schubert Search for this author in: * NPG journals * PubMed * Google Scholar
• Mysteries about drug metabolism in the obese weigh on doctors
  - Nat Med 17(10):1158 (2011)
  Article preview View full access options Nature Medicine | News Mysteries about drug metabolism in the obese weigh on doctors * Alisa OparJournal name:Nature MedicineVolume: 17,Page:1158Year published:(2011)DOI:doi:10.1038/nm1011-1158aPublished online11 October 2011 The surgery was a success, but a question loomed after the procedure: given that the patient was obese, what was the right antibiotic dose? "The thought was, well, she's twice as big as a normal person, so we'll give her twice the dose," says Aaron Cook, a clinical pharmacy specialist at the University of Kentucky in Lexington. "For that drug, levofloxacin, there's just no information to go on, no dosage recommendation for obese patients." The patient fared well, but such conundrums are becoming increasingly common as obesity rates rise around the globe. Just a month ago, researchers released new figures estimating that the US will see an additional 65 million obese individuals by 2030 (Lancet, 815–825, 2011). Already in the country approximately one in three adults and one in six children are obese—a condition that can precipitate heart disease, diabetes, respiratory failure and other illnesses that often require medication. But experts say that merely doubling the dose isn't the solution because the physiological changes that accompany obesity, such as increases in the volume of blood pumped by the heart and fat mass, can in turn lead to changes drug absorption and metabolism. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Alisa Opar Search for this author in: * NPG journals * PubMed * Google Scholar
• Ten years on from anthrax scare, analysis lags behind sequencing
  - Nat Med 17(10):1158-1159 (2011)
  Article preview View full access options Nature Medicine | News Ten years on from anthrax scare, analysis lags behind sequencing * Amber DanceJournal name:Nature MedicineVolume: 17,Pages:1158–1159Year published:(2011)DOI:doi:10.1038/nm1011-1158bPublished online11 October 2011 A decade ago this month, a microbiologist at Northern Arizona University, in Flagstaff, took a special delivery from the US government. Federal investigators wanted the scientist, Paul Keim, to identify the anthrax that appeared in letters mailed to news organizations and US lawmakers. Overnight, he used PCR to determine that the anthrax sent was the Ames strain, commonly used in research—but that was just the beginning of a scientific investigation that would catapult the still wet-behind-the-ears science of microbial forensics to the forefront of the criminal inquiry. Ten years on, Keim's PCR-based technique seems downright quaint in comparison with modern, speedy DNA sequencing. "In a lot of ways we've matured," says Bruce Budowle of the University of North Texas Health Science Center in Fort Worth. But there are challenges ahead, adds Budowle, who retired in 2009 from the US Federal Bureau of Investigation (FBI), where he was involved in the anthrax studies as a senior scientist in the laboratory division: "In a lot of ways, we've got a long way to go... We haven't grown in the interpretation of the results and what they might mean." Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Amber Dance Search for this author in: * NPG journals * PubMed * Google Scholar
• New fee structure proposed by FDA might lead to more talk
  - Nat Med 17(10):1159 (2011)
  Article preview View full access options Nature Medicine | News New fee structure proposed by FDA might lead to more talk * Hannah WatersJournal name:Nature MedicineVolume: 17,Page:1159Year published:(2011)DOI:doi:10.1038/nm1011-1159Published online11 October 2011 Ever since 1992, when US lawmakers passed the Prescription Drug User Fee Act (PDUFA) to accelerate review of new drugs by the US Food and Drug Administration, industry money has had an increasingly important role in fueling the regulatory agency. In the program's first year, drug companies paid less than $9 million total to the FDA through the initiative. But in the past two decades the amount has ballooned; this year, the agency anticipates receiving at least $619 million in user fees, composing roughly 65% of its budget for overseeing human drugs. Despite the torrent of funds, the FDA has still failed to meet its goal of completing the review of 90% of new drug applications within ten months. Industry isn't exactly pleased with this report card, and they have spent the past year in negotiations with the agency to plan how the fees can be used to make drug review more efficient. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Waters Search for this author in: * NPG journals * PubMed * Google Scholar
• Childhood tuberculosis treatment remains imprecise science
  - Nat Med 17(10):1160 (2011)
  Article preview View full access options Nature Medicine | News Childhood tuberculosis treatment remains imprecise science * Julie ManoharanJournal name:Nature MedicineVolume: 17,Page:1160Year published:(2011)DOI:doi:10.1038/nm1011-1160aPublished online11 October 2011 Last year, the World Health Organization released updated procedures on how best to tackle the global scourge of tuberculosis. The fourth edition of the "Treatment of tuberculosis: Guidelines" recommended, among other changes, increasing the dosage of tuberculosis medication required to treat children. But, in a sense, the new guidance provided a destination without a map: it failed to address the larger problem of how to improve the accuracy of pediatric dosing. In recent months, researchers have pointed to a host of problems plaguing the diagnosis and treatment of tuberculosis in children, especially those younger than age 5. For example, at a June workshop held by a taskforce of the US Centers for Disease Control and Prevention, Steve Graham of the Royal Children's Hospital in Melbourne, Australia called for new and better means of pediatric tuberculosis diagnosis, which can be complicated by concurrent ailments such as malnourishment, HIV infection and pneumonia. And, in September, scientists noted that a negative result from the new interferon-gamma release assays cannot definitively rule out tuberculosis in children (Pediatr. Infect. Dis. J., 817–818, 2011). Also in September, another group urged that animal models for tuberculosis "must be designed and utilized in a manner that is also pertinent to the pediatric population" by addressing age-related variance in drug metabolism (Pharmacol Res., 176–179, 2011). Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Julie Manoharan Search for this author in: * NPG journals * PubMed * Google Scholar
• NIH student training overhauled after HHMI pulls funding
  - Nat Med 17(10):1160-1161 (2011)
  Article preview View full access options Nature Medicine | News NIH student training overhauled after HHMI pulls funding * Hannah WatersJournal name:Nature MedicineVolume: 17,Pages:1160–1161Year published:(2011)DOI:doi:10.1038/nm1011-1160bPublished online11 October 2011 As an undergraduate student at the University of Wisconsin–Madison, Amanda Herzog worked in a number of different research laboratories, where she studied everything from cartilage growth to pediatric tonsillectomy techniques to fruit fly behavior. So, when she started medical school at UW's School of Medicine and Public Health in 2007, she figured she'd keep up with her research activities. Yet, other than a three-month stint assisting oncologist Mark Albertini in his melanoma lab for a summer research program between her first and second years, Herzog has struggled to fit research into her schedule. "I had to devote my time to my studies," she says. Herzog's passion for lab work inspired her to apply for a unique research program funded jointly by the US National Institutes of Health (NIH) and the Howard Hughes Medical Institute (HHMI). She succeeded and became one of the 42 students chosen from hundreds of applicants for the program's 2010–2011 academic year. As an HHMI-NIH Research Scholar, she was able to defer her third year of medical school and instead study an experimental head and neck cancer drug in cellular and animal models at the NIH in Bethesda, Maryland. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Waters Search for this author in: * NPG journals * PubMed * Google Scholar
• Businesses ready whole-genome analysis services for researchers
  - Nat Med 17(10):1161 (2011)
  Article preview View full access options Nature Medicine | News Businesses ready whole-genome analysis services for researchers * Trevor StokesJournal name:Nature MedicineVolume: 17,Page:1161Year published:(2011)DOI:doi:10.1038/nm1011-1161Published online11 October 2011 The cost of sequencing an individual's entire genome has fallen precipitously over the past five years, from around $100 million for the first personal genome to under $5,000 today when sequencing services are purchased in bulk. In response, a handful companies have started developing whole-genome annotation services that give clinical researchers lacking expertise in bioinformatics the ability to use genomic data for disease-discovery and drug-response testing. One company, Knome, based in Cambridge, Massachusetts, already offers a package deal. For about $5,000 it will sequence and annotate a genome—with a minimum order of ten genomes. Meanwhile, two California companies, Emeryville-based Omicia and Personalis in Palo Alto, are beta-testing annotation services in academic settings, with future plans to roll out their services in the clinic. Although neither of the two has set its pricing yet, Omicia is expected to release an annotation service for academics and clinicians in early 2012. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Trevor Stokes Search for this author in: * NPG journals * PubMed * Google Scholar
• Start-up tries bidding model to outsource academic research
  - Nat Med 17(10):1162 (2011)
  Article preview View full access options Nature Medicine | News Start-up tries bidding model to outsource academic research * Elie DolginJournal name:Nature MedicineVolume: 17,Page:1162Year published:(2011)DOI:doi:10.1038/nm1011-1162Published online11 October 2011 Marc Lippman was facing a tight deadline. His grant application to the Susan G. Komen for the Cure foundation was due on 20 August of this year, and, with a week to go before the closing date, he still hadn't gathered all of the requisite preliminary data. "I just needed to have the information," recalls Lippman, chair of the department of medicine at the University of Miami Miller School of Medicine. Fortunately, his former postdoc Elizabeth Iorns had just launched a new website called Science Exchange to help scientists outsource their research. Lippman posted that he needed someone to quickly analyze all the microRNAs involved in driving metastasis in breast cancer, and Emory University's Cancer Genomics Shared Resource agreed to take on the rushed job for just $7,000. Lippman mailed off his tumor samples, and, within a week, he had all the raw data and bioinformatics analysis back from the Atlanta laboratory. To his relief, he managed to get his grant completed and submitted on time. Although Lippman could have turned to a core facility at his own institution, "in this case, it was cheaper and a hell of a lot faster" to outsource the project, he says. "I haven't gotten data back this fast in my life, and they did a better analysis then I've ever seen. It was lovely." The problem, according to Lippman and others, is that it's often hard to find an academic institution with the expertise and equipment to take on projects on short turnaround times. Over the past two years, the US National Center for Research Resources (NCRR) has funded efforts to remedy the situation, including a nine-institution, shared resource repository called the eagle-i Consortium, a network of Institutional Development Awards–funded core laboratories to coordinate facilities in small states with fewer investigators and resources, and a national social network of biomedical scientists called VIVO. The Association for Biomolecular Resource Facilities (ABRF), an international collective of more than 140 core labs, also maintains searchable 'white pages' and 'yellow pages' directories of people and facilities associated with the organization. But all of these efforts often struggle to stay up to date without continuous curation. Dan Knox / Science Exchange Science Exchange founders Dan Knox (left), Elizabeth Iorns and Ryan Abbott. Enter Science Exchange. "Science Exchange may be able to have very fresh information as compared to what may be on an outdated website," says Gregory Farber, director of the Office of Technology Development and Coordination in the US National Institute of Mental Health. "It's all in front of me on one webpage, in one place," adds Megan Rieger, a postdoc and cancer researcher at the University of Southern California in Los Angeles who has used the new online marketplace to outsource some small histology projects. Plus, she adds, "I'm looking to save my lab some money, and here's an easy way to do it." Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar
• Straight talk with... Mahendra Rao
  - Nat Med 17(10):1163 (2011)
  Nature Medicine | News Straight talk with... Mahendra Rao * Elie DolginJournal name:Nature MedicineVolume: 17,Page:1163Year published:(2011)DOI:doi:10.1038/nm1011-1163Published online11 October 2011 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 October 2005, Mahendra Rao shocked the scientific community when he quit his job as head of the US National Institute on Aging's stem cell section and announced plans to go into industry. Rao felt that a ban at the time on federal funding for most human embryonic stem cell research hampered researchers in his division and prohibited him from doing the job he was hired to do. So he joined the research-tool giant Invitrogen (which later became Life Technologies) as vice president of regenerative medicine at the company's Maryland facility. Six years on, times have changed in the field of stem cell biology: rules governing taxpayer-backed research involving embryonic stem (ES) cells have been relaxed in the US, and induced pluripotent stem (iPS) cells have come into the fray. Prompted by those changes, Rao opted to return to the US National Institutes of Health (NIH) in August to head the new Intramural Center for Regenerative Medicine. The $52 million center was launched in early 2010 by the agency to develop new therapies using stem cell approaches. With a heightened focus at the NIH on translational medicine, spoke to Rao to find out how he plans to turn stem cell discoveries into cell-based therapies. View full text Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar
• News in brief: Biomedical briefing
  - Nat Med 17(10):1164-1165 (2011)
  Article preview View full access options Nature Medicine | News News in brief: Biomedical briefing Journal name:Nature MedicineVolume: 17,Pages:1164–1165Year published:(2011)DOI:doi:10.1038/nm1011-1164Published online11 October 2011 Thanks to a one-year pilot program announced on 16 September, biomedical entrepreneurs are more easily able to license technologies developed by US government scientists. Beginning this month, small startups that meet certain criteria can license patents for drugs and vaccines from intramural research laboratories at the country's Food and Drug Administration (FDA) and National Institutes of Health (NIH) for just $2,000 upfront and with royalty payments deferred until companies have enough cash to pay out. "Anything we can do to get technologies out to businesses for commercialization will speed their ultimate access to the public," says Mark Rohrbaugh, director of the NIH's Office of Technology Transfer. "And, as a byproduct of expediting such technology transfer, we will also hopefully create more jobs quicker." In a 30 August report, the Australian National Audit Office took the country's drug regulator to task for failing to obtain sufficient efficacy information from companies selling alternative medicines such as vitamins, herbal remedies and homoeopathic products. "It is clear from this report that consumers should be concerned at the current regulatory and legal framework for dealing with complementary medicines and that a major overhaul is needed," says Carol Bennett, chief executive officer of the Consumers Health Forum of Australia. The eight-month inquiry into the Therapeutic Goods Administration cited previous estimates that up to 90% of the goods sold in the country's A$1.2 billion ($1.2 billion) alternative-medicines industry do not meet health and safety rules. In the largest ever single cash injection into translational research in Britain, the UK government in August announced five-year funding totaling £800 million ($1.2 billion) to develop partnerships between National Health Service (NHS) hospitals, universities, industry and charities. "This sends out a very positive message and reflects a cultural shift for the NHS, showing that it has a critical role in innovative translational research," says Mark Downs, chief executive of the London-based Society of Biology. The money, which comes in the shape of 31 awards, will be coordinated by the National Institute of Health Research, the research arm of the NHS. The NIH issued new rules in August to tighten conflict-of-interest oversight, requiring federally funded researchers to disclose income from drug companies above $5,000 or any equity in non–publicly traded companies. Although the policy is similar to draft regulations released in May 2010, it shies away from a proposed requirement that institutions post their faculty's financial conflicts on publicly accessible websites. "This is a setback for the efforts that have been made to facilitate anyone who wants to from having easy access to this kind of information," says Sidney Wolfe, director of the Health Research Group at Public Citizen, a consumer advocacy organization based in Washington, DC. Starting next month, gay men in England, Scotland and Wales who have not had sexual contact with another man for at least one year will be eligible to donate blood, British health officials announced on 8 September. "The change brings the criteria for men who have sex with men in line with those for the majority of other groups that are deferred from blood donation for 12 months due to sexual behavior," says John Forsythe, a transplant surgeon at the Royal Infirmary of Edinburgh and chair of the advisory committee that recommended the policy change. The move follows the publication of a survey showing that the old policy wasn't working: one in ten British men who had sex with men donated blood even with the blanket ban (BMJ, d5604, 2011). Two protein-folding pioneers have won 2011 Lasker prizes along with the first ever Chinese awardee. Yale University's Arthur Horwich and the Max Planck Institute of Biochemistry's Franz-Ulrich Hartl took home the Albert Lasker Basic Medical Research Award for uncovering the action of chaperonins, protein complexes that assist the folding of other proteins. This year's Lasker~DeBakey Clinical Medical Research Award went to Youyou Tu of the China Academy of Chinese Medical Sciences for discovering artemisinin, now a staple of malaria therapy, from the leaves of the wormwood plant. And, in an unusual twist, an institution—the US National Institutes of Health Clinical Center—rather than any single person won the Lasker~Bloomberg Public Service Award. See page 1201 for commentaries from all the winners. Jackson Laboratory The world's largest mouse genetics research institute has a new big cheese. In August, the Jackson Laboratory announced that Edison Liu, founding executive director of the Genome Institute of Singapore, will succeed Rick Woychik as president and chief executive officer of the Bar Harbor, Maine facility. President of the Human Genome Organisation for the past four years, Liu also ran the clinical sciences division the US National Cancer Institute in Bethesda, Maryland from 1996 to 2001—experience that Liu says he plans to put to use in moving the Jackson Lab "beyond the mouse model into human genetics, translational medicine and genomics." Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Networking for new drugs
  - Nat Med 17(10):1166-1168 (2011)
  Nature Medicine | News | News Feature Networking for new drugs * Claire Ainsworth1Journal name:Nature MedicineVolume: 17,Pages:1166–1168Year published:(2011)DOI:doi:10.1038/nm1011-1166Published online11 October 2011 Many of today's most celebrated drugs are designed to hit only one biological target with great precision. But a novel clinical trial aims to turn this idea on its head by using 'network pharmacology' to more effectively tackle a common neurological disorder affecting limb movement. looks into how medicine's proverbial 'magic bullet' might soon give way to a more sophisticated arsenal. View full text Additional data Affiliations * Claire Ainsworth is a science journalist based in Southampton, UK. Author Details * Claire Ainsworth Search for this author in: * NPG journals * PubMed * Google Scholar
• Biomedicine in Brazil
  - Nat Med 17(10):1169 (2011)
  Nature Medicine | News Biomedicine in Brazil Journal name:Nature MedicineVolume: 17,Page:1169Year published:(2011)DOI:doi:10.1038/nm1011-1169Published online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Brazil may best be known for Carnival celebrations, golden-sand beaches and soccer players who go by one name—but research into disease theraputics is now also starting to put the country on the scientific map. The number of biomedical publications with at least one Brazil-based author nearly tripled over the past decade, from around 4,500 papers in 2000 to close to 13,000 last year, according to data compiled for Nature Medicine by the University of São Paulo's André Frazão Helene. And even though the country churns out only a little over 2% of the world's biomedical output at present, that small number belies a larger trend toward innovative drug development and translational science. In the pages that follow, we highlight some of the strengths of Brazilian biomedicine and many of the challenges that lie ahead. Additional data
• Laws hinder drug development inspired by Amazonian biodiversity
  - Nat Med 17(10):1170 (2011)
  Nature Medicine | News Laws hinder drug development inspired by Amazonian biodiversity * Carlos Henrique FioravantiJournal name:Nature MedicineVolume: 17,Page:1170Year published:(2011)DOI:doi:10.1038/nm1011-1170Published online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg SÃO PAULO — For her graduate work, Eliana Rodrigues spent several years splitting her time between two indigenous communities in the north of Brazil, studying their ritual healing practices and chronicling which plants they used for treatments. In a comprehensive survey of their practices, she identified 169 plant species used in 345 formulations to treat 68 different diseases (J. Psychoactive Drugs, 285–295, 2006). After detailing her initial findings, Rodrigues, now a professor at the Federal University of São Paulo, hoped to take some of the natural compounds found in these plants and turn them into commercial drug products, but legal hurdles paralyzed her work. In 2001, after a scandal involving the Swiss drug giant Novartis and a consortium of British researchers who hoped to patent new compounds derived from microbes found in the Amazon, the Brazilian government imposed a tough law called Provisional Measure 2186. The legislation required drug companies and universities to pay indigenous communities a part of any profits stemming from traditional knowledge. Although it was aimed at protecting the exploitation of native biodiversity, critics say the law has severely limited scientists' access to the country's biological resources—even for basic research—and has hindered the development of medicines derived from natural products. "The current legal framework is completely inadequate to a country that is said to value innovation," says Maria Celeste Emerick, science and technology management coordinator at the Oswaldo Cruz Foundation in Rio de Janeiro. "It doesn't suit the interest of university researchers, local pharmaceutical companies and traditional communities." Because of the provisional measure—which, despite its name, was made permanent within a month of its initial passage—"companies have now become unmotivated," says João Batista Calixto, a pharmacologist at the Federal University of Santa Catarina in Florianópolis. Creative Commons The government cracked down on a cream derived from the Passiflora passion fruit. Calixto describes the challenges he himself has faced in regards to two natural compounds developed in his lab. One is an ointment for muscular pain made from the leaves of a shrub used by fishermen along the southeastern coast of Brazil; the other is an antiaging cream based on antiinflammatory substances found in a type of passion fruit. Both compounds were successfully commercialized and marketed more than four years ago. But last year, the Council for Genetic Heritage Management (CGEN), a federal administrative body created by the Environment Ministry to apply the provisional measure, fined both products' manufacturers for not having the necessary authorizations to deal with the plants. This year, around 100 more companies and universities have similarly been penalized for collecting plants or other organisms without authorization, leading some firms to suspend all projects involving biodiversity until the legal framework is made more business friendly. At a meeting here in August, academics, drug company representatives and government officials met to discuss ways of simplifying access to genetic resources and easing legal provisions. "There is political will to make corrections in the provisional measure," CGEN President Braulio Dias told delegates of the meeting. "But to get congressional approval will not be easy." Navigating the labyrinth Brazilian lawmakers have tacked dozens of resolutions and amendments onto the provisional measure since its initial passage ten years ago. The result has been a labyrinth of rules that have slowed down scientific progress and weakened the interaction between universities and pharmaceutical companies, experts say. To make matters worse, scientists hoping to cement intellectual property also have to abide by Law 9279—the 1996 legislation that prohibits the patenting of living organisms. According to Calixto, the double whammy of Law 9279 and Provisional Measure 2186 has sidelined development of drugs based on natural compounds and rendered the field of ethnopharmacology into academic obsolescence. The bureaucracy is so demanding, in fact, that many scientists prefer to work solely with synthetic compounds. Against these odds, a handful of researchers have found ways of working within the system. "The provisional measure is absurd but feasible," says Luis Carlos Marques, a pharmacist at Bandeirantes University in São Paulo. In 2008, as director of São Paolo-based Apsen Pharmaceuticals, he helped develop a bedsore medicine originating from a small tree with large, circular bean pods native to Brazil, called barbatimão. And, by working with the regulatory authorities, Marques managed to get CGEN approval to market the new product within ten months of filing an application. Other Brazilian scientists still look to the natural world but try to find organisms that exist beyond the local ecosystem to skirt legal challenges. For instance, Farmabrasilis, a São Paulo–based nonprofit research network modeled after international development organizations such as OneWorld Health and the Global Alliance for TB Drug Development, is advancing two natural compounds—an immunomodulatory agent called P-MAPA that is produced by fungal fermentation and an antibiotic called violacein extracted from a particular bacterium. Neither of the organisms used to make these products are unique to Brazil and, so, are not protected by the provisional measure. "The current legal framework has not affected our research lines, because we don't work on material from Brazilian biodiversity," says Farmabrasilis chief executive Iseu Nunes. As for Rodrigues, she has opted to work within the system and secure all the necessary paperwork to continue the anthropological and botanical research she started more than ten years ago. She and her team are now studying the antianxiety effects of a native herb used in religious rituals and the painkilling effects of an Amazonian frog secretion on headaches. "Now I have all the bureaucratic authorizations required to do my work," Rodrigues says. Additional data Author Details * Carlos Henrique Fioravanti Search for this author in: * NPG journals * PubMed * Google Scholar
• Brazilian drug companies hope to benefit from foreign investment
  - Nat Med 17(10):1171 (2011)
  Nature Medicine | News Brazilian drug companies hope to benefit from foreign investment * Mike MayJournal name:Nature MedicineVolume: 17,Page:1171Year published:(2011)DOI:doi:10.1038/nm1011-1171aPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Brazil's drug industry is booming—and foreign drugmakers are hoping to cash in. Ever since France's Sanofi acquired Brazil's leading generic manufacturer Medley two years ago, a who's who of multinational companies has been pouring money into the country's biopharmaceutical firms. For instance, GlaxoSmithKline, Novartis and Takeda all now have major investments in the local drug industry. "Our clients are looking at Brazil as a key investment and growth area," says Kay Formanek, who leads the life science practice in Europe, Africa and Latin America for Accenture, a global management and consulting firm. Experts from big pharma agree. Brazil is "one of the main countries in [Merck's] emerging market strategy," says João Sanches, market access and external affairs director for the Whitehouse Station, New Jersey–based company. The most recent major takeover comes from the world's largest biotech company. In April, Thousand Oaks, California–based Amgen bought the Brazilian pharmaceutical firm Bergamo for a cool $215 million. The takeover comes less than a year after New York–based Pfizer, the biggest drugmaker in the world, paid $240 million for a 40% stake in Brazil's Teuto with an option to acquire the rest of the generic drug specialist's shares beginning in 2014. According to Ricardo Vicente, a pharmaceuticals analyst with UK-based Espicom Business Intelligence, all this foreign investment should help the country's annual drug sales grow from around $31 billion today to close to $39 billion by 2016. "Foreign investment will certainly fuel pharmaceutical market growth," he says. Indeed, at the current pace of growth, Brazil could become the world's fifth largest pharmaceutical market—behind only the US, Japan, China and Germany—by 2015. Brazil offers a suite of attractive features for multinational drug companies looking to invest. For one thing, the country's per capita gross domestic product (GDP) has nearly tripled over the past decade—from $3,696 in 2000 to $10,710 last year—with a large fraction of that being put toward drugs. According to Vicente, Brazil now spends approximately 9% of its GDP on health care, and about one-sixth of that goes toward pharmaceuticals. Chronic opportunities With a shift from infectious diseases to noncommunicable ailments, the country's epidemiological profile is also becoming increasingly attractive to big pharma. "Chronic and degenerative diseases are the leading causes of morbidity [in Brazil]," Vicente says. "Drugs to treat these diseases are expensive; therefore, the pharmaceutical market will remain attractive." What's more, recent actions by Brazil's government are enhancing opportunities for biopharma. In December of last year, for instance, the country's national health surveillance agency, ANVISA, provided guidelines for companies wishing to bring 'biosimilar' drugs to market—something that other countries, including the US, have been struggling to accomplish. The government also recently introduced several drug-safety measures and established public-private partnerships to help roll out essential medicines, many of them sold by foreign companies. According to Formanek, the growing investment by foreign drugmakers should rub off on locally owned companies, too. It will "create a ripple effect in terms of the requirements for clinical experts, physicians and so on," she says. For one thing, she expects to see research and development hubs popping up around the facilities being established by multinationals. "Brazil will be codependent on multinationals for innovative medicines for the near future, because local companies lack the capacity to produce key products like vaccines," Formanek adds. But, she notes, "with investments and acquisitions, also fostered by the government, this gap will be closing in the next few years." Additional data Author Details * Mike May Search for this author in: * NPG journals * PubMed * Google Scholar
• New framework needed to thwart Brazil's crippling bureaucracy
  - Nat Med 17(10):1171 (2011)
  Nature Medicine | News New framework needed to thwart Brazil's crippling bureaucracy * Luisa MassaraniJournal name:Nature MedicineVolume: 17,Page:1171Year published:(2011)DOI:doi:10.1038/nm1011-1171bPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg RIO DE JANEIRO — Scientists around the globe often dream about vacationing in Brazil to waste some time on the beaches near Rio and São Paolo. But for researchers who regularly work in the biomedical strongholds of the country there's a far more insidious time-waster: bureaucracy. To eliminate some of the red tape that has been blamed for stultifying research and innovation, an expert panel representing state funding agencies and science secretaries released a report on 26 August calling for a new code of conduct governing how scientific research is carried out in Brazil. "The present context discourages scientists and hinders the scientific and technological development of the country," says Mário Neto Borges, president of the National Council of State Research Funding Foundations (CONFAP). "If approved, the new code will be a big step for Brazilian science—a revolution," adds Odenildo Sena, president of the National Council of Secretaries for Science, Technology and Innovation Issues (CONSECTI). Currently, provisions relating to importing research equipment or grant spending, for example, are spread out across at least ten different pieces of legislation. The architects of the new report hope to bring all these measures together into one place and to lower some of the bureaucratic hurdles along the way. For instance, the 25-page proposal—coauthored by CONFAP and CONSECTI—calls for an elimination of import tariffs on research tools and reagents as well as outlines specific ways that scientists can get the lowest prices to stretch their grant money further. "The suggested code is a single, comprehensive, simple legislation specific to science," Borges says. Miguel Nicolelis, a Brazilian-born neuroscientist who works both at the Duke University Medical Center in Durham, North Carolina and at the Edmond and Lily Safra International Institute of Neuroscience of Natal in northeastern Brazil, hopes these measures will streamline biomedical research in his native country. "In the US, if I need to buy something for a research project, I just pick up the phone and buy it," he notes. "In Brazil, the rules are the same as for building a hydroelectric plant. The rules are not made for science." Luiz Antonio Elias, executive secretary of the Brazilian Ministry of Science, Technology and Innovation, welcomes the new proposal. "This is an original movement in which both state and national stakeholders joined for suggesting an improvement of the legislation," he says. According to Elias, the ministry now plans to work with the scientific community to address any outstanding concerns and to codify some of the report's proposals into law. Additional data Author Details * Luisa Massarani Search for this author in: * NPG journals * PubMed * Google Scholar
• In Brazil, basic stem cell research lags behind clinical trials
  - Nat Med 17(10):1172 (2011)
  Nature Medicine | News In Brazil, basic stem cell research lags behind clinical trials * Elie DolginJournal name:Nature MedicineVolume: 17,Page:1172Year published:(2011)DOI:doi:10.1038/nm1011-1172Published online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the 1990s, Richard Burt injected people in the US with their own bone marrow stem cells and successfully treated a variety of autoimmune disorders, including multiple sclerosis and lupus. So it seemed obvious to him that the same approach could be used to reset the overactive immune system in people with another autoimmune disease: type 1 diabetes. If the therapy was performed early enough in the disease course, Burt thought, it could help people go off insulin-replacement therapy by enabling them to generate insulin-producing beta cells again before irreversible damage to the pancreas set in. He drew up a clinical protocol and shopped it around to colleagues close to home, but "endocrinologists in America had no interest in it whatsoever," recalls Burt, head of immunotherapy at Northwestern University's Feinberg School of Medicine in Chicago. "The focus in America has always been intensive insulin therapy and embryonic stem cells [to treat diabetes], and this is neither one of those things." Burt did, however, find a receptive audience for his innovative therapy in Júlio Voltarelli at the University of São Paulo (USP) School of Medicine of Ribeirão Preto in Brazil. In 2003, he handed off the trial design to Voltarelli and his colleagues, and, with the blessing of ethics committees at both the local University Hospital and the federal Ministry of Health, the Brazilian team treated 23 people newly diagnosed with type 1 diabetes with stem cells taken from their own blood. As the researchers had hoped, the majority of the study subjects managed to maintain normal blood sugar levels for years after the treatment without having to continue taking insulin injections (JAMA301, 1573–1579, 2009). Off the back of that first trial success, Voltarelli and Burt are currently treating more young diabetics with their approach—in Ribeirão Preto, but, notably, in Chicago, St. Louis and Paris, too. The diabetes trial is not the only one using stem cells to have found its footing in Brazil. In 2007, for example, Brazilian doctors launched the world's largest stem cell clinical trial to date: a 1,200-person study testing whether bone marrow cells can reverse the damage found in four heart conditions. The first arm of the study, for people with Chagas disease, is now complete; according to trial investigator Antonio Carlos Campos de Carvalho of the Federal University of Rio de Janeiro (UFRJ), the treatment did not show clinical benefit. However, pioneering adult stem cell trials are ongoing in Brazil for respiratory disease, stroke, epilepsy, spinal cord injury and anemia, among other ailments. Meanwhile, the country has produced some noteworthy stem cell findings in the basic science arena. Last month, for example, UFRJ's Stevens Rehen reported the derivation of the first Brazilian reprogrammed cell lines from healthy donors and someone with schizophrenia, too (Cell Transplant. doi:10.3727/096368911X600957, 2011). Rehen and his colleagues also released software to help automate image counting of pluripotent stem cells, which has been downloaded close to 500 times to date (Integr. Comput. Aided Eng., 91–106, 2011). That may sound like the metrics of a vibrant research community. But, as Rehen point out, his lab alone produces around half of all papers in Brazil related to pluripotent stem cells, whereas most of the stem cell field continues to focus on applying adult stem cells. "Brazil has managed to advance a lot in the preclinical and clinical area," says Carvalho, who coordinates the National Cell Therapy Network, which counts 52 labs across five Brazilian states. "But we're lagging behind in the basic science." To remedy that, the Brazilian government introduced several measures to give the stem cell field a boost. In 2005, the country passed the Biosafety Act, which allowed researchers to derive human embryonic stem cells from donated surplus embryos stored for at least three years at assisted reproduction clinics. The measure was quickly challenged by religious factions, but, with backing from scientific groups and patient advocacy organizations, the Brazilian Supreme Court ultimately upheld the legislation in 2008. Confidence interval Legalizing human embryonic cell research "was a major event—not only in terms of shaping the research of the field and determining what the regulations were going to be, but also in terms of inspiring confidence and motivating the field," says Dominique McMahon, a postdoc at the University of Toronto who wrote a paper last year surveying the regenerative medicine landscape in Brazil (Regen. Med., 863–876, 2010). Ana M. Fraga The BR-1 cell line. Yet, despite the legislation, "most of the groups continue to work with adult, not embryonic, stem cells," says Mayana Zatz, director of USP's Human Genome Research Center. Indeed, it wasn't until August of this year that researchers, led by the USP's Lygia Pereira, reported the first establishment of an embryonic stem cell line, called BR-1, from a Brazilian person (Cell Transplant., 431–440, 2011). Pereira says she has since derived three more embryonic stem cell lines, but because all four come from private fertility clinics, which tend to cater to Brazil's ethnically European elite, they do not come close to representing the rich genetic diversity of the country. That doesn't bode well for translational research, she says, because "we want to work now to make a library of cells that will represent the Brazilian genetic diversity that can be used in pharmacogenomic studies and drug screening programs." For that, Pereira, in collaboration with Rehen, hopes to create several hundred induced pluripotent stem (iPS) cell lines through the Brazilian National Laboratory of Embryonic Stem Cells (LaNCE). The lab, which has sites at UFRJ and USP, was established in 2009 as part of the government's 25-million-Brazilian-real ($15 million) initiative to create eight Cell Technology Centers across the country. Parts of LaNCE—which, despite its name, focuses on all pluripotent stem cells—are operational, thanks to state and federal grants. But plans to build a new facility for the center have been delayed, in part because the Brazilian Development Bank (BNDES) only delivered on its promised 6-million-real contribution earlier this year. "Everything took a very long time," says Pereira, who expects construction to begin later this month. "It was frustrating, but we're now celebrating the arrival of the money." The Brazilian stem cell community faces other budget issues, however, especially following the presidential election last year in which long-time stem cell supporter and former health minister José Serra lost to current President Dilma Rousseff. "We are not sure what the priority of stem cell research is under the new administration," says Pereira. That's a problem, adds Rehen, because "we need to guarantee constant funding opportunities for the stem cell field in Brazil in the next years. If not, everything regarding stem cells—both adult and pluripotent—built in the last years will not stay." Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar
• Brazilians lured back home with research funding and stability
  - Nat Med 17(10):1173 (2011)
  Nature Medicine | News Brazilians lured back home with research funding and stability * Anna PetherickJournal name:Nature MedicineVolume: 17,Page:1173Year published:(2011)DOI:doi:10.1038/nm1011-1173Published online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg "Basically, I'm in limbo," says cancer biologist Fabricio Costa. After growing up and studying in Brazil, Costa moved to the US in 2004 for a postdoctoral fellowship at Massachusetts General Hospital in Boston. Two years later, he became a research scientist at Northwestern University's Feinberg School of Medicine in Chicago, where he continues to work today. And, in 2008, he further put down US roots, founding a Chicago-based consulting company called Genomic Enterprise. Lately, however, Costa has grown increasingly disillusioned with the US system. Over the past few years, he has applied for a handful of grants from the US National Institutes of Health and hundreds of US-based faculty positions but so far has had no luck. Meanwhile, job offers in Brazil have started to trickle in. Although none of them have proven attractive enough to lure him back quite yet, Castro figures it's now only a matter of time before he returns to his native country. "Maybe I'll go next year," he shrugs. With Brazil's economy taking off and the country's academic centers on the rise, more and more young scientists like Castro who went abroad for their training are toying with the idea of continuing their careers back home. Indeed, many have already returned. Déborah Schechtman moved south several years ago, after spending 13 years working on vaccine development and signal transduction pathways in labs outside of Brazil, first at the Weizmann Institute of Science in Israel and then at the Stanford University School of Medicine in California. "I think a lot of things are much better now, comparing when I left and when I came back," says Schechtman, who has worked in various institutes of the University of São Paulo (USP) since 2005. "It is certainly possible to do world-class research in Brazil now." Fabricio Costa After seven years away, Fabricio Costa is thinking of moving back to Brazil. Brazilian-born researchers contemplating where to base their scientific careers can rattle off a common list of push and pull factors. Brazil wins on job security and the availability of money for new projects, scientists say, particularly in wealthy São Paulo, where almost 10% of the state's sales tax is funneled into the region's three largest universities. In contrast, North America and Europe enjoy much better relations between academia and industry, and spinning off companies in those regions is much easier than in Brazil. The US in particular is also better at creating incentives for people to do their best work, with higher salaries and a generally more competitive research environment (although Brazil is catching up on both fronts). Slowly by surely Among the challenges in Brazil, the availability and pricing of reagents is probably the most frequently bemoaned bugbear. "Basically, a product that would arrive the next day in the US or Europe or Japan takes several months to get to Brazil," says Schechtman. In addition, labor laws make it hard to employ people for just a few years, so Brazilian universities tend not to hire technicians, which forces researchers to spend longer learning to operate lab gadgets themselves. Both problems slow the pace of research and make projects less flexible in Brazil than abroad, notes Ricardo DeMarco, a USP parasitologist who spent a year as a postdoc at the UK's York University. "Sometimes this leads you to spend more money," he says. "One time I bought a lot of reagents at once because I didn't want to have to wait for a second order, and then we changed the protocol and didn't use them." Programs such as the Young Investigator Awards from the São Paulo Research Foundation (FAPESP) have attempted to make it easier for scientists to adjust to the Brazilian system by providing funding to early-career returnees. However, not all award recipients have had a smooth transition upon their homecoming. For example, William Festuccia, who held postdoc positions in Quebec and Massachusetts before returning to USP, where he completed his PhD, now has a fellowship but no dedicated lab space to do his research. Understandably, he is frustrated with the situation, which he sees as less likely to arise in more established science institutes in the Northern Hemisphere. Even after six years away, Festuccia says, "a lot of the same old problems still exist." Yet, what are often passed off as country-wide problems are really just issues specific to particular institutes in Brazil, argues neurobiologist Sidarta Ribeiro, who returned from the US in 2005 after a decade of graduate and postdoc work to help set up the Edmond and Lily Safra International Institute for Neuroscience of Natal. This past summer, Ribeiro and nine other principal investigators walked out en masse from the Natal center, complaining of various policies that impeded their research. Nonetheless, Ribeiro, who now directs a new institute at the nearby Federal University of Rio Grande do Norte, is strikingly upbeat about his academic future in Brazil. "My generation in science in Brazil has a strongly utopian view of our country," he says. "We were teenagers when Brazil was becoming a democracy again, so we know that countries can change quickly and for the better, and we feel that at this moment we can really make a difference." Despite struggling with his limbo, Costa shares this optimism. Last year, he even cofounded a Rio de Janeiro–based social media company called Datagenno aimed at connecting researchers, physicians and others with an interest in molecular and clinical genetics. "People are telling me that new spinoff companies are appearing out of academia in Brazil," he says. "That never happened before." Additional data Author Details * Anna Petherick Search for this author in: * NPG journals * PubMed * Google Scholar
• After years of neglect, Brazil takes aim at Chagas disease
  - Nat Med 17(10):1174 (2011)
  Nature Medicine | News After years of neglect, Brazil takes aim at Chagas disease * Anna PetherickJournal name:Nature MedicineVolume: 17,Page:1174Year published:(2011)DOI:doi:10.1038/nm1011-1174aPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In the 1990s, Brazil made headlines for cutting HIV infection rates to well below projected levels, but the country has not mobilized itself as energetically against other public health challenge since then. Chagas disease, a parasitic infection first described over a century ago by the Brazilian physician Carlos Chagas, is carried by 3 million Brazilians—more than are now afflicted by HIV—with close to 5,000 of those people dying from the disease each year. Yet Chagas has remained sorely neglected, and, to this day, most people with the disease are treated with decades-old drugs that fail to completely eliminate the Trypanosoma cruzi protozoan responsible for the chronic condition. The sluggish pace of development for Chagas treatments stems in part from the relative lack of support from public health authorities. Unlike HIV, for which Brazil's Ministry of Health eventually leapt into action, progress for combating Chagas has been far more gradual, largely falling on the research community, which has pushed for government programs. Five years ago, for example, parasite transmission by the Southern Cone's most common Chagas vector, the blood-sucking triatoma bug, was effectively halted in Brazil through a sustained national campaign of insecticide spraying and housing improvement projects. And research into innovative therapies is starting to bear fruit. "The intense effort of the scientific Brazilian community called attention to a large health problem and generated epidemiological and clinical data," says Tania Araújo-Jorge, director of the Oswaldo Cruz Institute in Rio de Janeiro. For instance, Brazil is now home to two ongoing phase 3 trials for Chagas drugs: the so-called BENEFIT trial, a study involving more than 1,000 participants that aims to determine whether benznidazole, one of the two currently used antiparasitic drugs for the disease, helps people with chronic as well as the more serious, acute form of Chagas; and a smaller, 130-person trial testing whether the micronutrient selenium prevents the heart problems associated with chronic infection. Both are due to finish in 2013. (Elsewhere, a small crop of earlier-stage trials for new compounds against Chagas have been launched in the US, Spain, Argentina and Bolivia.) In addition, attention is now finally being paid to the problem of HIV-Chagas co-infection. In 2006, a network of Brazilian scientists was set up to find answers to basic research questions such as how many people with Chagas also carry HIV. And, in a paper published in August, a team led by Maria Shikanai-Yasuda, an infectious disease researcher at the University of São Paulo, reported that these co-infected individuals tend to have T. cruzi levels that are several orders of magnitude greater than those who are HIV negative (PLoS Negl. Trop. Dis., e1277, 2011). This suggests that co-infected people may particularly benefit from taking benznidazole to keep their parasite loads in check—and, as such, provides some hope where there was little before. Additional data Author Details * Anna Petherick Search for this author in: * NPG journals * PubMed * Google Scholar
• Hopes build that new infrastructure can aid drug discovery
  - Nat Med 17(10):1174-1175 (2011)
  Nature Medicine | News Hopes build that new infrastructure can aid drug discovery * Bernardo EstevesJournal name:Nature MedicineVolume: 17,Pages:1174–1175Year published:(2011)DOI:doi:10.1038/nm1011-1174bPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg RIO DE JANEIRO — Four years ago, Carlos Morel, a biophysicist at the Oswaldo Cruz Foundation (Fiocruz), penned an opinion piece urging Brazil to improve its research infrastructure to address the country's public health challenges (Nature449, 180–182, 2007). Now that call to action is close to a reality, thanks to the nearly complete, five-story Center for Technological Development in Health (CDTS), which is slated to open here on the Fiocruz campus next year. "This building represents a dream come true for the entire Fiocruz community," says Morel, beaming like an expectant father. CDTS/Fiocruz and PAAL Fiocruz's near-finished building aims to boost translational research. Fiocruz is one of the most prominent biomedical research institutions in all of Latin America. Like a geographically dispersed version of the US National Institutes of Health, the public organization employs thousands of scientists at 15 units throughout Brazil. Fiocruz is also a major producer of therapeutics, capable of delivering 2 billion drug units and 200 million doses of vaccines every year. Nevertheless, it suffers from a problem common to Brazilian science as a whole: it fails to convert the knowledge gleaned by its researchers into biotech solutions. "Fiocruz researchers publish over 1,600 papers in indexed journals every year," says Morel, director of the CDTS. "Yet, the institution owns only a hundred patents." Morel and his colleagues—not to mention his financial backers—hope this building can help foster translational innovation and drive commercial success. When it opens next fall, the CDTS, built with a 140-million-Brazilian-reais ($84 million) investment from the federal government, will be equipped with state-of-the-art molecular biology labs for basic research, a high-throughput screening core for biotech prospection and animal facilities for preclinical proof-of-concept studies. What's more, a whole floor of the new center will be devoted to so-called 'flexible laboratories', where government scientists will work alongside collaborators from the private sector. For now, however, the entire 150,000-square-foot building—including the spiral staircase leading to the main entrance and the curved, aerofoil-shaped roof—remains shrouded in scaffolding. Some of the first projects likely to benefit from the new facility are partnerships between Fiocruz and foreign drug companies, including Britain's GlaxoSmithKline and the US's Genzyme, to develop treatments for a range neglected diseases such as dengue fever. In addition, CDTS scientists will focus on cancer, diabetes, hypertension and other noncommunicable diseases that represent a source of growing concern for public health in Brazil. "We are undergoing an epidemiological transition, and Fiocruz is reorienting its actions to act in both fronts," Morel says. According to Reinaldo Guimarães, an independent consultant in public health management and a former executive of the Ministry of Health, this new center should help Brazilian biomedical researchers cross the so-called 'valley of death' of drug development. "But," he notes, "it will not solve this problem alone. Brazil needs other centers like this if we want to foster translational science." Although no other Brazilian institution has built anything as ambitious—or as architecturally distinct—as CDTS, many academic centers throughout the country are improving their laboratories for translational research. For example, in 2009 the René Rachou Research Center, a Fiocruz unit in Belo Horizonte, inaugurated its Excellence Center in Bioinformatics, which is aimed at attracting private companies to use its facilities for computationally driven drug development. In a similar strategy, the University of São Paulo's Institute of Biomedical Sciences (ICB) is about to launch a new core facility with the latest proteomics and genomics technologies. "Our purpose," says ICB director Rui Curi, "is to provide access to huge expensive facilities that could not be afforded by individual projects from our university scientists and from other institutions, including private partners." Now the research community is waiting to see whether once they build it, the drug discoveries will come. Additional data Author Details * Bernardo Esteves Search for this author in: * NPG journals * PubMed * Google Scholar
• Hard line take on public health gives Brazil soft political power
  - Nat Med 17(10):1175 (2011)
  Nature Medicine | News Hard line take on public health gives Brazil soft political power * Anna PetherickJournal name:Nature MedicineVolume: 17,Page:1175Year published:(2011)DOI:doi:10.1038/nm1011-1175Published online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg With its booming economy and surging population, Brazil is expected to emerge as one of the major global powerhouses of the 21st century. But the country's growing influence on the world stage is driven by more than just raw economic and demographic might. Brazil has been particularly adept among so-called BRIC (Brazil, Russia, India and China) nations at using a gently persuasive form of 'soft power' diplomacy—and the country is using public health issues in particular to leverage long-term economic and political gain. Brazil's HIV/AIDS policy is probably the best example of what some experts have dubbed the country's 'health industrial complex'. In 1990, when more than 10,000 new AIDS cases were reported in the country, the World Bank famously predicted that Brazil would have 1.2 million infections by 2000. But thanks to universal provision of antiretroviral drugs, starting in 1996, and government social programs aimed at encouraging condom use and needle exchange, the number of people now living with HIV in Brazil sits at around 730,000, with a prevalence rate on par with that of the US. Buoyed by its successful track record, Brazil has used its international reputation and expertise to help several sub-Saharan African countries confront their ongoing HIV epidemics. Over the past decade, for example, Brazil has sent delegates to Mozambique, Nigeria and Angola to build up pharmaceutical plants and then hosted workshops back in the capital city, Brasilia, for African health officials to gain technical training for analytical techniques and strategies for drug production. "The ministry of foreign affairs has always wanted to market Brazil's success with AIDS," says Eduardo Gómez, a political scientist at Rutgers University in Camden, New Jersey who has studied the geopolitical incentives that led Brazilian officials to help Africa combat the disease (Geo. Public Poly. Rev., 15, 2009). "Brazil's not so focused on money, like the US, but on knowledge about how to build sustainable pharmaceutical companies, for example. Equally, it doesn't ask for access to oil [in return], like China, or try to redeem itself in some way like the US. It is trying to increase its standing." Brazil has similarly demonstrated global leadership when it comes to tobacco control, notes Kelley Lee, a health policy researcher at the London School of Hygiene and Tropical Medicine. Although the country is one of the biggest producers and exporters of tobacco in the world, Brazil established a strict national tobacco control program that led to a 35% drop in the adult smoking rate between the start of the effort in 1989 and 2003. As chronicled by Lee and her colleagues, Brazilian officials then promoted these advances and used their diplomatic channels to help build international consensus for the World Health Organization's Framework Convention of Tobacco Control, signed in 2003 (PLoS Med., e1000232, 2010). As a result, Brazilian diplomats have been appointed to a number of key positions within the World Health Organization (WHO) and the UN. Most recently, the country was also chosen to host the new South American Institute of Government in Health (ISAGS), which opened its doors in July in Rio de Janeiro as a place to facilitate the sharing of skills and ideas on public health between Latin American countries. But tobacco control and HIV are not the only arenas for Brazil's soft but powerful diplomatic approach. "Whenever there are formal negotiations on global health issues"—for example, the WHO's April 2011 agreement on sharing influenza-virus samples or last month's UN High-Level Meeting on Non-Communicable Diseases—"Brazil is an active participant," says Lee. "The country is certainly not a two-trick pony." In unpublished work, Gómez has examined how Brazilian diplomats are capitalizing on the country's policies toward tuberculosis and obesity. And João Augusto de Castro Neves, a political consultant based in Washington, DC, points to Brazil's leadership of the UN peacekeeping mission in Haiti as a demonstration to larger powers that it can handle affairs in its neighborhood of Latin America. "That will give it better credentials to become a permanent member of the UN Security Council," Castro Neves says. "Health is an area Brazil has a comparative advantage. It has a problem with military resources, some fiscal problems. But for the past 20 years Brazil has developed a lot of expertise in health." By lending others a hand, it seems, health diplomacy is slowly helping Brazil to help itself. Additional data Author Details * Anna Petherick Search for this author in: * NPG journals * PubMed * Google Scholar
• The NIH translational research center might trade public risk for private reward
  - Nat Med 17(10):1176 (2011)
  Nature Medicine | News | Opinion The NIH translational research center might trade public risk for private reward * Jerry Avorn1 * Aaron S Kesselheim1 * AffiliationsJournal name:Nature MedicineVolume: 17,Page:1176Year published:(2011)DOI:doi:10.1038/nm1011-1176Published online11 October 2011 The new National Center for Advancing Translational Sciences planned for the US National Institutes of Health intends to help transform biological findings into new therapeutic products. But if taxpayer funding of risky biomedical research translates into lucrative new medicines, the public should share in the economic benefits as well. View full text 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 Affiliations * Jerry Avorn is a professor of medicine and Aaron S. Kesselheim is an assistant professor of medicine at Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA. Author Details * Jerry Avorn Search for this author in: * NPG journals * PubMed * Google Scholar * Aaron S Kesselheim Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• An epidemic and social taboos
  - Nat Med 17(10):1177 (2011)
  Article preview View full access options Nature Medicine | Book Review An epidemic and social taboos * Stanley A. Plotkin1Journal name:Nature MedicineVolume: 17,Page:1177Year published:(2011)DOI:doi:10.1038/nm.2509Published online11 October 2011 Dangerous Pregnancies Leslie J. Reagan University of California Press, 2010 392 pp., hardcover, $29.95 ISBN: 9780520259034 Buy this book: USUKJapan Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Perhaps the most accurate view of current events is through the 'retrospectoscope,' for those who live through them often do not perceive their long-term importance. At least that thought came to mind while I was reading Dangerous Pregnancies by Leslie Reagan. The author is a historian who writes about the repercussions of the rubella epidemic that struck the US in 1964–1965. At the time, I had just returned from the UK to open a laboratory at the Wistar Institute in Philadelphia, expressly to work on rubella. Two preceding events had opened the way for me: the isolation of the virus in tissue culture by Thomas Weller, and separately by Paul Parkman and his associates and my clinical experience of the disease, which was frequent in the UK during the spring of 1963. By that time, the discovery by Norman Gregg of the teratogenic power of rubella virus in infected pregnant women was two decades old and common knowledge. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Affiliations * Stanley A. Plotkin is Emeritus Professor of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Stanley A. Plotkin Author Details * Stanley A. Plotkin Contact Stanley A. Plotkin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Fractional synthesis and clearance rates for amyloid β
  - Nat Med 17(10):1178-1179 (2011)
  Nature Medicine | Correspondence Fractional synthesis and clearance rates for amyloid β * Steven D Edland1, 2 * Douglas R Galasko2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1178–1179Year published:(2011)DOI:doi:10.1038/nm.2495Published online11 October 2011 To the Editor: We are writing regarding the 2006 publication of a new stable isotope labeling kinetics (SILK) protocol for assessing the fractional synthesis and clearance rates of amyloid β (Aβ) in the brain1. This protocol has been used to demonstrate the relatively accelerated rate of turnover of Aβ peptides in the brain1 and to assess the effect of drugs on Aβ production2. However, we are concerned that aspects of the protocol have not been fully appreciated, raising questions about the interpretation of findings from the protocol. View full text Author information * Author information * Supplementary 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 * Department of Family Preventive Medicine, University of California–San Diego, La Jolla, California, USA. * Steven D Edland * Department of Neuroscience, University of California–San Diego, La Jolla, California, USA. * Steven D Edland & * Douglas R Galasko Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Steven D Edland Author Details * Steven D Edland Contact Steven D Edland Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas R Galasko Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (353K) Supplementary Results, Supplementary Methods and Supplementary Figures 1 and 2 Additional data
• Reply to: Fractional synthesis and clearance rates for amyloid β
  - Nat Med 17(10):1179-1180 (2011)
  Nature Medicine | Correspondence Reply to: Fractional synthesis and clearance rates for amyloid β * Donald L Elbert1 * Bruce W Patterson2 * Lindsay Ercole3 * Vitaliy Ovod4 * Tom Kasten4 * Kwasi Mawuenyega4 * Kevin Yarasheski5 * John C Morris4, 6 * Tammie Benzinger3 * David M Holtzman4, 6, 7 * Randall J Bateman4, 6, 7 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1179–1180Year published:(2011)DOI:doi:10.1038/nm.2496Published online11 October 2011 Elbert et al. reply: We appreciate the motivation for the theoretical model proposed by Edland et al.1 on the basis of our report in Nature Medicine2 and subsequent studies showing decreased amyloid-β (Aβ) production with pharmacologic treatment3 and decreased Aβ clearance in Alzheimer's disease4. We use 'clearance' to refer to any process that removes the measured analyte (for example, Aβ), including cellular and enzymatic degradation processes and blood-brain barrier and cerebrospinal fluid (CSF) transport mechanisms. The authors propose that clearance of labeled proteins, as measured by in vivo stable isotope–labeled kinetics (SILK), does not reflect brain processes of clearance1. Although they do not provide additional experimental data, they do propose a theoretical model to support their assertions. However, in reviewing the proposed model, we suggest the model and the assertions made are not an accurate interpretation of our data or the physiology of CSF production and clearance (fo! r additional description, please see Supplementary Figs. 1–8). Furthermore, we are concerned that the suggestion to not measure clearance of labeled proteins will lead to less understanding of CNS protein metabolism. We suggest that the proposed model of Edland et al.1 is limited because it does not fit the experimental data, is not physiologically compatible with known CNS structure and physiology, and implements incorrect assumptions that are not supported by established isotope tracer methods. We thank Edland et al.1 for proposing their model and support the modeling of CNS Aβ generation, transport and degradation mechanisms, combined with additional experimental measures (including clearance measures) to provide major insights into the normal physiological and pathological roles of Aβ and other CNS proteins. View full text Author information * Author information * Supplementary 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 * Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA. * Donald L Elbert * Center for Human Nutrition, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. * Bruce W Patterson * Department of Radiology, Washington University School of Medicine, St. Louis, Missouri, USA. * Lindsay Ercole & * Tammie Benzinger * Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, USA. * Vitaliy Ovod, * Tom Kasten, * Kwasi Mawuenyega, * John C Morris, * David M Holtzman & * Randall J Bateman * Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA. * Kevin Yarasheski * Charles F. and Joanne Knight Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, Missouri, USA. * John C Morris, * David M Holtzman & * Randall J Bateman * Hope Center for Neurological Disorders, Washington University School of Medicine, St Louis, Missouri, USA. * David M Holtzman & * Randall J Bateman Competing financial interests R.J.B. and D.M.H. are cofounders of a company (C2N Diagnostics) that has licensed a Washington University patent on the technology described in the original article. Corresponding author Correspondence to: * Randall J Bateman Author Details * Donald L Elbert Search for this author in: * NPG journals * PubMed * Google Scholar * Bruce W Patterson Search for this author in: * NPG journals * PubMed * Google Scholar * Lindsay Ercole Search for this author in: * NPG journals * PubMed * Google Scholar * Vitaliy Ovod Search for this author in: * NPG journals * PubMed * Google Scholar * Tom Kasten Search for this author in: * NPG journals * PubMed * Google Scholar * Kwasi Mawuenyega Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Yarasheski Search for this author in: * NPG journals * PubMed * Google Scholar * John C Morris Search for this author in: * NPG journals * PubMed * Google Scholar * Tammie Benzinger Search for this author in: * NPG journals * PubMed * Google Scholar * David M Holtzman Search for this author in: * NPG journals * PubMed * Google Scholar * Randall J Bateman Contact Randall J Bateman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (601K) Supplementary Figures 1–8 Additional data
• The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species
  - Nat Med 17(10):1180-1181 (2011)
  Nature Medicine | Correspondence The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species * Alexis Kaushansky1 * Stefan H I Kappe1, 2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1180–1181Year published:(2011)DOI:doi:10.1038/nm.2456Published online11 October 2011 To the Editor: Protozoan parasites of the genus Plasmodium are the vector-borne causative agents of malaria, responsible for more than 350 million clinical cases and one million deaths annually. Studies using rodent malaria models have shown that during the bite of an infected Anopheles mosquito, Plasmodium sporozoites are transmitted and subsequently traverse a variety of cell types, wounding their membranes on the way1. This allows sporozoites to travel through the dermis into the bloodstream and then pass through the cell layer that lines blood vessels in the liver as well as several hepatocytes before taking up residence in host hepatocytes for further development as exoerythrocytic forms (EEFs)2. Although cell traversal provides a means to cross tissue barriers, it is unclear whether this constitutes its only biological role. A novel function for parasite host cell traversal was proposed by Carrolo et al.3, who showed that cell traversal by sporozoites of the rodent malaria parasite P! lasmodium berghei induces secretion of hepatocyte growth factor (HGF). HGF is a soluble factor that activates the receptor tyrosine kinase MET, which is capable of initiating a cascade of signaling events that result in hepatocyte proliferation and survival. Carrolo et al.3 further showed that this signaling cascade is crucial to promote early development of EEFs and thus substantially contributes to transmission success. More recently, the same group demonstrated, again using P. berghei, that MET signaling events are important for the parasite to protect itself from host hepatocyte apoptosis4 and subsequently showed that a dietary supplement, genistein, which inhibits MET activation, can treat liver-stage malaria infection5. A fundamental question, then, is whether this unique function for cell traversal is broadly conserved among Plasmodium species and whether it is found in human malaria parasites. View full text Author information * Author information * Supplementary 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 * Seattle Biomedical Research Institute, Seattle, Washington, USA. * Alexis Kaushansky & * Stefan H I Kappe * Department of Global Health, University of Washington, Seattle, Washington, USA. * Stefan H I Kappe Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stefan H I Kappe Author Details * Alexis Kaushansky Search for this author in: * NPG journals * PubMed * Google Scholar * Stefan H I Kappe Contact Stefan H I Kappe Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (601K) Supplementary Figures 1 and 2 and Supplementary Methods Additional data
• The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species
  - Nat Med 17(10):1181 (2011)
  Article preview View full access options Nature Medicine | Correspondence The crucial role of hepatocyte growth factor receptor during liver-stage infection is not conserved among Plasmodium species * Ana Rodriguez1 * Maria M Mota2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Page:1181Year published:(2011)DOI:doi:10.1038/nm.2487Published online11 October 2011 Rodriguez and Mota reply: We previously showed that migration of Plasmodium berghei sporozoites through hepatocytes causes cell wounding and leads to the release of hepatocyte growth factor (HGF) and the activation of its tyrosine kinase receptor, MET. We also showed that MET activation boosts P. berghei infection in mice, whereas its depletion leads to a reduction in infection1, 2. Now, Kaushansky and Kappe, although fully confirming our data with P. berghei, further show that neither Plasmodium yoelii nor Plasmodium falciparum sporozoite migration through hepatocytes activates MET. The authors use this system to highlight the similarities between P. falciparum and P. yoelii versus P. berghei. As the authors clearly state, P. berghei is less selective for its host cell than other Plasmodium species, as it is able to infect several hepatoma cell lines as well as several nonhepatocytic cells in a CD81-dependent or CD81-independent manner in vitro3. In contrast, P. yoelii and P. falciparum require CD81! to invade cells and are much more restricted in the range of cells they can infect in vitro. Nevertheless, P. falciparum and P. yoelii also differ quite markedly from each other. Although neither of them can infect HepG2 cells, expression of CD81 makes these cells susceptible to P. yoelii but not to P. falciparum infection. In contrast, certain aspects of the P. berghei model of liver stage infection resemble another important human pathogen, Plasmodium vivax. Indeed, like P. berghei, P. vivax readily infects HepG2 cells, indicating that both parasites are able to infect cells in a CD81-independent manner4. Altogether, although these studies emphasize the limitations of the currently available Plasmodium rodent models, these models are nevertheless very useful and often constitute the only possible approach to reveal and dissect the mechanisms underlying Plasmodium infection. Furthermore, all of these studies also highlight how the different Plasmodium species have evolved! different mechanisms to infect their hosts. Understanding how! these mechanisms were acquired and their role in virulence and infection will certainly contribute to improved design of future strategies to combat malaria. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine, New York, New York, USA. * Ana Rodriguez * Unidade de Malária, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal. * Maria M Mota Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Maria M Mota Author Details * Ana Rodriguez Search for this author in: * NPG journals * PubMed * Google Scholar * Maria M Mota Contact Maria M Mota Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Memory in disguise
  - Nat Med 17(10):1182-1183 (2011)
  Article preview View full access options Nature Medicine | Article A human memory T cell subset with stem cell–like properties * Luca Gattinoni1, 9 * Enrico Lugli2, 9 * Yun Ji1 * Zoltan Pos3, 4 * Chrystal M Paulos5, 6 * Máire F Quigley7, 8 * Jorge R Almeida8 * Emma Gostick7 * Zhiya Yu1 * Carmine Carpenito5, 6 * Ena Wang3, 4 * Daniel C Douek8 * David A Price7, 8 * Carl H June5, 6 * Francesco M Marincola3, 4 * Mario Roederer2, 9 * Nicholas P Restifo1, 9 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:1290–1297Year published:(2011)DOI:doi:10.1038/nm.2446Received29 April 2011Accepted19 July 2011Published online18 September 2011 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Immunological memory is thought to depend on a stem cell–like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human st! em cell–like memory T cell population is of direct relevance to the design of vaccines and T cell therapies. Figures at a glance * Figure 1: Identification of TSCM cells in human blood. () Flow cytometry analysis of sorted human CD45RO−CD62L+ naive CD8+ T cells before and 14 d after stimulation with α-CD3/CD28-coated beads and IL-2 in the presence or absence of 5 μM TWS119. Numbers indicate the percentage of cells in the CD45RO−CD62L+ gate. () Flow cytometry analysis of TWS119-generated CD45RO−CD62L+ naive-like CD8+ T cells overlaid with CD45RO−CD62L+ naive and memory (non-CD45RO−CD62L+) cells from a healthy donor (HD). () Flow cytometry analysis of PBMC from a healthy donor. Dot plots show the gating strategy to identify CD95+, IL-2Rβ+ TSCM cells. () Percentages of circulating CD8+ T cell subsets in 29 healthy donors. () Flow cytometry analysis of PBMC from a representative healthy donor. Overlaid histogram plots show expression levels of a given molecule in different CD8+ T cell subsets. CD8+ T cell subsets were defined as follows: TN cells, CD3+CD8+CD45RO−CCR7+CD45RA+CD62L+CD27+CD28+IL7Rα+CD95−; TSCM cells, CD3+CD8+CD45RO–CCR7+CD45RA+! CD62L+CD27+CD28+IL7Rα+CD95+; TCM cells, CD3+CD8+CD45RO+CD45RA−CCR7+CD62L+; TEM cells, CD3+CD8+CD45RO+CD45RA−CCR7−CD62L−. * Figure 2: TSCM cells possess attributes of conventional memory T cells. () TREC copy number in sorted CD8+ T cell subsets relative to TN cells. Data are represented as means ± s.e.m. of four donors. () Intracellular cytokine staining of PBMCs from a representative healthy donor after stimulation with SEB. Graphs show naive-like (NL) gated T cells. NL, CD45RO−CCR7+CD45RA+CD27+CD28+. Numbers represent the percentage of CD95+ (TSCM) and CD95− (TN) cells producing a single cytokine. () Percentages of CD8+ T cell subsets producing IFN-γ, IL-2 and TNF-α in six healthy donors (obtained as described in ). () Pie charts depicting the quality of the cytokine response in CD8+ T cell subsets in six healthy donors as determined by the Boolean combination of gates identifying IFN-γ+, IL-2+ and TNF-α+ cells. () Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution in sorted CD8+ T cell subsets after stimulation with 25 ng ml−1 of IL-15 for 10 d. Data are shown after gating on CD8+ cells. PD, percentage divided; PI, proliferation index. SSC! , side scatter. (,) Percentage of divided cells () and proliferation index of different CD8+ T cell subsets (), after stimulation as in panel . Data are represented as means ± s.e.m. of 9 donors. () Flow cytometry analysis of PBMC from HLA-A2+ donors. Graphs show tetramer-binding cells versus CD95 expression in the NL (CD45RO−CCR7+CD45RA+CD27+IL7Rα+) gate. () Percentage of tetramer-binding cells expressing CD95 in the NL gate, determined as in panel . Data represent the donors tested for tetramer specificity. HD, healthy donor; MP, melanoma patient. () Frequency of two immunodominant CMV-specific TCRβ clonotypes relative to all CMV-specific TCRβ clonotypes in pp65-specific T cell subsets isolated over a period of 22 months from a representative donor. The CDR3β amino acid sequences are shown. Changes in the frequencies of immunodominant clonotypes are depicted as the thickness of the bars, with the magnitude scale shown on the right. *P < 0.05; **P < 0.01; ***P < 0.0! 01; NS, not significant (t test, ,,, and χ2 permutation test,! ). * Figure 3: TSCM cells represent a distinct, less-differentiated T cell memory subset. () Heat map of differentially expressed genes (P < 0.01; one-way repeated measures analysis of variance (ANOVA), false discovery rate <5%, Benjamini-Hochberg's method) among CD8+ T cell subsets. Red and blue colors indicate increased and decreased expression, respectively. () Robust multichip analysis (RMA)-normalized intensity of selected genes progressively downregulated (naive-associated genes) or upregulated (effector-associated genes) from TN cells TSCM cells TCM cells TEM cells. Data are represented as means ± s.e.m. of three donors. () MDS analysis of differentially expressed genes (P < 0.01, false discovery rate <5%). Numbers represent the differentially regulated genes among each CD8+ T cell subset (P < 0.01 (t test) and > twofold change in expression). () Heat map of differentially expressed genes among TSCM and TCM cells (P < 0.01 (t test) and > twofold change in expression). Red and blue colors indicate increased and decreased expression, respectively. Full gene! names are listed in the Supplementary Methods. * Figure 4: Enhanced self-renewal and multipotency of TSCM cells. () Percentage of CD8+ T cells expressing CCR7 and CD62L (right) and CD45RA (left) relative to cell division after exposure to 25 ng ml−1 of IL-15 for 10 d. Slopes were compared using a Wilcoxon rank test, *P = 0.0391. Pre, the phenotype of sorted CD8+ T cell subsets before stimulation. () Percentage of CFSE-diluted CD8+ T cells that retained the parental phenotype after stimulation with 25 ng ml−1 of IL-15 for 10 d. *P < 0.05; **P < 0.01 (t test). () Percentage of CD8+ T cells expressing CCR7 and CD62L (right) and CD45RA (left) relative to cell division after stimulation with α-CD3/CD2/CD28-coated beads for 6 d. Pre, the phenotype of sorted CD8+ T cell subsets before stimulation. () Percentage of CFSE-diluted CD8+ T cells with a given phenotype after stimulation with α-CD3/CD2/CD28-coated beads for 6 d. () Stemness index of CD8+ memory T cell subsets. *P < 0.05 (t test). Data are represented as means ± s.e.m. of eight (,), six (,) or four () donors. * Figure 5: Increased proliferative capacity, survival and antitumor activity of TSCM cells. () 3H-thymidine incorporation by sorted CD8+ T cell subsets after stimulation with α-CD3/CD2/CD28-coated beads. Data are represented as means ± s.e.m. of ten donors. Results are normalized to the number of seeded cells, as different cell numbers were obtained from different sorts. c.p.m., counts per min. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). () Flow cytometry analysis of human T cells in the spleen, lymph nodes (LN) and liver of a representative NSG mouse at 4 weeks after adoptive transfer of CD4+ T cells (5 × 106) with or without sorted CD8+ T cell subsets (106). Graphs show T cells after gating on human CD45+ cells. Numbers indicate the percentage of cells in the CD4+CD8− or CD4−CD8+ gates. () Total human CD8+ T cell recovery in the spleens, LN and livers from six NSG mice 4 weeks after adoptive transfer of CD4+ T cells with or without sorted CD8+ T cell subsets. A total of six mice per T cell subset from two independent experiments (three replicate mice per! T cell subset per experiment) are shown. Horizontal lines indicate median values. *P < 0.05; **P < 0.01 (t test). (–) In vivo bioluminescent imaging (), percentage change of body weight (), and survival of NSG mice () bearing M108-luciferase mesothelioma after adoptive transfer of CD4+ T cells (106) with or without sorted CD8+ T cell subsets (3 × 106) expressing a mesothelin-specific chimeric antigen receptor. ***P < 0.001, one-way repeated measures ANOVA () and log-rank (Mantel-Cox) test (). Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE23321 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Luca Gattinoni, * Enrico Lugli, * Mario Roederer & * Nicholas P Restifo Affiliations * Center for Cancer Research, National Cancer Institute, US National Institutes of Health (NIH), Bethesda, Maryland, USA. * Luca Gattinoni, * Yun Ji, * Zhiya Yu & * Nicholas P Restifo * ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Enrico Lugli & * Mario Roederer * Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, US National Institutes of Health, Bethesda, Maryland, USA. * Zoltan Pos, * Ena Wang & * Francesco M Marincola * Center for Human Immunology, US National Institutes of Health, Bethesda, Maryland, USA. * Zoltan Pos, * Ena Wang & * Francesco M Marincola * Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Chrystal M Paulos, * Carmine Carpenito & * Carl H June * Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Chrystal M Paulos, * Carmine Carpenito & * Carl H June * Department of Infection, Immunity and Biochemistry, Cardiff University School of Medicine, Heath Park, Cardiff, UK. * Máire F Quigley, * Emma Gostick & * David A Price * Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Máire F Quigley, * Jorge R Almeida, * Daniel C Douek & * David A Price Contributions L.G., E.L., Y.J., Z.P., C.M.P., J.R.A., Z.Y. and C.C. carried out experiments; L.G., E.L., Y.J., Z.P., C.M.P. and J.R.A. analyzed experiments; L.G., E.L., C.M.P., E.W., D.C.D., D.A.P., C.H.J., F.M.M., M.R. and N.P.R. designed experiments; E.G., M.F.Q. and D.A.P. contributed reagents; E.L. and M.R. edited the manuscript; and L.G. and N.P.R. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Luca Gattinoni or * Nicholas P Restifo Author Details * Luca Gattinoni Contact Luca Gattinoni Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico Lugli Search for this author in: * NPG journals * PubMed * Google Scholar * Yun Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Zoltan Pos Search for this author in: * NPG journals * PubMed * Google Scholar * Chrystal M Paulos Search for this author in: * NPG journals * PubMed * Google Scholar * Máire F Quigley Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge R Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Emma Gostick Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiya Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Carmine Carpenito Search for this author in: * NPG journals * PubMed * Google Scholar * Ena Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel C Douek Search for this author in: * NPG journals * PubMed * Google Scholar * David A Price Search for this author in: * NPG journals * PubMed * Google Scholar * Carl H June Search for this author in: * NPG journals * PubMed * Google Scholar * Francesco M Marincola Search for this author in: * NPG journals * PubMed * Google Scholar * Mario Roederer Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas P Restifo Contact Nicholas P Restifo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–15, Supplementary Tables 1–9 and Supplementary Methods Additional data
• Epigenetic tumor suppression by BRCA1
  - Nat Med 17(10):1183-1185 (2011)
  Article preview View full access options Nature Medicine | Article Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155 * Suhwan Chang1 * Rui-Hong Wang2 * Keiko Akagi3 * Kyung-Ae Kim1 * Betty K Martin4 * Luca Cavallone5, 6 * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab)7 * Diana C Haines8 * Mark Basik5 * Phuong Mai9 * Elizabeth Poggi10 * Claudine Isaacs10 * Lai M Looi11 * Kein S Mun11 * Mark H Greene9 * Stephen W Byers10 * Soo H Teo12, 13 * Chu-Xia Deng2 * Shyam K Sharan7 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1275–1282Year published:(2011)DOI:doi:10.1038/nm.2459Received13 October 2010Accepted03 August 2011Published online25 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg BRCA1, a well-known tumor suppressor with multiple interacting partners, is predicted to have diverse biological functions. However, so far its only well-established role is in the repair of damaged DNA and cell cycle regulation. In this regard, the etiopathological study of low-penetrant variants of BRCA1 provides an opportunity to uncover its other physiologically important functions. Using this rationale, we studied the R1699Q variant of BRCA1, a potentially moderate-risk variant, and found that it does not impair DNA damage repair but abrogates the repression of microRNA-155 (miR-155), a bona fide oncomir. Mechanistically, we found that BRCA1 epigenetically represses miR-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miR-155 promoter. We show that overexpression of miR-155 accelerates but the knockdown of miR-155 attenuates the growth of tumor cell lines in vivo. Our findings demonstrate a new mode of tumor suppression by BRC! A1 and suggest that miR-155 is a potential therapeutic target for BRCA1-deficient tumors. Figures at a glance * Figure 1: R1699Q mutant ES cells show reduced survival and differentiation defects. () Schematics of generation of R1699Q ES cells using PL2F8 cells containing a null and a conditional allele of Brca1. Two halves of human HPRT1 minigenes (HP and RT) flanking the two loxP sites (shaded triangles) of the conditional allele. Cre recombinants are HAT resistant (HATR). () Southern hybridization of HATR colonies from experiments without BAC (NO BAC), wild-type (WT) BRCA1 BAC and R1699Q BRCA1 BAC. Bottom band, null allele (MT); top band, conditional allele (cko). Rescue rate, percentage Brca1ko/ko clones. Asterisk, Brca1ko/ko ES cell. () Whole mount of embryoid bodies generated from ES cells expressing wild-type (left) and R1699Q (right) BRCA1 at day 14 in culture. Bottom, higher magnification of embryoid bodies. Scale bars, 50 μm, top; 20 μm, bottom. () H&E staining of embryoid bodies generated from ES cells expressing wild-type (left) and R1699Q (right) BRCA1 at day 14 in culture. Scale bars, 100 μm, top; 50 μm, bottom. () TUNEL staining of embryoid bodies. ! Arrow, TUNEL+ cells. Scale bars, 50 μm. () Teratoma growth of one wild-type and two R1699Q clones were examined in mice (n = 5 for each group). Values are means ± s.e.m. (P = 0.007). () H&E staining of teratomas dissected 15 d after injection. Top, section of the whole teratoma; middle, magnified images of the regions indicated at top. Arrows, neurorosette structures. Bottom, neural cells immature in wild-type (left) and more differentiated in R1699Q (right) teratomas. Scale bars, 2 μm, top; 0.2 μm, middle; 50 μm, bottom. * Figure 2: Identification of miR-155 upregulation in R1699Q mutant cells and its effect on ES cell differentiation. () Quantification of miR-155 by rtPCR in wild-type (WT), R1699Q (RQ) and M1652I (MI) ES cells and embryoid bodies (EB cells) on day 7 of culture. U6 small nuclear RNA (snRNA) was used for normalization. Top, BRCA1 protein expression. () Representative pictures of miR-155 in situ hybridization in wild-type and R1699Q embryoid bodies using DIG-labeled antisense LNA of miR-155. Top, no-probe controls showing background signal (scale bar, 50 μm). Right, relative average signal. Error bars indicate s.d. () Schematic of miR-155 inducible system in ES cells. miR-155 is induced by tetracycline, which then represses the luciferase reporter (luc) containing miR-155-binding sites at the 3′ end. () miR-155 overexpression in ES cells after tetracycline (tet) induction, with U6 snRNA as control. Top, overexpression by northern hybridization; bottom, quantification by rtPCR. Error bars indicate s.d. () Representative pictures of embryoid bodies generated from wild-type ES cells with ind! uced expression of miR-155. Top, whole mount of embryoid bodies; bottom, H&E-stained sections. Scale bars, 100 μm. () Teratoma growth of wild-type ES cells with (Tet1–Tet5) and without (Con1–Con5) miR-155 induction. Left, teratoma growth. Right, average tumor volume on day 18 after injection. Minimum value of each group was excluded (n = 4; values are means ± s.e.m.). () Representative picture of teratomas in mice injected with control cells (−Tet, right) and Tet-induced cells (+Tet, left). Bottom, enlarged view. * Figure 3: BRCA1 negatively controls miR-155 expression. () Quantification of miR-155 in tumors from the Brca1ko/+;Trp53ko/+;TgR1699Q mice (RQ, n = 9). Normal liver (RQ93 nor), mammary gland (RQ515 nor) and tumors from Brca1ko/+;Trp53ko/+;TgM1652I mice (MI, n = 4) used as control. () Quantification of miR-155 in a breast tumor from an R1699Q mutation carrier. Normal, normal breast tissue. () Expression analysis of miR-155 by northern hybridization in four human breast cancer cell lines of different BRCA1 genotypes. Positive control, HEK293 cell line overexpressing miR-155; loading control, U6 snRNA; asterisk, miR-155 signal. Right, quantification of signal, normalized by U6 snRNA. () rtPCR of miR-155 in four Brca1 deficient tumors (42, 572, 907 and M161) from Brca1cko/cko;p53ko/+;MMTV Cre mice and four tumors (Con 112X, Con 172X, Con I107 and Con I224) from Her2/Neu transgenic mice. MCF7 and HCC1937 were negative and positive control, respectively. () rtPCR of miR-155 in MECs from two Brca1cko/cko;K14-Cre mice. Control, MECs from ! K14 Cre only and Brca1cko/cko. () Knockdown of BRCA1 in two clones (75 and 80) of HEK293 cells stably expressing BRCA1 short hairpin RNA (top). Loading control, β-actin (middle). Bottom, rtPCR quantification of miR-155 in the BRCA1 knockdown clones (75 and 80). Con, control. () Real-time quantification of miR-155 after ectopic expression of wild-type (WT) or R1699Q BRCA1 in BRCA1-deficient MDA-MB-436 cells. Top, expression of wild-type and R1699Q BRCA1. () miR-155 reporter assay in BRCA1-deficient HCC1937 cells using increasing amounts of wild-type BRCA1 cDNA (Con, untransfected cells and BRCA50, 50 ng; BRCA100, 100 ng). Error bars indicate s.d. * Figure 4: Mechanism of miR-155 repression by BRCA1. () Binding of BRCA1 to miR-155 promoter at two potential regions (BRCA1-1 and BRCA1-2). ES cells and embryoid bodies (EB) at day 7 in culture with wild-type and R1699Q (RQ). BRCA1 by ChIP assay. Input, 5% of total chromatin. () Effect of mutation of putative BRCA1-binding sites (Mut1, Mut2 or double mutant) on miR-155 promoter activation measured by luciferase assay (*P < 0.05, **P < 0.01, ***P < 0.005). () Effect of HDAC inhibitors on miR-155 expression measured by rtPCR in BRCA1+ MDA-MB-468 (left) and BRCA1-deficient MDA-MB-436 cells (right). Con, no treatment; Aph, apicidin. () Effect of HDAC inhibitors on wild-type miR-155 promoter (left) and mutant promoter (right) activation in MDA-MB-468 cells measured by luciferase assay (*P < 0.02, **P < 0.01, ***P < 0.005). Con, no treatment; Aph, apicidin. () ChIP assay to quantify acetylation level of histones H2A and H3 on miR-155 promoter, in wild-type and R1699Q EB cells by rtPCR. () Effect of wild-type, RQ and wild-type + RQ ! BRCA1 on acetylation of histones H2A and H3 on miR-155 promoter in MDA-MB-436 cells measured by ChIP assay and rtPCR. Con, transfection with vector only (*P < 0.02, **P < 0.01). Western blot (WB) analysis of ectopic protein expression using antibody to hemagglutinin (HA). () Association of HDAC2 on miR-155 promoter in wild-type and R1699Q EB cells analyzed by ChIP assay and rtPCR. IgG, negative control. () Co-immunoprecipitation (Co-IP) between HDAC2 and wild-type or M1652I or R1699Q BRCA1 in MECs from mice with wild-type or R1699Q BAC transgenes (left) or M1652I transgene (right). Top, IP with HDAC2 antibody; bottom, IP with human-specific BRCA1 antibody. () MDA-MB 468 cells transfected with luciferase reporter plasmid with mouse wild-type or double mutant (MT) miR-155 promoter (pro). ChIP assay was carried out with indicated antibodies (Ab). Association of BRCA1-HDAC2 complex and acetylation of histones H2A and H3 on the transfected mouse miR-155 promoter (mir155, top) an! d endogenous miR-155 promoter (MIR155). BRCA1 binding on ESRRG! , CCNB1 and STAT5A promoters with putative binding sites. Error bars indicate s.d. * Figure 5: Physiological relevance of miR-155 upregulation in BRCA1-deficient tumors. () Xenograft tumor growth of MDA-MB-468 cells with stable expression of miR-155 (P = 4.12 × 10−4, ANCOVA test). Values are mean ± s.e.m. () Growth of two clones (C9 and D6) with miR-155 stable knockdown and the parental cells (Con) orthotropically transplanted in mice (values are means ± s.e.m., *P = 0.031, **P = 0.025). () Average mass of tumors in (values are means ± s.e.m.). () Representative pictures of 66 human breast tumors in tumor tissue microarray probed with antibody to BRCA1 (Ab-1) or mouse IgG (negative control) and DIG-labeled antibody to miR-155. Scale bars, 50 μm. () rtPCR quantification of miR-155 in 28 tumors from non-BRCA1 controls and 14 tumors from BRCA1 mutation (mut) carriers (see Supplementary Table 6 for mutation description). RNU5A was used for normalization. Shaded area, low miR-155 based on twofold cut-off (dashed horizontal line). Text box, number of miR-155 high and low tumors in each group. () Schematic of role for BRCA1 in epigenetic con! trol of miR-155 promoter. In wild-type BRCA1–containing cells, miR-155 is silenced by the BRCA1-HDAC2 complex, which deacetylates H2A and H3 histones. Without functional BRCA1, the interaction of BRCA1-HDAC2 complex is disrupted, which in turn increases acetylated (Ac) H2A and H3, which activates the miR-155 promoter. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Affiliations * Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Suhwan Chang & * Kyung-Ae Kim * Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA. * Rui-Hong Wang & * Chu-Xia Deng * Human Cancer Genetics Program, Departments of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio, USA. * Keiko Akagi * Science Applications International Corporation (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Betty K Martin * Lady Davis Institute, Jewish General Hospital, Montréal, Quebec, Canada. * Luca Cavallone & * Mark Basik * Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montréal, Quebec, Canada. * Luca Cavallone * The Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia. * $affiliationAuthor & * Shyam K Sharan * Pathology Histotechnology Laboratory, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Diana C Haines * Clinical Genetics Branch, National Cancer Institute, Rockville, Maryland, USA. * Phuong Mai & * Mark H Greene * Georgetown-Lombardi Comprehensive Cancer Center Georgetown University, Washington, DC, USA. * Elizabeth Poggi, * Claudine Isaacs & * Stephen W Byers * Department of Pathology, University Malaya Medical Center, Kuala Lumpur, Malaysia. * Lai M Looi & * Kein S Mun * Department of Surgery University Malaya Cancer Research Institute, University Malaya Medical Centre, Kuala Lumpur, Malaysia. * Soo H Teo * Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Kuala Lumpur, Malaysia. * Soo H Teo Consortia * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) Contributions S.C. conceived the idea, conducted all the experiments and wrote the manuscript, R.-H.W. and C.-X.D. helped with xenograft experiment, provided mouse tumor samples and cell lines, K.A. carried out bioinformatics analysis, K.-A.K. helped with experiments, B.K.M. helped with mouse work, D.C.H. performed histopathological analysis, L.C. analyzed human tumor samples, M.B., P.M., M.H.G., KConFab, L.M.L., K.S.M., S.H.T., E.P., C.I. and S.W.B. provided human tumor samples, S.K.S. conceived the idea, supervised the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shyam K Sharan Author Details * Suhwan Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Rui-Hong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Keiko Akagi Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung-Ae Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Betty K Martin Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Cavallone Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) * Diana C Haines Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Basik Search for this author in: * NPG journals * PubMed * Google Scholar * Phuong Mai Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Poggi Search for this author in: * NPG journals * PubMed * Google Scholar * Claudine Isaacs Search for this author in: * NPG journals * PubMed * Google Scholar * Lai M Looi Search for this author in: * NPG journals * PubMed * Google Scholar * Kein S Mun Search for this author in: * NPG journals * PubMed * Google Scholar * Mark H Greene Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen W Byers Search for this author in: * NPG journals * PubMed * Google Scholar * Soo H Teo Search for this author in: * NPG journals * PubMed * Google Scholar * Chu-Xia Deng Search for this author in: * NPG journals * PubMed * Google Scholar * Shyam K Sharan Contact Shyam K Sharan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10, Supplementary Tables 1–6, Supplementary Methods and Supplementary Discussion Additional data
• Tuberculosis vaccine promises sterilizing immunity
  - Nat Med 17(10):1185-1186 (2011)
  Article preview View full access options Nature Medicine | Article A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis * Kari A Sweeney1, 2 * Dee N Dao1, 2, 6 * Michael F Goldberg2, 6 * Tsungda Hsu1, 2 * Manjunatha M Venkataswamy2 * Marcela Henao-Tamayo3 * Diane Ordway3 * Rani S Sellers4 * Paras Jain1, 2 * Bing Chen1, 2 * Mei Chen1, 2 * John Kim1, 2 * Regy Lukose1, 2 * John Chan2, 5 * Ian M Orme3 * Steven A Porcelli2, 5 * William R Jacobs Jr1, 2, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1261–1268Year published:(2011)DOI:doi:10.1038/nm.2420Received18 April 2011Accepted14 June 2011Published online04 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosisesx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4+ memory T cells and involved a di! stinct shift in the pattern of cytokine responses by CD4+ cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis. Figures at a glance * Figure 1: Role of the esx-3 region in evasion of innate immunity by M. smegmatis in a high-dose infection model. () Serum concentrations of IL-12 p40, IL-6 and IFN-γ in mice (C57BL/6) infected intravenously with 5 × 107 CFU per mouse of parental Msmeg (strain mc2155), IKE (Δesx-3) or Δesx-1 strains. () Survival after intravenous inoculation of 5 × 107 CFU parental Msmeg or IKE in C57BL/6 (n = 10), Rag1−/− (n = 5) or Myd88−/− (n = 5) mice. Survival was significantly different for IKE versus parental Msmeg in C57BL/6 and Rag1−/− mice (P < 0.001, log-rank test), but not in Myd88−/− mice. () CFU in lungs and kidneys of C57BL/6 mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE infection. () CFU in lungs and kidneys of Myd88−/− mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. For , and , data are mean ± s.e.m. of three mice per group. † indicates de! ath or killing owing to extreme morbidity of all mice in a group; *P < 0.01 (analysis of variance (ANOVA)). Data for all panels are representative of six independent experiments. * Figure 2: Characterization of innate immune responses against the Msmeg IKEPLUS strain. () Serum concentrations of IL-12 p40, IL-12p70 and IFN-γ in C57BL/6 mice infected with IKE or IKEPLUS (*P < 0.01, ANOVA with Bonferroni post-test). () Growth of bacteria (CFU) in the lungs (left) and kidneys (right) of Rag1−/− mice after intravenous inoculation of Msmeg parental, IKE or IKEPLUS strains. Differences in CFU were statistically significant (P < 0.001, two-way ANOVA) for Msmeg versus either IKE or IKEPLUS at day 3. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE and IKEPLUS. () Average time to death of mice after intravenous inoculation of IKEPLUS in C57BL/6 (n = 10), Rag1−/− (n = 5) and Myd88−/− (n = 5) mice. Survival of wild-type C57BL/6 and Rag1−/− strains was significantly different than that of Myd88−/− mice (P < 0.05, log-rank test). All inoculations were done at a high intravenous dose (5 × 107 CFU per mouse). Data from and are mean ± s.e.m. of three mice per group! . Data for all panels are representative of four independent experiments. * Figure 3: Bactericidal immunity against Mtb in mice vaccinated with IKEPLUS. () Survival of C57BL/6 mice that were sham immunized (intravenous (i.v.) PBS injection; n = 21) or immunized by intravenous infection with IKEPLUS (5 × 107 CFU per mouse; n = 20) or by subcutaneous (s.c.) infection with BCG (1 × 107 CFU per mouse; n = 18) and subsequently challenged 8 weeks later with a high intravenous dose (1 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival were significant for PBS versus BCG (P = 0.0389, log-rank test), PBS versus IKEPLUS (P < 0.0001, log-rank test) or BCG versus IKEPLUS (P < 0.0001, log-rank test). () Measurement of CFU from the lungs, spleens and livers of C57BL/6 mice in a separate experiment in which mice were immunized and challenged as described in . Each symbol represents one mouse. # at base of the y axis for the liver CFU plot indicates that no colonies were obtained from IKEPLUS-vaccinated mice at day 202, consistent with clearance of Mtb infection in that tissue (entire organs were plated to give a limit of detecti! on of 1 CFU). The CFU at day 100 was not significantly different between BCG- or IKEPLUS-immunized mice in any organ (P > 0.05, ANOVA). Data shown are pooled from two independent experiments. () Survival and lung CFU of C57BL/6 mice that were sham immunized (i.v. PBS; n = 6) or immunized by intravenous infection with IKE (5 × 107 CFU per mouse; n = 6) or IKEPLUS (5 × 107 CFU per mouse; n = 6) and subsequently challenged 6 weeks later with a high intravenous dose (5 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival curves were significant for PBS versus IKEPLUS (P = 0.0007, log-rank test), PBS versus IKE (P = 0.0049, log-rank test) and IKEPLUS versus IKE (P = 0.0246, log-rank test). For lung CFU, asterisks indicate significant differences (P < 0.05, two-way ANOVA) compared to the PBS control group. Results shown are representative of four independent experiments. * Figure 4: Improvement of histopathology in IKEPLUS-immunized mice during resolution of Mtb infection. () Representative images are shown for lungs (left) and livers (right) of C57BL/6 mice immunized with IKEPLUS and challenged with Mtb as described in Figure 3. Top and middle, H&E staining; bottom, AFB staining. Scale bars correspond to 650 nm, 40 nm and 10 nm in top, middle and bottom rows, respectively. The asterisk indicates an area of residual dense granulomatous infiltrate. Filled arrowheads indicate occasional macrophages in open alveoli in the lungs of mice with resolving inflammation at day 202. Open arrowheads mark granulomatous foci in the liver, which were also substantially resolved by day 202. AFBs were visualized in macrophages in lung and liver sections at both time points. () Quantitative scoring of histopathology confirming that all three groups showed similar pathology in the lungs and liver at day 100 (P > 0.05, two-way ANOVA). However, at day 202 the survivors from the IKEPLUS-immunized group showed significantly reduced pathology scores (P < 0.05 compare! d to all other groups at day 100; one-way ANOVA with Tukey post-test). * Figure 5: Protection from Mtb challenge by subcutaneous immunization with IKEPLUS. () Survival (left) of C57BL/6 mice (n = 10–14 per group) immunized subcutaneously with PBS, IKEPLUS (1 × 108 CFU per mouse), or BCG (1 × 106 CFU per mouse) and challenged 5 weeks later with a high intravenous dose (1 × 108 CFU per mouse) of Mtb H37Rv. CFU (right) data from the same experiment show means of three mice per group. The survival curve for IKEPLUS-immunized mice was significantly different from that of the BCG-immunized mice (P < 0.001, log-rank test). The CFU levels in lungs of IKEPLUS-immunized mice were significantly different from those of surviving saline-immunized mice at days 14 and 20 (P < 0.01, two-way ANOVA) and compared to BCG-immunized mice at day 20 (P < 0.01, two-way ANOVA). () Survival (left) of mice immunized subcutaneously with PBS (n = 10), 1 × 106 CFU of BCG (n = 10) or 1 × 108 CFU of IKEPLUS (n = 9) and challenged 1 month later with ~100 CFU of aerosolized Mtb H37Rv. The survival curves for IKEPLUS- and BCG-immunized mice were significan! tly different from that of PBS-immunized mice (P < 0.05, log-rank test). The survival curve for IKEPLUS was not significantly different from that of BCG-immunized mice (P = 0.0898, log-rank test). Lung CFU (right) from three mice per group from the same experiment showed that the IKEPLUS- and BCG-vaccinated groups had significantly reduced CFU compared to saline-immunized mice at days 21, 56 and 112 (P < 0.05, two-way ANOVA). IKEPLUS-immunized mice also showed lower CFU versus saline- or BCG-immunized mice at day 175 (*P < 0.05, two-way ANOVA). Results are representative of three independent experiments. * Figure 6: Role of CD4+ T cells in IKEPLUS-induced protective immunity. () Contributions of CD4+ and CD8+ subsets, as assessed by adoptive transfer of T cells from IKEPLUS-immunized mice. Naive recipient mice (n = 5 per group) were challenged 1 d after T cell transfer with M. tuberculosis (H37Rv, 1 × 107 CFU i.v. per mouse). Left, lung CFU of Mtb for mice killed 3 weeks after Mtb challenge. The dashed line indicates mean CFU in the lungs of five mice that were directly immunized with IKEPLUS (5 × 107 CFU per mouse i.v.) at 3 weeks before challenge. A negative control group of five sham-immunized mice that did not receive T cell transfer before challenge had CFU levels that were not significantly different from those of recipients of T cells from saline-immunized donors (not shown). *P < 0.05 compared to negative control group (ANOVA). This experiment was performed three times with similar results. Right, survival curves from similarly treated groups (n = 5) of mice from one experiment. IKEPLUS CD4+, adoptive transfer of CD4+ T cells from IKEPL! US-immunized donors; BCG CD4+, transfer of CD4+ T cells from BCG-immunized donors. Mice immunized intravenously with IKEPLUS directly or injected with PBS only were included as controls. The IKEPLUS CD4+ group was significantly different from the PBS group (P = 0.035, log-rank test) but not significantly different from the directly IKEPLUS-immunized group (P = 0.3177). () Cytokine production by CD4+ T cells in the lungs of mice immunized intravenously with PBS (sham) or IKEPLUS (5 × 107 CFU per mouse) or subcutaneously with BCG (1 × 106 CFU per mouse) and challenged 8 weeks later with Mtb H37Rv (1 × 107 CFU i.v.) (n = 5 mice per time point). The graphs indicate the absolute numbers of CD4+ T cells staining positively for three, two or one of the cytokines analyzed (IFN-γ, TNF and IL-2) after re-stimulation in vitro with a peptide representing the immunodominant epitope of TB9.8. Significant differences between the IKEPLUS- and BCG-vaccinated groups are indicated (*P < 0! .05, **P < 0.01, ***P < 0.001, two-way ANOVA). Results shown a! re representative of two similar experiments. () Same as in except that the cells were re-stimulated with plate-bound monoclonal antibodies to CD3 and CD28 before analysis. () Pie charts showing the relative fractions of CD4+ T cells at 14 d after Mtb challenge producing three, two or one of the cytokines with either specific antigen re-stimulation in vitro (TB9.8 peptide) or polyclonal activation (anti-CD3). Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Dee N Dao & * Michael F Goldberg Affiliations * Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA. * Kari A Sweeney, * Dee N Dao, * Tsungda Hsu, * Paras Jain, * Bing Chen, * Mei Chen, * John Kim, * Regy Lukose & * William R Jacobs Jr * Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA. * Kari A Sweeney, * Dee N Dao, * Michael F Goldberg, * Tsungda Hsu, * Manjunatha M Venkataswamy, * Paras Jain, * Bing Chen, * Mei Chen, * John Kim, * Regy Lukose, * John Chan, * Steven A Porcelli & * William R Jacobs Jr * Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA. * Marcela Henao-Tamayo, * Diane Ordway & * Ian M Orme * Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA. * Rani S Sellers * Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. * John Chan, * Steven A Porcelli & * William R Jacobs Jr Contributions K.A.S. constructed bacterial strains, performed or contributed to the design of most experiments, and analyzed and interpreted data. D.N.D. carried out portions of the infection and challenge experiments. M.F.G. contributed to the T cell adoptive transfer studies and designed, performed and analyzed all flow cytometry analyses. T.H. participated in design and construction of bacterial strains and in the performance of mouse infection and challenge experiments. P.J. contributed to construction of bacterial strains. M.M.V. and M.H.-T. assisted with experiments analyzing responding T cell populations. R.S.S. analyzed and scored the histopathology samples. B.C., M.C., J.K. and R.L. carried out mouse infections, organ harvesting and quantification of bacilli in tissues. D.O., J.C., I.M.O., S.A.P. and W.R.J. Jr. designed and interpreted experiments. K.A.S., S.A.P. and W.R.J. Jr. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * William R Jacobs Jr Author Details * Kari A Sweeney Search for this author in: * NPG journals * PubMed * Google Scholar * Dee N Dao Search for this author in: * NPG journals * PubMed * Google Scholar * Michael F Goldberg Search for this author in: * NPG journals * PubMed * Google Scholar * Tsungda Hsu Search for this author in: * NPG journals * PubMed * Google Scholar * Manjunatha M Venkataswamy Search for this author in: * NPG journals * PubMed * Google Scholar * Marcela Henao-Tamayo Search for this author in: * NPG journals * PubMed * Google Scholar * Diane Ordway Search for this author in: * NPG journals * PubMed * Google Scholar * Rani S Sellers Search for this author in: * NPG journals * PubMed * Google Scholar * Paras Jain Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Mei Chen Search for this author in: * NPG journals * PubMed * Google Scholar * John Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Regy Lukose Search for this author in: * NPG journals * PubMed * Google Scholar * John Chan Search for this author in: * NPG journals * PubMed * Google Scholar * Ian M Orme Search for this author in: * NPG journals * PubMed * Google Scholar * Steven A Porcelli Search for this author in: * NPG journals * PubMed * Google Scholar * William R Jacobs Jr Contact William R Jacobs Jr Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Methods Additional data
• Turning up the heat on colorectal cancer
  - Nat Med 17(10):1186-1188 (2011)
  Article preview View full access options Nature Medicine | Article Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis * Coralie Dorard1, 2 * Aurélie de Thonel3, 4 * Ada Collura1, 2 * Laetitia Marisa5 * Magali Svrcek1, 2, 6 * Anaïs Lagrange1, 2 * Gaetan Jego3, 4 * Kristell Wanherdrick1, 2 * Anne Laure Joly3, 4 * Olivier Buhard1, 2 * Jessica Gobbo3, 4 * Virginie Penard-Lacronique7 * Habib Zouali8 * Emmanuel Tubacher8 * Sylvain Kirzin9 * Janick Selves9 * Gérard Milano10 * Marie-Christine Etienne-Grimaldi10 * Leila Bengrine-Lefèvre11 * Christophe Louvet11 * Christophe Tournigand11 * Jérémie H Lefèvre2, 12 * Yann Parc2, 12 * Emmanuel Tiret2, 12 * Jean-François Fléjou1, 2, 6, 13 * Marie-Pierre Gaub14 * Carmen Garrido3, 4, 15, 16 * Alex Duval1, 2, 16 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:1283–1289Year published:(2011)DOI:doi:10.1038/nm.2457Received23 February 2011Accepted01 August 2011Published online25 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and tre! atment response in CRC. Figures at a glance * Figure 1: Identification of HSP110 as a new target gene for frequent mutation in MSI CRC cell lines and primary tumors. () Principle of the study we performed using RT-PCR to find exon-skipping events due to MSI in CRC cell lines. n = the number of analyzed exons. IR, intronic repeat. () BET staining of RT-PCR product in agarose gel detecting the presence of a specific, additional HSP110 band in MSI CRC cell lines. Its size implied exon 9 skipping in HSP110 mRNA. We did not detect its expression in all MSS cell lines tested. The results were confirmed in a series of primary CRCs (T) that were MSI or MSS, respectively, and their matching normal mucosa (N). () Allelic profiles for several MSI CRC cell lines and primary tumors and controls (MSS CRC and LBL cell lines) at the intronic HSP110 T17. MSS samples were weakly polymorphic, whereas MSI CRC cell lines and primary tumors always showed aberrant alleles that fell outside the polymorphic zone (in orange). Numbers indicate the size of the HSP110 T17 deletion in MSI tumor samples (in base pairs). () Mutation of the intronic HSP110 T17 repeat wa! s more frequent in MSI primary CRCs (100%) and in premalignant adenomas (53%) than that of any known coding microsatellite alteration. Numbers indicate the size of the HSP110 T17 deletion in MSI tumor or adenoma samples. * Figure 2: Expression of HSP110ΔE9 relatively to mutational status of HSP110 T17 sequence in MSI CRC. () Amplification plots corresponding to HSP110wt and HSP110ΔE9 RT-PCR products. Results are expressed (E) as n-fold difference in HSP110ΔE9 relative to HSP110wt expression (ΔCt), where ΔCt was determined in each case by subtracting the average Ct value of the HSP110ΔE9 mRNA from the average Ct value of the HSP110wt mRNA. () Values of E ratio are shown for the 13 MSI CRC cell lines and 43 primary tumor samples. MSS LBLs (n = 20), MSS CRC cell lines (n = 7; ALA, COLO320, SW480, FET, GLY, FRI, EB) and MSS primary CRCs (n = 20) were also tested as controls. A significant difference was observed between MSI and MSS primary CRCs with respect to HSP110ΔE9 expression (P = 8.5 × 10–5; Student's t test). Medium E values are indicated by red bars in each case. () Black and red bars correspond to CRCs in which HSP110ΔE9 mRNA expression was low or high, respectively (below or above the median value calculated in our tumor series; see also Supplementary Table 2). The size of the! T17 deletion was significantly different between these two groups of tumors (P = 1.1 × 10–13; Student's t test). () Two different HSP110 antibodies (Abs) were used in western blots—one recognizing the C-terminal part of the protein and detecting the α and β HSP110 isoforms (upper blot) and the other targeting the N-terminal part and detecting HSP110ΔE9 (this Ab detects HSP110βE9 only weakly). Error bars correspond to the s.d. of measured densitometric values. *P < 0.05. a.u., arbitrary units. * Figure 3: HSP110ΔE9 is a dominant-negative mutant that binds to HSP110 and blocks its chaperone function. () Protein aggregation was evaluated in heated extracts from MEF HSF1− cells transfected with HSP70, HSP110wt, HSP110ΔE9, and HSP110wt and HSP110ΔE9 combined, as reported26. Each bar is the mean value of four different experiments. *P < 0.05. () Immunoprecipitation (IP) with HA antibody (HSPs) was followed by immunoblotting with GFP antibody (HSP110 proteins) in lysates from HCT116 cells co-transfected with a GFP-tagged HSP110wt or HSP110ΔE9 (left) or an empty GFP vector (right) together with the indicated hemagglutinin (HA)-tagged HSP. () Immunoprecipitation of HSP110wt, using an antibody that recognizes the C terminus of HSP110, was followed by immunoblotting with GFP antibody in lysates from HCT116 cells transfected with GFP-tagged HSP110wt and/or HSP110ΔE9. Inputs, protein level in total cell lysates. () Top, fluorescence microscopic analysis in HCT116 cells of GFP-tagged HSP110wt or HSP110ΔE9 (green) and nuclei (blue). Scale bars, 0.5 μm. Bottom, cell fractionat! ion studies in HCT116 cells transfected with GFP-tagged HSP110wt or HSP110ΔE9. HSP90 serves as cytosolic marker. () We did cell fractionation studies on HCT116 cells transfected with GFP-tagged HSP110wt, HSP110ΔE9 or both (HSP11wt + HSP110ΔE9). Exp1 and Exp2 constitute two independent transfection experiments. HSP90 serves as a cytosolic marker and histone H3 as a nuclear marker. * Figure 4: HSP110ΔE9 has an antitumor effect in xenografts, blocks the HSP110 antiapoptotic effect and sensitizes cancer cells to die. () Antitumor effect of HSP110ΔE9 in xenografts. Mean tumor volumes were measured in nude mice (± s.d., six mice per group. *P < 0.05) injected with HCT116 cells transfected either with a GFP vector (open circles) or GFP-tagged HSP110ΔE9 (filled circles). Inset, expression of the transfected proteins. HSP70 serves as a loading control. () Apoptosis was measured by immunodetection of caspase 8 (CASP8) and PARP cleavage (left), or by fluorescence-activated cell sorting (FACS) analysis of caspase 3 activity (right), in HCT116 cells transfected with GFP-tagged HSP110wt, HSP110ΔE9 or an empty vector (GFP) and treated with recombinant TRAIL ligand. () FACS analysis of apoptosis in GFP-positive (GFPpos, transfected with a GFP-empty vector or HSP110ΔE9) and GFP-negative (GFPneg, non transfected) HCT116 cells treated with TRAIL. () The percentage of apoptosis (chromatin condensation) induced by TRAIL was determined in HCT116 cells transfected with GFP-tagged HSP110wt and increase! d doses of HSP110ΔE9 or an empty vector (GFP). () Immunoblot of caspase 8 (CASP8) and PARP cleavage in LoVo cells transfected as described in and treated, when indicated, with TRAIL ligand. () HCT116 (MSI) or SW480 (MSS) cells transfected with HSP110wt (1.5 μg) were co-transfected with two different doses (c1, 0.5 μg and c2, 1 μg) of either GFP-empty vector or HSP110ΔE9. After treatment with oxaliplatin (Oxa, 40 μM, 48 h), apoptosis was assessed by FACS analysis as in . *P < 0.05. a.u., arbitrary units. * Figure 5: Clinical impact of HSP110ΔE9 expression in people with MSI CRC. Kaplan-Meier univariate analyses of disease-free survival (DFS) in persons with stage 2 or stage 3 MSI CRC are shown according to their HSP110ΔE9 expression. For statistical analyses, only the first 5 years are shown. Subjects from both series were combined using series-specific cutoff points to define HSP110ΔE9 expression classes (75% and 50% for first and second series, respectively). Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Carmen Garrido & * Alex Duval Affiliations * Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche Saint-Antoine, Equipe 'Instabilité des Microsatellites et Cancers', Paris, France. * Coralie Dorard, * Ada Collura, * Magali Svrcek, * Anaïs Lagrange, * Kristell Wanherdrick, * Olivier Buhard, * Jean-François Fléjou & * Alex Duval * Université Pierre et Marie Curie Paris, Paris, France. * Coralie Dorard, * Ada Collura, * Magali Svrcek, * Anaïs Lagrange, * Kristell Wanherdrick, * Olivier Buhard, * Jérémie H Lefèvre, * Yann Parc, * Emmanuel Tiret, * Jean-François Fléjou & * Alex Duval * INSERM, Dijon, France. * Aurélie de Thonel, * Gaetan Jego, * Anne Laure Joly, * Jessica Gobbo & * Carmen Garrido * University of Burgundy, Dijon, France. * Aurélie de Thonel, * Gaetan Jego, * Anne Laure Joly, * Jessica Gobbo & * Carmen Garrido * Programme 'Cartes d'Identité des Tumeurs', Ligue Nationale Contre le Cancer, Paris, France. * Laetitia Marisa * Assistance Publique–Hôpitaux de Paris (AP-HP), Hôpital Saint-Antoine, Service d'Anatomie et Cytologie Pathologiques, Paris, France. * Magali Svrcek & * Jean-François Fléjou * INSERM, Institute Gustave Roussy, Villejuif, France. * Virginie Penard-Lacronique * Centre d'Etude du Polymorphisme Humain, Fondation Jean Dausset, Institut de Génétique Moléculaire, Paris, France. * Habib Zouali & * Emmanuel Tubacher * INSERM, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France. * Sylvain Kirzin & * Janick Selves * Laboratoire d'Oncopharmacologie, Centre Antoine Lacassagne, Nice, France. * Gérard Milano & * Marie-Christine Etienne-Grimaldi * Service d'Oncologie Médicale, Hôpital Saint-Antoine, AP-HP, Paris, France. * Leila Bengrine-Lefèvre, * Christophe Louvet & * Christophe Tournigand * AP-HP, Service de Chirurgie Générale et Digestive, Hôpital Saint-Antoine, Paris, France. * Jérémie H Lefèvre, * Yann Parc & * Emmanuel Tiret * AP-HP, Hôpital Saint-Antoine, Tumorothèque Cancer Est, Paris, France. * Jean-François Fléjou * INSERM, Développement et Physiopathologie de l'Intestin et du Pancréas, Strasbourg, France. * Marie-Pierre Gaub * Centre Hospitalier Universitaire Dijon, Dijon, France. * Carmen Garrido Contributions C.D. carried out analyses of aberrant splicing events due to MSI in CRC and genetic study of HSP110 in CRC cells and primary tumors. A.d.T. carried out analyses of wild-type and mutated HSP110 chaperone functions in CRC cells. A.C., M.S., A.L., K.W. and O.B. assisted with the mutational screening of primary CRC. J.G. (with G.J. and A.L.J.) carried out mouse work. L.M. carried out the clinical study and survival analyses. H.Z. and E. Tubacher assisted with the in silico search of candidate genes containing intronic microsatellite sequences. V.P.-L., S.K., J.S., G.M., M.-C.E.-G., L.B.-L., C.L., C.T., J.H.L., Y.P., E. Tiret, J.-F.F. and M.-P.G. provided CRC samples and clinical data. A.D. and C.G. conceived the project, coordinated and directed the study, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Carmen Garrido or * Alex Duval Author Details * Coralie Dorard Search for this author in: * NPG journals * PubMed * Google Scholar * Aurélie de Thonel Search for this author in: * NPG journals * PubMed * Google Scholar * Ada Collura Search for this author in: * NPG journals * PubMed * Google Scholar * Laetitia Marisa Search for this author in: * NPG journals * PubMed * Google Scholar * Magali Svrcek Search for this author in: * NPG journals * PubMed * Google Scholar * Anaïs Lagrange Search for this author in: * NPG journals * PubMed * Google Scholar * Gaetan Jego Search for this author in: * NPG journals * PubMed * Google Scholar * Kristell Wanherdrick Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Laure Joly Search for this author in: * NPG journals * PubMed * Google Scholar * Olivier Buhard Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica Gobbo Search for this author in: * NPG journals * PubMed * Google Scholar * Virginie Penard-Lacronique Search for this author in: * NPG journals * PubMed * Google Scholar * Habib Zouali Search for this author in: * NPG journals * PubMed * Google Scholar * Emmanuel Tubacher Search for this author in: * NPG journals * PubMed * Google Scholar * Sylvain Kirzin Search for this author in: * NPG journals * PubMed * Google Scholar * Janick Selves Search for this author in: * NPG journals * PubMed * Google Scholar * Gérard Milano Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Christine Etienne-Grimaldi Search for this author in: * NPG journals * PubMed * Google Scholar * Leila Bengrine-Lefèvre Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Louvet Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Tournigand Search for this author in: * NPG journals * PubMed * Google Scholar * Jérémie H Lefèvre Search for this author in: * NPG journals * PubMed * Google Scholar * Yann Parc Search for this author in: * NPG journals * PubMed * Google Scholar * Emmanuel Tiret Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-François Fléjou Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Pierre Gaub Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen Garrido Contact Carmen Garrido Search for this author in: * NPG journals * PubMed * Google Scholar * Alex Duval Contact Alex Duval Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (3M) Supplementary Figures 1–5 and Supplementary Tables 1–4 Additional data
• EGFR signaling in podocytes at the root of glomerular disease
  - Nat Med 17(10):1188-1189 (2011)
  Article preview View full access options Nature Medicine | Article Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis * Guillaume Bollée1, 2, 21 * Martin Flamant3, 4, 21 * Sandra Schordan5 * Cécile Fligny1, 2 * Elisabeth Rumpel5 * Marine Milon1, 2 * Eric Schordan5 * Nathalie Sabaa3, 4 * Sophie Vandermeersch6, 7 * Ariane Galaup8, 9 * Anita Rodenas10 * Ibrahim Casal11, 12 * Susan W Sunnarborg13 * David J Salant14 * Jeffrey B Kopp15 * David W Threadgill16 * Susan E Quaggin17 * Jean-Claude Dussaule6, 7, 18 * Stéphane Germain8, 9 * Laurent Mesnard6, 7 * Karlhans Endlich5 * Claude Boucheix11, 12 * Xavier Belenfant19 * Patrice Callard7, 10 * Nicole Endlich5 * Pierre-Louis Tharaux1, 2, 20 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1242–1250Year published:(2011)DOI:doi:10.1038/nm.2491Received22 February 2011Accepted11 August 2011Published online25 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF–deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started! 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF–EGFR pathway could also be beneficial in treatment of human RPGN. Figures at a glance * Figure 1: Induction of renal HB-EGF synthesis leads to glomerular activation of EGFR during RPGN. () Representative images of in situ hybridization study in nephrotoxic serum (NTS)-injected WT animals, showing proHB-EGF expression in glomeruli (G), especially in parietal glomerular epithelial cells (Pec), in podocytes (day 4) and in crescents (Cr; day 8). White arrow, abundant proHB-EGF mRNA expression in areas where tuft-capsular podocyte bridges were present. Scale bars (orange), 50 μm. () Quantification by real-time RT-PCR of proHB-EGF mRNA in freshly isolated podocytes on day 6 after NTS injection (NTS) and in podocytes from noninjected control mice (CT; n = 3 per group). *P < 0.05 versus controls. Values are mean ± s.e.m. (n = 3 mice per group). () Western blot analysis of pEGFR and total EGFR in the renal cortex from nonchallenged controls (CT), WT mice infused with NTS ((+/+)NTS), HB-EGF–deficient mice infused with NTS ((−/−)NTS), and WT mice given intraperitoneal injections of EGFR tyrosine kinase inhibitor AG1478 ((+/+)NTS+AG1478). Values are mean ± s.e! .m. (n = 6–8 per group). *P < 0.05 versus controls at baseline; **P < 0.01 versus controls at baseline; ##P < 0.01 versus mice treated with vehicle only. () Immunofluorescence staining for pEGFR in renal cortex from CT, (+/+)NTS, (−/−)NTS and (+/+)NTS+AG1478 mice on day 8 after NTS administration. Scale bars (orange), 25 μm. * Figure 2: HB-EGF induces a migratory phenotype in podocytes in vitro. () Podocyte outgrowth over 6 d from decapsulated glomeruli of WT (+/+) or Hbegf−/− (−/−) mice (arrow). Cultures were incubated with either AG1478 (AG) or HB-EGF or vehicle. Cells are stained for WT-1 expression. () Outgrowth area from glomeruli of mice carrying functional Hbegf alleles (light blue bar) was suppressed by 500 nM of AG (dark blue bar). Sparse outgrowth from glomeruli of Hbegf−/− mice (light gray bar) was rescued by addition of 30 nM HB-EGF (black bar). () Schematic of podocyte outgrowth from isolated glomeruli, used as migration-proliferation assay to assess crescent formation in vitro. Podocytes are in a stationary state (blue) on the surface of capillary loops (gray circle) when glomeruli are plated. Subsequently, podocytes assume a migratory phenotype (orange) characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. () Representative image of F-actin reorganizatio! n and formation of RiLiSs induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, with or without AG (500 nM). Induction of apical protrusions by HB-EGF is abrogated with AG (500 nM). () Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added without (Ctl) or with inhibitors AG (500 nM), Wortmannin (Wo, 100 nM), LY294002 (LY, 30 μM), PD98059 (PD, 25 μM) and SB203580 (SB, 25 μM). () BrdU incorporation in differentiated podocytes over 48 h. () Distance of migration of differentiated podocytes within 8 h in wound assay. Data are mean ± s.e.m. (n = 3 or 4 experiments). *P < 0.05 versus untreated WT glomeruli in and versus HB-EGF alone in –. Scale bar, 300 μm in and 30 μm in . * Figure 3: Deletion of Hbegf gene prevents fatal renal destruction. () Survival curve for challenged WT and Hbegf−/− mice. () Masson's trichrome staining of kidneys and proportion of crescentic glomeruli in control mice and in NTS-injected WT and Hbegf−/− mice (day 8 after NTS; Cr, crescents; G, glomeruli; Tc, tubules with proteinaceous casts). Scale bars, 50 μm. (–) Ascites score as index of albumin plasma loss and water and sodium retention (), albuminuria () and blood urea nitrogen concentrations () in NTS-challenged WT and Hbegf−/− animals on day 8 after NTS, and in unchallenged controls (CT). Values are mean ± s.e.m. (n = 9–12 per group). *P < 0.05 versus controls at baseline; **P < 0.01 versus baseline; ***P < 0.001 versus baseline. #P < 0.05 versus NTS-treated+/+; ##P < 0.01 versus NTS-treated+/+. * Figure 4: Selective deletion of Egfr from podocytes protects from RPGN. (–) Albuminuria () blood urea nitrogen concentration () and proportion of crescentic glomeruli () in Pod-Tet on-Cre Egfrwt/wt and Pod-Tet on-Cre EgfrloxP/loxP mice at 8 d after NTS-induced RPGN (*P < 0.05 for all comparisons). () Survival curve of challenged Pod-Tet on-Cre EgfrloxP/loxP and littermate control mice in a severe model of RPGN. *P < 0.01. () Ultrastructural analysis of podocytes by transmission electron microscopy (TEM) in NTS-treated Pod-Tet on-Cre Egfrwt/wt and Pod-Tet on-Cre EgfrloxP/loxP mice. Scale bars, top, 2 μm; bottom, 1 μm. * Figure 5: Delayed EGFR tyrosine kinase inhibition stops development of crescentic RPGN. () Quantification by western blot analysis of pEGFR and total EGFR in renal cortex from nonchallenged controls (CT), NTS-injected mice treated with vehicle alone and NTS-injected mice treated with erlotinib either started 12 h before administration of NTS (days 0–14) or in a curative protocol, started 4 d later (days 4–14). Mice were killed after 14 d of RPGN. (,) Blood urea nitrogen concentration () and proportion of crescentic glomeruli in CT () in groups of mice as in . Data are mean ± s.e.m., n = 9 mice per group. **P < 0.01 versus controls at baseline (CT); ***P < 0.001 versus CT; ##P < 0.01 versus vehicle; ###P < 0.001 versus vehicle. () Ultrastructural analysis of podocytes by TEM in erlotinib-treated and vehicle-treated mice at 5 d after injection of NTS. Scale bars, 2 μm. () Masson's trichrome staining of renal cortex from a mouse treated with erlotinib (days 4–14; left) and a vehicle-treated mouse (right) on day 14. Ne, necrotic glomerular lesions; Cr, cell! ular crescents; Tc, tubular proteinaceous casts; Infilt, diffuse CD3+ cell infiltrates in vehicle-treated mice. Scale bars, 100 μm. * Figure 6: HB-EGF expression is induced in human crescentic glomerulonephritis. Representative images of immunostaining for HB-EGF using monoclonal sc-74526 antibody in sections of kidney biopsies from eight random subjects diagnosed with noncrescentic glomerulopathies (top), including diabetic nephropathy, amyloidosis, minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN) and membranous nephropathy (MN). Bottom, immunostaining for HB-EGF in renal biopsies from eight random subjects with RPGN of various etiologies, including lupus nephritis, microscopic polyangiitis (MP), endocarditis (End), Goodpasture disease (Gp) and Wegener disease (Wg). Scale bars, 50 μm. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Guillaume Bollée & * Martin Flamant Affiliations * Unité Mixte de Recherche (UMR) 970, Paris Cardiovascular Research Centre, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. * Guillaume Bollée, * Cécile Fligny, * Marine Milon & * Pierre-Louis Tharaux * Université Paris Descartes, Sorbonne Paris Cité, Paris, France. * Guillaume Bollée, * Cécile Fligny, * Marine Milon & * Pierre-Louis Tharaux * Université Paris Diderot, Sorbonne Paris Cité, Paris, France. * Martin Flamant & * Nathalie Sabaa * Service de Physiologie–Explorations Fonctionnelles, Hôpital Bichat, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France. * Martin Flamant & * Nathalie Sabaa * Institut für Anatomie und Zellbiologie, Universitätsmedizin Greifswald, Greifswald, Germany. * Sandra Schordan, * Elisabeth Rumpel, * Eric Schordan, * Karlhans Endlich & * Nicole Endlich * INSERM UMR702, Hôpital Tenon, Paris, France. * Sophie Vandermeersch, * Jean-Claude Dussaule & * Laurent Mesnard * Université Pierre et Marie Curie, Sorbonne Universités, Paris, France. * Sophie Vandermeersch, * Jean-Claude Dussaule, * Laurent Mesnard & * Patrice Callard * Centre for Interdisciplinary Research in Biology, Centre National de la Recherche Scientifique UMR 7241, INSERM U1050, Paris, France. * Ariane Galaup & * Stéphane Germain * Chaire de Médecine Expérimentale, Collège de France, Paris, France. * Ariane Galaup & * Stéphane Germain * Service d'Anatomie et de Cytologie Pathologiques, AP-HP, Hôpital Tenon, Paris, France. * Anita Rodenas & * Patrice Callard * INSERM UMR 1004, Institut André Lwoff, Hôpital Paul Brousse, Villejuif, France. * Ibrahim Casal & * Claude Boucheix * Université Paris-Sud, Villejuif, France. * Ibrahim Casal & * Claude Boucheix * The Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. * Susan W Sunnarborg * Renal Section, Boston University Medical Center, Boston, Massachusetts, USA. * David J Salant * Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA. * Jeffrey B Kopp * Department of Genetics, North Carolina State University, Raleigh, North Carolina, USA. * David W Threadgill * St. Michael's Hospital, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. * Susan E Quaggin * Service de Physiologie-Exploration Fonctionnelles, AP-HP, Hôpital Saint-Antoine, Paris, France. * Jean-Claude Dussaule * Service de Néphrologie et Dialyse, Centre Hospitalier Intercommunal André Grégoire, Montreuil, France. * Xavier Belenfant * Service de Néphrologie, Hôpital Européen Georges Pompidou, Assistance Publique–Hôpitaux de Paris, Paris, France. * Pierre-Louis Tharaux Contributions M.F., G.B. and P.-L.T. conceived the project and experiments. P.-L.T. and N.E. supervised the project. S.S., C.F., M.M., S.V. and E.S. developed methods to culture and analyze primary podocytes and conceived experiments for gene expression analysis. E.R. and M.M. carried out electron microscopy (EM) studies. S.W.S., S.E.Q., J.B.K., D.W.T., I.C. and C.B. helped generate mice with targeted deficiency of HBEGF, TGF-α, epiregulin and Egfr. A.G. and S.G. carried out in situ hybridization studies. D.J.S. and L.M. provided nephrotoxic serum and discussed data with P.-L.T. K.E., C.B. and J.-C.D. also discussed experiments with P.-L.T. and N.E. P.-L.T., G.B., M.M., C.F. and N.S. carried out all in vivo studies. M.F., A.R. and P.C. analyzed human kidney biopsies collected by X.B. G.B. and M.F. contributed equally to the study. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Pierre-Louis Tharaux Author Details * Guillaume Bollée Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Flamant Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Schordan Search for this author in: * NPG journals * PubMed * Google Scholar * Cécile Fligny Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Rumpel Search for this author in: * NPG journals * PubMed * Google Scholar * Marine Milon Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Schordan Search for this author in: * NPG journals * PubMed * Google Scholar * Nathalie Sabaa Search for this author in: * NPG journals * PubMed * Google Scholar * Sophie Vandermeersch Search for this author in: * NPG journals * PubMed * Google Scholar * Ariane Galaup Search for this author in: * NPG journals * PubMed * Google Scholar * Anita Rodenas Search for this author in: * NPG journals * PubMed * Google Scholar * Ibrahim Casal Search for this author in: * NPG journals * PubMed * Google Scholar * Susan W Sunnarborg Search for this author in: * NPG journals * PubMed * Google Scholar * David J Salant Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey B Kopp Search for this author in: * NPG journals * PubMed * Google Scholar * David W Threadgill Search for this author in: * NPG journals * PubMed * Google Scholar * Susan E Quaggin Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Claude Dussaule Search for this author in: * NPG journals * PubMed * Google Scholar * Stéphane Germain Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Mesnard Search for this author in: * NPG journals * PubMed * Google Scholar * Karlhans Endlich Search for this author in: * NPG journals * PubMed * Google Scholar * Claude Boucheix Search for this author in: * NPG journals * PubMed * Google Scholar * Xavier Belenfant Search for this author in: * NPG journals * PubMed * Google Scholar * Patrice Callard Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Endlich Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre-Louis Tharaux Contact Pierre-Louis Tharaux Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–8 and Supplementary Methods Additional data
• Glioma-related seizures: glutamate is the key
  - Nat Med 17(10):1190-1191 (2011)
  Article preview View full access options Nature Medicine | Article Glutamate release by primary brain tumors induces epileptic activity * Susan C Buckingham1 * Susan L Campbell1 * Brian R Haas1 * Vedrana Montana1 * Stefanie Robel1 * Toyin Ogunrinu1 * Harald Sontheimer1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1269–1274Year published:(2011)DOI:doi:10.1038/nm.2453Received05 May 2011Accepted27 July 2011Published online11 September 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into severe combined immunodeficient mice. Within 14–18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system xc− cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration–approved drug that blocks system xc−. We found that acute administration of SAS at concentrations equivalent to those used to treat Crohn's disease in humans red! uced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans. Figures at a glance * Figure 1: Tumor-bearing mice show abnormal spontaneous EEG events indicative of epileptic activity. () Representative EEG recordings from three glioma-implanted mice, juxtaposing abnormal events and baselines for each. () A power spectrum from one representative event (inset) and the corresponding baseline from the same mouse. () Frequency of epileptic activity in 13 tumor-bearing mice quantified over 10 consecutive d; hourly event frequency is plotted as a function of time. () A U251-GFP tumor identified in cortical brain by EGFP fluorescence before conducting glutamate release assays. Scale bar, 1 mm. () Extracellular glutamate, released in the presence of 100 μM cystine (Cys), comparing acute cortical brain slices from sham-operated and U251-GFP-implanted mice in the presence and absence of 250 μM SAS. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01. * Figure 2: Acute cortical slices from tumor-bearing mice show spontaneous epileptiform activity. (,) Cresyl violet–stained brain slices from a sham () and a glioma-implanted mouse (). Recording (Rec) and stimulating (Stim) electrodes were placed in peritumoral regions and in corresponding regions of sham slices as shown. Scale bars, 350 μm. () Higher-magnification image of the tumor shown in (, box). Scale bar, 150 μm. () Extracellular field recordings conducted in the presence of Mg2+ comparing spontaneous activity in a slice from a representative U251-GFP–bearing mouse to a slice from a sham animal. * Figure 3: Acute cortical slices from tumor-bearing mice are hyperexcitable. (,) Whole-cell recordings from representative neurons in a cortical slice from a sham-operated () versus tumor-bearing mouse () before (ACSF) and after removal of Mg2+ (Mg2+-free). Individual events are shown on an expanded time scale below each recording. () Mean latency to development of epileptiform activity comparing neurons from sham mice, to peritumoral neurons in animals implanted with U251-GFP, GBM12 and GBM22 tumors (*P < 0.05). () Mean event duration recorded in sham, U251, GBM12 and GBM22-containing brain slices. Error bars represent means ± s.e.m. * Figure 4: Cortical slices from glioma-bearing mice show increased cortical network activity and hyperexcitable layer 2 and 3 peritumoral pyramidal cells. () Representative examples of optical recordings comparing a slice from a sham and a slice from a U251-GFP–bearing mouse incubated in the voltage dye RH414 and then field stimulated with 80 μA. Each image is a pseudocolored representation of activity measured using a Neuroplex 464-diode array. Adjacent frames were recorded 1.8 ms apart. () The spread of voltage responses were measured by the number of activated diodes within the array. (,) Summary of RMP () and input resistance () recorded using whole-cell patch clamp in peritumoral neurons from U251-GFP, GBM12 and GBM22-implanted mice. () Examples of voltage responses to increasing amplitude current injections, from −100 pA to +80 pA (in 20-pA steps) in whole-cell current clamped pyramidal peritumoral neurons in glioma-bearing and in sham slices (pulse duration, 500 ms). () The average action potential number obtained in response to 20, 40, 60 and 80 pA depolarizing current pulses was plotted as a function of applied c! urrent yielding the input-output curves shown. Error bars show means ± s.e.m. *P < 0.05, ***P < 0.01. * Figure 5: SAS application reduces epileptiform activity in cortical slices from glioma-bearing mice. (,) Recordings from peritumoral neurons before (ACSF) and after removal of Mg2+ (Mg2+-free) followed by application of SAS and APV in mice bearing GBM12 () or GBM22 () tumors. () Mean response duration for peritumoral neurons recorded in U251, GBM12 and GBM22 containing brain slices comparing recordings in Mg2+-free medium to those in Mg2+-free plus SAS and Mg2+- free plus SAS and APV. Error bars show means ± s.e.m. () A cresyl violet–stained slice containing GBM22 shows the proximity of biocytin-filled recorded neuron (right inset, middle box) to the darker-stained tumor. Scale bars, 150 μm (left and middle); 20 μm (right inset). * Figure 6: Sulfasalazine reduces frequency of epileptic activity in tumor-bearing mice. () The average number of epileptic events for eight SAS-treated and six PBS-treated tumor bearing mice is shown for the 4-h period before and the 4-h period after treatment. () The average hourly event frequency is plotted for eight SAS-treated mice compared with six PBS-treated mice before and after treatment. Injection time is indicated by '0'. () The number of events that occurred for each hour during the 8-h SAS treatment block (4 h before and 4 h after) is shown for one mouse; hash marks separate 3 consecutive d and arrowheads indicate the time of SAS injection. Error bars show means ± s.e.m. **P < 0.05; ***P < 0.01. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA. * Susan C Buckingham, * Susan L Campbell, * Brian R Haas, * Vedrana Montana, * Stefanie Robel, * Toyin Ogunrinu & * Harald Sontheimer Contributions S.C.B. and S.L.C. acquired the majority of the data presented. B.R.H. was instrumental in mouse surgeries and statistical analyses of data. V.M. (supported by an American Brain Tumor Association Basic Research Fellowship) carried out glutamate release assays. S.R. assisted in electrophysiological recordings. T.O. did western blotting and glutamate uptake assays. H.S. designed experiments, supervised all research and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harald Sontheimer Author Details * Susan C Buckingham Search for this author in: * NPG journals * PubMed * Google Scholar * Susan L Campbell Search for this author in: * NPG journals * PubMed * Google Scholar * Brian R Haas Search for this author in: * NPG journals * PubMed * Google Scholar * Vedrana Montana Search for this author in: * NPG journals * PubMed * Google Scholar * Stefanie Robel Search for this author in: * NPG journals * PubMed * Google Scholar * Toyin Ogunrinu Search for this author in: * NPG journals * PubMed * Google Scholar * Harald Sontheimer Contact Harald Sontheimer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 and Supplementary Methods Additional data
• Zinc fingers hit off target
  - Nat Med 17(10):1192-1193 (2011)
  Article preview View full access options Nature Medicine | Community Corner Zinc fingers hit off target Journal name:Nature MedicineVolume: 17,Pages:1192–1193Year published:(2011)DOI:doi:10.1038/nm1011-1192Published online11 October 2011 Linzhao Cheng These two studies1, 2 are important and timely, providing a sobering assessment of the reality of ZFN-mediated gene targeting. Although it is disappointing (but not surprising) to realize the inherent imperfections of this method, there are several ways we could avoid off-target effects and optimize targeting in human cells. For example, screening in vitro1 or in a model cell line2 is better than in silico predictions of all the possible ZFN cleavage sites on the basis of biophysical and biochemical principles, although not all the potential sites will be cut in a given cell type, especially if the ZFN concentration is optimized. Therefore, the most relevant cell types should be used for gene targeting, and the targeted cells should be analyzed before conducting important biological experiments or putting the targeted cells into patients. It is unlikely that we will be able to completely avoid off-target events in the near future, even if the specificity of ZFNs or related technologies such as homing endonucleases or transcription activator–like effector nucleases (TALENs) is further improved. However, for most applications, we will be able to select correctly targeted cells without needing to ensure correct targeting in every cell. In fact, current gene targeting technologies using homologous recombination rely on the ability to select and expand rare cells that have been correctly gene targeted, because the homologous recombination rate in nontransformed human cells is still low (1 × 10–4 or less), even when aided by ZFNs. Therefore, it would be desirable to use cell types that can be substantially expanded in culture, such as stem cells. Human pluripotent stem cells make it feasible to select a correctly targeted clone3, 4. With the improved efficiency of gene targeting achieved by ZFNs and other tool! s, and the improved capacity for whole-genome DNA sequencing of several selected clones, we should be confident that it will be possible to derive precisely edited human cells for basic research and gene therapy. . Bruce Blazar ZFNs are reagents that allow precise gene targeting and offer an advantage over gene therapy vectors that are prone to semirandom genomic integration. Given that the majority of the genome is transcribed5 and thus may produce either protein-coding or regulatory transcripts, it is of the utmost importance that only the intended genomic sequence is targeted. "Even these precise tools can cause unintended genomic modification." Two recent manuscripts analyzed the off-target effects of ZFNs and show that even these precise tools can cause unintended genomic modification1, 2. The papers overlap in their analysis of a ZFN targeting the chemokine receptor gene CCR5 that has already entered clinical trials as an anti-HIV therapy. Using an in vitro DNA-binding assay, Pattanayak et al.1 identified 37 sites in the human genome cleaved by the CCR5 ZFN. Analysis in intact human cells, however, showed that only ten of the sites were actually modified in K562 cells. Using the same cell type and ZFN but a different approach, whereby an integrase-deficient lentiviral vector cassette can be 'trapped' in a ZFN-induced double-stranded DNA break, Gabriel et al.2 mapped the genomic sites at which the ZFN was active. They showed that 93.8% of the insertion events were at the CCR5 locus. Interestingly, in only one case did both studies identify the same off-target site, CCR2. Given that in vitro DNA binding does not fu! lly predict in vivo binding and given the high homology between the two genes, the finding that the CCR2 locus was affected is not surprising. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Competing financial interests K.H. is a co-inventor on a pending patent using zinc finger nucleases to do genome editing in hemophilia. Additional data
• Moving ahead an HIV vaccine: Use both arms to beat HIV
  - Nat Med 17(10):1194-1195 (2011)
  Nature Medicine | Between Bedside and Bench Moving ahead an HIV vaccine: Use both arms to beat HIV * Bruce D Walker1 * Rafi Ahmed2 * Stanley Plotkin3 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1194–1195Year published:(2011)DOI:doi:10.1038/nm.2529Published online11 October 2011 Despite remarkable advances in managing disease progression in people infected with HIV, an effective vaccine to prevent infectivity and stop the HIV epidemic remains an unmet clinical need. The genetic variability of the virus and the poor natural immune response—humoral and cellular—generated against HIV are hurdles that pose challenges to vaccine development. In 'Bench to Bedside', Bruce Walker, Rafi Ahmed and Stanley Plotkin examine a study in rhesus macaques where a vector-based viral vaccine that elicits a persistent and rapid T effector cell response to SIV antigens results in control of the infection. Although only 50% of the rhesus macaques controlled the infection, this in vivo finding stresses how outdoing the natural immune cellular response can prove effective to clear systemic viruses. But a humoral response will still remain the 'holy grail' to avoid HIV infection and transmission. In 'Bedside to Bench' Tom Hope peruses recent vaccine trials to propose how! to best achieve an effective antibody response against HIV by discussing the perks and perils of non-neutralizing versus broadly neutralizing antibodies. 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 * Bruce D. Walker is at the Ragon Institute of Massachusetts General Hospital, Massachusetts Instiute of Technology and Harvard, Massachusetts General Hospital, Boston, Massachusetts, USA and Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Rafi Ahmed is at Emory Vaccine Center, Emory University School of Medicine, Emory University, Atlanta, Georgia, USA. * Stanley Plotkin is at the University of Pennsylvania and Vaxconsult, Doylestown, Pennsylvania, USA. Competing financial interests S.P. consults for all major manufacturers of vaccines, including Sanofi Pasteur, sponsor of the RV144 trial. Corresponding author Correspondence to: * Bruce D Walker Author Details * Bruce D Walker Contact Bruce D Walker Search for this author in: * NPG journals * PubMed * Google Scholar * Rafi Ahmed Search for this author in: * NPG journals * PubMed * Google Scholar * Stanley Plotkin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Moving ahead an HIV vaccine: To neutralize or not, a key HIV vaccine question
  - Nat Med 17(10):1195-1197 (2011)
  Article preview View full access options Nature Medicine | Between Bedside and Bench Moving ahead an HIV vaccine: To neutralize or not, a key HIV vaccine question * Thomas J Hope1Journal name:Nature MedicineVolume: 17,Pages:1195–1197Year published:(2011)DOI:doi:10.1038/nm.2528Published online11 October 2011 Bedside to Bench The goal of developing a vaccine to decrease the sexual transmission of HIV remains one of the highest priorities of public health worldwide. The prevention of new HIV infections would clearly have a tremendous impact on the ability of physicians to deliver quality healthcare. Recently, there have been a number of developments in our understanding of how the immune system responds to HIV and how such responses might be harnessed to develop an effective vaccine1, 2. But the interpretation of these observations is subject to debate—what is viewed as encouraging by some will lead others to the opposite viewpoint. An active debate of the importance of these results will be invaluable in making sure that future investments in vaccine development advance the field. In the past few years, the results of two vaccine trials have transformed much the HIV vaccine field by generating positive and negative results3, 4. First came the disappointment of the STEP trial vaccine, which used an Ad5 vector to deliver a T cell–based vaccine3, which generated robust cytotoxic T cell responses but failed to show any protection. This was followed by the potential protection from HIV acquisition observed in the RV144 Thai vaccine trial, a result that shocked many in the field. The surprise was due to the fact that it used a combination of vaccines that failed when tested individually4. Unlike the STEP trial vaccine, this vaccine generated robust responses of antibodies that bind the HIV envelope protein but did not generate strong cytotoxic T cell responses. However, the antibodies generated did not show the crucial ability to neutralize varied strains of HIV. A vaccine that generates broadly neutralizing antibodies (bnAbs) has been the 'holy grail' of! HIV vaccine research since the virus was discovered more than 25 years ago. The promise of bnAbs was clear. Monoclonal antibodies with broadly neutralizing activity could protect rhesus macaques from vaginal challenge after intravenous injection5. Although many years of research yielded only a small number of monoclonal bnAbs, recent advances in technologies have led to an explosion in the identification of new ones. Some of these recently identified bnAbs have the ability to disrupt the infectivity of more that 90% of the HIV variants known worldwide in standard neutralization assays6. bnAbs targeting one of several specific sites on the HIV envelope protein can be isolated from the 1% of HIV-infected individuals, the so-called elite neutralizers, who show evidence of broadly neutralizing activity in their blood7. This proliferation in the number of example bnAbs has facilitated comparative studies revealing the characteristics that give broadly neutralizing function, which generally fall into several classes that interact with different regions of! the envelope protein8. Comparison of the binding specificity of monoclonal bnAbs revealed that they focus their binding activity on a small number of regions of the viral envelope and often are polyreactive (sticky)—a beneficial property, as it can increase binding avidity when the low number of envelope spikes in HIV virions prevents simultaneous binding of both arms of the antibody9. Sequencing of the genes encoding bnAbs also revealed another rather remarkable characteristic—extensive hypermutation of the variable regions of the antibodies that mediate binding specificity10. Hypermutation is a normal function of the antibody response that allows for maturation to achieve optimal binding. Most of the bnAbs contain 80 or more amino acid changes within the heavy and light chains. The extensive hypermutation can lead to the convergent evolution of related sequence10, structure and envelope-binding specificity from two different heavy chain genes. It is therefore clear that an effective vaccine ! would need only to present an epitope that will select antibodies with the defined bnAb specificities. Unfortunately, selecting binding specificities requiring such extensive hypermutation will clearly require multiple rounds of injections followed by selection for increased affinity in vaccinated individuals. This affinity maturation through selection requires continuous exposure to antigen over extended periods of time during the vaccination protocol. The study of the temporal development of bnAbs in infected individuals reveals that it takes, on average, 2.5 years to develop these protective antibodies2. The type of vaccination regimen that can recapitulate the continuous, daily systemic exposure to billions of virions that drives hypermutation in infected individuals is not clear. And only 10–30% of infected individuals will actually develop any of these bnAbs during infection2. If bnAbs do not arise within the first 3 years of infection, it does not seem that they can be developed subsequently2. This is not good news in the efforts to develop a vaccine that is focused on the goal of generating bnAbs. The identification and characterization of bnAbs has showed the field what humoral response a vaccine strategy must generate while revealing that stimulating such a response seems insurmountable at first glance. The vaccine field has been focused on neutralizing antibodies since the beginning, and rightly so, as this effort has the greatest promise and must therefore continue. However, there may be an alternative. The recent RV144 Thai vaccine trial generated robust binding antibodies that did not show broadly neutralizing activity in standard assays4. On the basis of the outcome of the RV144 Thai vaccine trial, these binding yet non-neutralizing antibodies can potentially inhibit HIV transmission at the barriers of the sexual mucosa by alternative mechanisms (Fig. 1). First, they can potentially induce particle crosslinking into large complexes that cannot penetrate the mucosal barriers. Second, they might also potentially trap viral particles within superficial epithelial barriers and the protective mucus of the female reproductive tract. Third, binding antibodies can mediate antigen-specific targeting and killing of infected cells through antibody-directed cell cytoxicity and rel! ated mechanisms11. Figure 1: Broadly binding antibodies against HIV infection may efficiently block HIV infection. Although bnAbs could potently block numerous HIV virus strains and prevent infection, their production can be tedious and highly variable among controllers. Despite these pitfalls, a broadly binding Ab vaccine may generate antibodies capable of providing efficacy through different means. * Full size image (132 KB) There may be other ways binding antibodies might prove to confer protection at mucosal barriers. Such binding antibody responses are readily generated by vaccination with antigens of the type used in the RV144 vaccine trial but have generally been dismissed because they have not protected vaccinated individuals in previous trials4, 12. But not all antibody responses are the same, and maybe a binding antibody response associated with the right functional activity could generate binding antibodies that provide protection. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Thomas J. Hope is in the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Thomas J Hope Author Details * Thomas J Hope Contact Thomas J Hope Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• The card players of Caravaggio, Cézanne and Mark Twain: tips for getting lucky in high-stakes research
  - Nat Med 17(10):1201-1205 (2011)
  Article preview View full access options Nature Medicine | Commentary 2011 Albert Lasker Medical Research Awards Focus issue: October 2011 Volume 17, No 10 The card players of Caravaggio, Cézanne and Mark Twain: tips for getting lucky in high-stakes research * Joseph L Goldstein1Journal name:Nature MedicineVolume: 17,Pages:1201–1205Year published:(2011)DOI:doi:10.1038/nm.2465Published online11 October 2011 To the scientist, the thrill of discovery is like the thrill of a royal flush to the poker player. Scientists who receive Lasker Awards and Nobel Prizes share many things in common with poker superstars, both of whom take risks and gamble for high stakes. Scientists pit their wits against Nature's puzzles, betting that their efforts will ferret out those rare nuggets of truth embedded in a vast mountain of artifacts. Poker players pit their wits against mathematical probabilities, wrestling with the fact that a deck of 52 cards can be shuffled into 52! sequences, which is 52 × 51 × 50 × 49 and so on down to 1. This comes to 8.1 × 1067 possible card permutations—a number 53 orders of magnitude greater than the 1014 synaptic connections in the human brain and 45 orders of magnitude greater than the 1023 stars in the universe. Given the mathematical odds of being dealt a good card hand and the tightness with which Nature guards her secrets, a key question is whether success, in poker or in science, depends predominantly on luck or on skill. Luck is difficult to define. Perhaps the best definition comes from the great film producer Samuel Goldwyn: "The harder I work, the luckier I get." The skill element in poker depends on acquiring expertise in statistics and probability and on mastering the complexities of betting and bluffing—knowing when to bet, fold or raise at any decision point and knowing how many chips to put on the table. The skill element in science also depends on being bold and knowing when to take risks—not to mention learning the art of asking the right questions and pursuing experimentation passionately and fearlessly. Philosophers of science have paid little attention to the relative roles of luck versus skill. But in card-playing circles, the luck-versus-skill question has been debated for hundreds of years. Some of the best insights come not from card-playing experts, but from artists such as Caravaggio and Cézanne and from writers such as Mark Twain. Caravaggio's Cardsharps In the late 1300s, when card playing first became a popular form of entertainment in Italy and France, card cheats and crooked gamblers dominated the game, minimizing the skill factor of the player. This state of affairs is wonderfully captured in The Cardsharps, a 1594 painting by the Italian artist Michelangelo Merisi da Caravaggio (Fig. 1a). Art historians consider The Cardsharps the most influential gambling-themed painting in the history of art. The composition is simple and elegant. An innocent-looking young man has been lured into a card game by a sinister, middle-aged man. Stealing a peek at his victim's cards, the older man signals with his fingers to an accomplice, who holds the 5 of hearts tucked in his belt behind his back. The object of the conspiracy—a stack of coins—sits at the edge of the table. Figure 1: Two gambling-themed paintings. () Caravaggio. The Cardsharps, c. 1594. Oil on canvas, 94.2 × 120.9 cm. Kimbell Art Museum, Fort Worth, Texas, USA. () Georges de La Tour. The Cheat with the Ace of Clubs, c. 1630–1634. Oil on canvas, 97.8 × 156.2 cm. Kimbell Art Museum, Fort Worth, Texas, USA. * Full size image (293 KB) * Figures index * Next figure Caravaggio's genius is the creation of a dramatic scene of concealment in which all three characters are hiding something. The tension and drama are heightened by details such as the split glove that allows the older man to easily feel the marked cards, the black hat of the innocent boy that hides the peering right eye of the older man, and the older man's left hand that seems to come out of nowhere to rest close to the younger cardsharp's dagger. The whole scene keeps the viewer on tenterhooks: any slight movement might reveal the trickery. The young boy may be skillful at his game, but we'll never know—he's the victim of bad luck. Figures at a glance * Figure 1: Two gambling-themed paintings. () Caravaggio. The Cardsharps, c. 1594. Oil on canvas, 94.2 × 120.9 cm. Kimbell Art Museum, Fort Worth, Texas, USA. () Georges de La Tour. The Cheat with the Ace of Clubs, c. 1630–1634. Oil on canvas, 97.8 × 156.2 cm. Kimbell Art Museum, Fort Worth, Texas, USA. * Figure 2: Paul Cézanne. The Card Players. Three of the five paintings in this series are shown: () c. 1890–1892. Oil on canvas, 65.4 × 81.9 cm. The Metropolitan Museum of Art, New York. () c. 1892–1896. Oil on canvas, 47 × 56 cm. Musée d'Orsay, Paris. () c. 1892–1896. Oil on canvas, 60 × 73 cm. The Courtauld Gallery, London. * Figure 3: John Chamberlain. AWESOMEMEATLOAF. 2011. Painted and chrome-plated sheet. 106 × 118 3/4 × 82 in. Gagosian Gallery, New York. * Figure 4: Damien Hirst. Medicine Cabinet: Problems. 1989–2010. Glass, faced particle board, ramin, wooden dowels, plastic, aluminum and pharmaceutical packaging. 54 × 40 × 9 in. White Cube Gallery, London. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Joseph L. Goldstein is Chair of the Lasker Awards jury. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Joseph L Goldstein Author Details * Joseph L Goldstein Contact Joseph L Goldstein Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Chaperone-assisted protein folding: the path to discovery from a personal perspective
  - Nat Med 17(10):1206-1210 (2011)
  Article preview View full access options Nature Medicine | Commentary 2011 Albert Lasker Medical Research Awards Focus issue: October 2011 Volume 17, No 10 Chaperone-assisted protein folding: the path to discovery from a personal perspective * F Ulrich Hartl1Journal name:Nature MedicineVolume: 17,Pages:1206–1210Year published:(2011)DOI:doi:10.1038/nm.2467Published online11 October 2011 Protein folding is the process by which newly synthesized polypeptide chains acquire the three-dimensional structures necessary for biological function. For many years, protein folding was believed to occur spontaneously, on the basis of the pioneering experiments of Christian Anfinsen, who showed in the late 1950s that purified proteins can fold on their own after removal from denaturant1. Anfinsen had discovered the fundamental principle that the linear amino acid sequence holds all the information necessary to specify a protein's three-dimensional structure. But it soon became apparent that test-tube folding experiments work mostly for small, single-domain proteins, often only in conditions far removed from those encountered in a cell. Large proteins frequently fail to reach native state under these experimental conditions, forming nonfunctional aggregates instead. Despite these problems, protein folding was of little interest to cell biologists until the mid- and late 19! 80s, when the chaperone story began to unfold. As a result, we now know that in cells, many (perhaps most) proteins require molecular chaperones and metabolic energy to fold efficiently and at a biologically relevant rate. Here I describe, from a personal perspective, the developments leading to this new view. Setting the stage After completing my doctoral thesis at Heidelberg University in 1985, I joined the laboratory of renowned biochemist Walter Neupert at the University of Munich. Walter's group studied how cell organelles such as mitochondria import newly synthesized proteins from the cytosol. The move to Munich turned out to be crucial for both my professional and personal future—the latter because Walter allowed me to attend a molecular biology summer school on a Greek island, where I met my future wife Manajit (facilitated by the chaperone-free, Mediterranean atmosphere, I might add). Around the time of my arrival in Walter's department, it was becoming clear that proteins need to adopt an unfolded state to cross the mitochondrial double membrane2, and definitive evidence for this came in 1986 from the group of Gottfried Schatz3. In 1988, two papers, one from Günter Blobel and the other from Randy Schekman and Elizabeth Craig, showed that the cytosolic precursors of mitochondrial and secretory proteins interact with so-called heat-shock proteins, such as Hsp70, before membrane translocation4, 5. These proteins had already been described in the 1970s, on the basis of their striking tendency to increase in abundance upon exposure of cells to heat stress6. Hugh Pelham had suggested in 1985 that these proteins are involved in dissociating protein aggregates that form under such stress conditions7. On the basis of the two 1988 papers, it was now plausible that Hsp70 also functioned in stabilizing precursor proteins in an unfolded state for translocation. How,! then, would proteins fold after import into the mitochondria? The atmosphere was ripe for something new, and I found myself in the right place at the right time. I became completely fascinated by the possibility of using the mitochondrial system to study how proteins fold in a physiological environment. The Hsp60 story In pursuing the problem of protein folding, I was fortunate that Walter introduced me to Art Horwich from Yale, and this led to an exciting and very productive collaboration (Fig. 1). Art had conducted a genetic screen in yeast to identify cellular machinery involved in mitochondrial protein uptake. The first temperature-sensitive mutant we analyzed in detail had a defect in the mitochondrial precursor protease8, but another mutant had a far more interesting and puzzling phenotype. The mitochondria of this mutant strain, called mif4, were still capable of importing and proteolytically processing proteins at the nonpermissive temperature, but the proteins failed to assemble into their respective oligomeric complexes9. This included the trimeric enzyme ornithine transcarbamylase and the β-subunit of the F1-ATPase. Other proteins that undergo complex intramitochondrial sorting, such as the Rieske Fe/S protein10, were incompletely processed. Interestingly, the mif4 mutation map! ped to the nuclear gene encoding the mitochondrial heat-shock protein Hsp60 (ref. 9), a protein that had just been identified as the homolog of Escherichia coli GroEL and of the Rubisco large subunit–binding protein (RBP) of chloroplasts11, 12. These proteins were known to form large macromolecular complexes, for which John Ellis and Costa Georgopoulos had coined the name 'chaperonin'12 in 1988, to denote a special class of molecular chaperone13. E. coli GroEL had already been discovered in the 1970s to function with GroES as a host factor in phage assembly14, 15, 16, and John Ellis had observed that large subunits of the enzyme Rubisco in chloroplasts interact with RBP before assembly17. Figure 1: Art Horwich (right) and I (left) in March 1991 taking a walk near my parents' village in the northern part of the Black Forest. Photograph by Manajit Hayer-Hartl. * Full size image (285 KB) * Figures index * Next figure We interpreted our findings with mif4 mitochondria in terms of a similar role for yeast Hsp60 in oligomeric assembly9. However, the discovery of the basic function of Hsp60 in polypeptide chain folding was just around the corner. To investigate its possible role in folding, we used the monomeric protein dihydrofolate reductase (DHFR) for import into mitochondria of the fungus Neurospora crassa18. Although denatured DHFR refolded spontaneously in vitro, this was not observed in mitochondria. Instead, we found that newly imported DHFR associated with Hsp60 in a highly protease-sensitive, unfolded state. Formation of folded, protease-resistant DHFR occurred concomitantly with release from Hsp60 in an ATP-dependent manner18. We thus concluded that the chaperonins mediated protein folding. Hence, the defects in oligomeric assembly observed in the mif4 mitochondria resulted from the failure of protein subunits to fold. These findings in 1989 established the new paradigm of chapero! ne-assisted protein folding. Figures at a glance * Figure 1: Art Horwich (right) and I (left) in March 1991 taking a walk near my parents' village in the northern part of the Black Forest. Photograph by Manajit Hayer-Hartl. * Figure 2: Averaged electron microscopic images. () GroEL alone. () GroEL-unfolded rhodanese. () GroEL-unfolded rhodanese-GroES. () GroEL-GroES complexes from the collaboration with Wolfgang Baumeister22. End-on views are shown in , and and side views in and . In , GroES sits like a lid on the GroEL cavity, causing a conformational change in the outer domains of the interacting GroEL subunits (also see ref. 31). * Figure 3: The current model for protein folding in the GroEL-GroES chaperonin cage. Substrate binding to GroEL (upon transfer from the upstream chaperone Hsp70; see Fig. 4) may result in local unfolding57. ATP binding then triggers a conformational rearrangement of the GroEL apical domains. This is followed by the binding of GroES (forming the cis complex) and substrate encapsulation for folding. At the same time, ADP and GroES dissociate from the opposite (trans) GroEL ring, allowing the release of substrate that had been enclosed in the former cis complex (omitted for simplicity). The substrate remains encapsulated, free to fold, for the time needed to hydrolyze the seven ATP molecules in the newly formed cis complex (~10 s). Binding of ATP and GroES to the trans ring causes the opening of the cis complex. Diagram modified from ref. 58. * Figure 4: Model from 1993 for the pathway of chaperone-assisted protein folding in the E. coli cytosol, shown for a GroEL-dependent protein (reproduced from ref. 25). The nascent chain is stabilized in a folding-competent state during translation by the Hsp70 chaperone system (DnaK, DnaJ) (1 and 2). These chaperones bind hydrophobic segments exposed by the extended chain that will later be buried within the folded structure. Upon completion of translation, the protein is unable to fold using the Hsp70 chaperone system and must be transferred into the central cavity of GroEL. This step requires GrpE, the nucleotide exchange factor of DnaK (3). After binding of the protein in a molten globule–like conformation into the open ring of GroEL (4), the protein is encapsulated by GroES in the folding cage (5). Folded protein emerges from the cage as GroES unbinds (6). The model was later extended to include the cooperation of DnaK with the ribosome-bound chaperone trigger factor and the finding that the Hsp70 system mediates the folding of proteins that do not require the physical environment of the chaperonin cage47, 55. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * F. Ulrich Hartl is at the Max Planck Institute of Biochemistry, Martinsried, Germany. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * F Ulrich Hartl Author Details * F Ulrich Hartl Contact F Ulrich Hartl Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Protein folding in the cell: an inside story
  - Nat Med 17(10):1211-1216 (2011)
  Article preview View full access options Nature Medicine | Commentary 2011 Albert Lasker Medical Research Awards Focus issue: October 2011 Volume 17, No 10 Protein folding in the cell: an inside story * Arthur L Horwich1Journal name:Nature MedicineVolume: 17,Pages:1211–1216Year published:(2011)DOI:doi:10.1038/nm.2468Published online11 October 2011 The final step of information transfer from DNA to effector protein involves the folding of newly translated polypeptide chains into characteristic three-dimensional active structures. In the fall of 1972, while working in a biochemistry laboratory as an undergraduate at Brown University, I heard about an astonishing experiment for which Christian Anfinsen was receiving the Nobel Prize in Chemistry. Anfinsen and his co-workers had unfolded purified RNase A by reducing its disulfide bonds and exposing it to a denaturing agent, and then asked whether the protein could find its way back to the enzymatically active native state upon removal of the reducing agent and denaturant1. Amazingly, it did. I was utterly haunted by the beauty of this observation and by the profound conclusion that the primary structure of a protein contains all the information necessary to direct its folding to the native functional state. I could never have imagined that, many years later and in a comple! tely unexpected fashion, I would have something to add to something so fundamentally beautiful. I finished my undergraduate and medical training at Brown and went to Yale for pediatric residency, but, by the second year of training, I was missing the laboratory. I became captivated by the problem of malignant transformation mediated by single viral genes, and, by the end of my residency, I had amassed on my nightstand a stack of several hundred papers on the topic. Contributors to the discovery of chaperonin action and to understanding of the chaperonin mechanism. Top left: Ulrich Hartl, c. 1988; bottom left: Ming Cheng, 1988. Top center: Andrzej Joachimiak, Paul Sigler and Zbyszek Otwinowski, c. 1990; bottom center: Kerstin Braig, c. 1993, and Zhaohui Xu, c. 1996. Top right: the 1996 team, members of which dissected the topology and reaction cycle. Shown left to right in top row, Krystyna Furtak, Jonathan Weissman, George Farr; in middle row, Matt Goldberg, Hays Rye, Oleg Kovalenko; in bottom row, myself, Steve Burston, Suwon Kim, David Boisvert. Bottom right: Wayne Fenton, timeless, and Helen Saibil, c. 2003. I then moved to the Salk Institute to work with two masters of the field, Walter Eckhart and Tony Hunter, who taught me molecular biology and biochemistry. I watched Tony discover tyrosine phosphorylation, a fortuitous finding that resulted from his using an aged electrophoresis buffer for separating phosphoamino acids (but as Pasteur noted, "...chance favors the prepared mind"). Observing Tony carry out his own experiments day by day as a role model was already quite an experience, but it went further. He took the time to teach me how to think about a problem, he taught me good technique directly and his tutelage solidified my love of science and of being at the bench. Figures at a glance * Figure 1: Mitochondrial protein import and the demonstration that a folding machine assists refolding of newly-imported proteins. () Most mitochondrial proteins are encoded in the nucleus, translated in the cytosol and targeted by N-terminal signal peptides (orange). It had been shown that, in order to cross the mitochondrial membranes, proteins had to occupy an unfolded conformation6. During and after translocation across the membranes, the signal peptide is cleaved by a protease inside the organelle (green 'PacMan'). The question we asked concerned refolding of the imported mature-sized proteins to the native state. Did they spontaneously refold, as models at that time suggested (top), or were they assisted by a putative molecular machine (bottom)? () Proteins that matured to their native, active form in the mitochondrial matrix compartment were affected by the mif4 mutation in the identified molecular machine, called Hsp60 (heat-shock protein 60). The studies of Rieske iron-sulfur protein (Fe/S) and dihydrofolate reductase showed a monomeric polypeptide folding step was involved, as opposed to oligo! meric assembly of already-folded subunits. Studies of α-ketoglutarate dehydrogenase (KGDH) and dihydrolipoamide dehydrogenase (LPDH) were reported in ref. 35. Figure adapted from ref. 36. * Figure 2: Cutaway views of crystallographic models of unliganded GroEL and an asymmetric GroEL-GroES-ADP7 complex. () The model of GroEL immediately demonstrated two back-to-back rings with three domains in each of the seven subunits per ring: an apical domain, the collective of which forms the terminal portions of the GroEL cylinder, with hydrophobic polypeptide binding sites (yellow) on the inside aspect; an intermediate hinge-like domain; and an equatorial domain, the collective of which forms the base of the assembly and houses a nucleotide binding site facing the cavity (see nucleotides colored red in ). () In the GroES-bound state, the bound GroEL ring has undergone large rigid-body movements that result in elevation of the apical domains to form contacts with GroES through the same hydrophobic surface that bound polypeptide. Thus, the hydrophobic binding surface is elevated and twisted away from the large, now GroES-encapsulated cavity, forming a cavity with a hydrophilic, electrostatic surface. (Note the absence of hydrophobic side chains exposed to the cavity in the upper, GroES! -bound ring.) In the course of these movements, the polypeptide substrate initially bound is released into the large folding chamber, where it folds in solitary confinement without the chance of aggregation. * Figure 3: The GroEL-GroES reaction cycle. The cycle is directed via the binding and hydrolysis of ATP in respective GroEL rings (red T, ATP; red D, ADP). Horovitz and co-workers showed early that ATP binds cooperatively within a GroEL ring but anticooperatively between rings37, such that when one ring has seven ATPs, the other does not bind ATP. Binding of GroES (blue 'lid') is dependent on the binding of ATP to the GroES-associated ring38, resulting in the formation of asymmetric complexes, in which folding proceeds inside the GroES-bound GroEL ring. A cycle begins with polypeptide (green 'squiggle') binding to the ATP-bound ring of an asymmetric complex (1) and is immediately followed by GroES collision with that ring, producing a state (2) in which both polypeptide and GroES are bound to the apical domains (this prevents escape of the polypeptide). Once GroES docks, there are further large rigid-body movements that produce the domed folding chamber (3). Folding proceeds in this longest-lived of the GroEL states. ! After ~10 s, ATP hydrolysis occurs (4), weakening the assembly and permitting entry of ATP into the trans ring (5)39. This sends an allosteric signal that ejects the ligands from what had been the folding-active ring (6), such that GroES, polypeptide (whether folded or not) and ADP depart, while a new folding cycle commences on the newly ATP-bound ring40. The machine thus alternates back and forth, using one round of seven ATPs at a time to nucleate a folding-active ring and dispatch the previous one. (Figure adapted from ref. 41.) Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Arthur L. Horwich is at the Yale University School of Medicine, New Haven, Connecticut, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Arthur L Horwich Author Details * Arthur L Horwich Contact Arthur L Horwich Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine
  - Nat Med 17(10):1217-1220 (2011)
  Article preview View full access options Nature Medicine | Commentary 2011 Albert Lasker Medical Research Awards Focus issue: October 2011 Volume 17, No 10 The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine * Youyou Tu1Journal name:Nature MedicineVolume: 17,Pages:1217–1220Year published:(2011)DOI:doi:10.1038/nm.2471Published online11 October 2011 Joseph Goldstein has written in this journal that creation (through invention) and revelation (through discovery) are two different routes to advancement in the biomedical sciences1. In my work as a phytochemist, particularly during the period from the late 1960s to the 1980s, I have been fortunate enough to travel both routes. I graduated from the Beijing Medical University School of Pharmacy in 1955. Since then, I have been involved in research on Chinese herbal medicine in the China Academy of Chinese Medical Sciences (previously known as the Academy of Traditional Chinese Medicine). From 1959 to 1962, I was released from work to participate in a training course in Chinese medicine that was especially designed for professionals with backgrounds in Western medicine. The 2.5-year training guided me to the wonderful treasure to be found in Chinese medicine and toward understanding the beauty in the philosophical thinking that underlies a holistic view of human beings and the universe. Discovery of antimalarial effect of qinghao Malaria, caused by Plasmodium falciparum, has been a life-threatening disease for thousands of years. After the failure of international attempts to eradicate malaria in the 1950s, the disease rebounded, largely due to the emergence of parasites resistant to the existing antimalarial drugs of the time, such as chloroquine. This created an urgent need for new antimalarial medicines. In 1967, a national project against malaria was set up in China under the leadership of the Project 523 office. My institute quickly became involved in the project and appointed me to be the head of a malaria research group comprising both phytochemical and pharmacological researchers. Our group of young investigators started working on the extraction and isolation of constituents with possible antimalarial activities from Chinese herbal materials. During the first stage of our work, we investigated more than 2,000 Chinese herb preparations and identified 640 hits that had possible antimalarial activities. More than 380 extracts obtained from ~200 Chinese herbs were evaluated against a mouse model of malaria. However, progress was not smooth, and no significant results emerged easily. The turning point came when an Artemisia annua L. extract showed a promising degree of inhibition against parasite growth. However, this observation was not reproducible in subsequent experiments and appeared to be contradictory to what was recorded in the literature. Seeking an explanation, we carried out an intensive review of the literature. The only reference relevant to use of qinghao (the Chinese name of Artemisia annua L.) for alleviating malaria symptoms appeared in Ge Hong's A Handbook of Prescriptions for Emergencies: "A handful of qinghao immersed with 2 liters of water, wring out the juice and drink it all" (Fig. 1). This sentence gave me the idea that the heating involved in the conventional extraction step we had used might have destroyed the active components, and that extraction at a lower temperature might be necessary to preserve antimalarial activity. Indeed, we obtained much better activity after switching to a lower-temperature procedure. Figure 1: A Handbook of Prescriptions for Emergencies by Ge Hong (284–346 CE). () Ming dynasty version (1574 CE) of the handbook. () "A handful of qinghao immersed with 2 liters of water, wring out the juice and drink it all" is printed in the fifth line from the right. (From volume 3.) * Full size image (158 KB) * Figures index * Next figure We subsequently separated the extract into its acidic and neutral portions and, at long last, on 4 October 1971, we obtained a nontoxic, neutral extract that was 100% effective against parasitemia in mice infected with Plasmodium berghei and in monkeys infected with Plasmodium cynomolgi. This finding represented the breakthrough in the discovery of artemisinin. Figures at a glance * Figure 1: A Handbook of Prescriptions for Emergencies by Ge Hong (284–346 CE). () Ming dynasty version (1574 CE) of the handbook. () "A handful of qinghao immersed with 2 liters of water, wring out the juice and drink it all" is printed in the fifth line from the right. (From volume 3.) * Figure 2: Artemisia annua L. () A hand-colored drawing of qinghao in Bu Yi Lei Gong Pao Zhi Bian Lan (Ming Dynasty, 1591 CE). () Artemisia annua L. in the field. * Figure 3: Artemisinin. () Molecular structure of artemisinin. () A three-dimensional model of artemisinin. Carbon atoms are represented by black balls, hydrogen atoms are blue and oxygen atoms are red. The Chinese characters underneath the model read Qinghaosu. * Figure 4: Delegates at the fourth meeting of the Scientific Working Group on the Chemotherapy of Malaria in Beijing in 1981. Professor Ji Zhongpu (center, first row), president of the Academy of Traditional Chinese Medicine, delivered the opening remarks to the meeting. The author is in the second row (fourth from the left). Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Youyou Tu is at the Qinghaosu Research Center, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Youyou Tu Author Details * Youyou Tu Contact Youyou Tu Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• The NIH Clinical Center and the future of clinical research
  - Nat Med 17(10):1221-1223 (2011)
  Article preview View full access options Nature Medicine | Commentary 2011 Albert Lasker Medical Research Awards Focus issue: October 2011 Volume 17, No 10 The NIH Clinical Center and the future of clinical research * John I Gallin1Journal name:Nature MedicineVolume: 17,Pages:1221–1223Year published:(2011)DOI:doi:10.1038/nm.2466Published online11 October 2011 In 1947, the US Congress gave the American public a special gift—the Clinical Center at the National Institutes of Health (NIH). This gift aligned with the vision of Albert and Mary Lasker, who had successfully advocated for the US government to support clinical research. President Harry Truman laid the cornerstone of the NIH Clinical Center on January 22, 1951, saying, "Modern medicine must find ways of detecting . . . diseases in their early stages and of stopping their destructive force. That will be the major work of this clinical research center"1 (Fig. 1). Figure 1: President Harry S. Truman applying the first trowel of mortar to the NIH Clinical Center cornerstone, June 22, 1951. * Full size image (110 KB) * Figures index * Next figure At its opening on July 2, 1953, the Clinical Center was the largest hospital ever built specifically for clinical research, and it remains so today. It was truly a hospital within a community of laboratories. The criteria for patient admission were "how their presence will aid scientists and clinicians in basic laboratory investigations and clinical studies of the problems of cancer, mental health, heart disease and other longer term illnesses"2. Figures at a glance * Figure 1: President Harry S. Truman applying the first trowel of mortar to the NIH Clinical Center cornerstone, June 22, 1951. * Figure 2: The Clinical Center in 1954. * Figure 3: The NIH Clinical Center in 2005. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * John I. Gallin is Director of the Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * John I Gallin Author Details * John I Gallin Contact John I Gallin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• Isolation and in vitro expansion of human colonic stem cells
  - Nat Med 17(10):1225-1227 (2011)
  Nature Medicine | Brief Communication Isolation and in vitro expansion of human colonic stem cells * Peter Jung1, 9 * Toshiro Sato2, 3, 9 * Anna Merlos-Suárez1 * Francisco M Barriga1 * Mar Iglesias4 * David Rossell5 * Herbert Auer6 * Mercedes Gallardo7 * Maria A Blasco7 * Elena Sancho1 * Hans Clevers2 * Eduard Batlle1, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1225–1227Year published:(2011)DOI:doi:10.1038/nm.2470Received25 February 2011Accepted14 August 2011Published online04 September 2011Corrected online20 September 2011 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 describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population. View full text Figures at a glance * Figure 1: Characterization of epithelial colon cells purified from human mucosa samples according to different EPHB2 surface levels. () Immunohistochemistry analysis for EPHB2, proliferation marker MKI67 and pan-differentiation marker KRT20 on serial sections of normal human colonic mucosa obtained from a colectomy sample from a subject with colorectal cancer disease. Scale bars, 50 μm. Black arrows indicate specific staining, and white arrowheads indicate nonspecific background staining (see Supplementary Fig. 1b). () Top, FACS profile of single-cell suspensions from normal human colonic crypts staining with antibody to EPHB2. Nonepithelial cell lineages were identified as CD11b, CD3+ or CD45+ and discarded from the analysis. The epithelial marker EpCAM was used to select positively for epithelial cells. Bottom, IgG control staining was performed to define the EPHB2− cell population. () Quantitative RT-PCR (qRT-PCR) analysis of expression levels of ISC, proliferation or differentiation genes on colon epithelial cells purified by FACS according to different EPHB2 surface levels. Error bars indicate s.d! . (n = 3). CEACAM7, carcinoembryonic antigen–related cell adhesion molecule 7; FOXM1, forkhead box M1; MYC, Myc oncogene; AURKB, aurora kinase B. () Percentage of cells with relatively short telomeres as measured by fluorescence in situ hybridization (FISH) in each EPHB2-purified cell population. Cells with short telomeres were defined according to three different ranges of average telomere fluorescence arbitrary units (AU); cells with ≤65 AU, ≤70 AU or ≤75 AU according to the quantitative FISH histograms depicted in Supplementary Figure 2. () Average telomere (Tel) length in EPHB2-sorted colon cell populations. The observed differences in average telomere length are highly significant (P < 0.01) except for the EPHB2medium to EPHB2low comparison (P = 0.488). Error bars indicate s.d. * Figure 2: Single EPHB2high human colonic crypt cells form in vitro spheroids with long-term proliferation and multilineage differentiation capacity. () Microscopy images documenting the growth of a representative spheroid derived from a single EPHB2high human colonic crypt cell from day 0 to day 13. Days 0–9: 40× objective, days 11–13: 20× objective. Scale bar, 50 μm. () Left, representative images of colonic spheroid cultures 2 weeks after seeding the same number cells of each EPHB2-purified cell population. Samples were derived from four individuals suffering either from cancer (P1–P3) or from a diverticulosis (P4). Each dashed black circle represents the boundary of a Matrigel drop. Right, quantification of relative number of spheroids generated in each sample. Scale bars, 1 mm. () Microscopic images of serially passaged in vitro organoids derived from single EPHB2high sorted cells after more than 100 d in culture. One 50 μl Matrigel droplet is depicted (left; scale bar, 1 mm). Single spheroids maintained their microscopic appearance after 16 weeks of culture and sequential passaging (right; 10× objective; ! scale bar, 100 μm). () Analysis of expression of stem cell proliferation and differentiation marker genes by qRT-PCR in human colonic spheroid cultures. Data are represented as relative changes of expression between spheroids cultured in stem cell medium compared to spheroids maintained in differentiation media for 7 d. Error bars indicate s.d. (n = 3). () Detection of MKI67, KRT20, chromogranin A (CHGA), EPHB2 or mucrosecreting cells (periodic acid Schiff (PAS) staining) on colonic sphere cultures maintained in stem cell medium (top) or in differentiation medium over 7 d (bottom). MKI67, KRT20 and CHGA (green label) are shown as confocal images (counterstaining with DAPI in magenta), whereas EPHB2 (brown staining) was detected by immunohistochemistry (counterstaining with hematoxylin). Scale bars, 50 μm (confocal images) and 10 μm (immunohistochemistry). () Morphological features of cells obtained by EM from in vitro–cultured human colon organoids either cultured in s! tem cells or differentiation media (7 d). L, sphere lumen; M, ! Matrigel; E, enterocyte; G, globet cells. Areas encompassed by squares are magnified in the right images and highlight microvilli in enterocytes and secretory granules in goblet cells. () Confocal images showing double detection of enterocytes (FABP1+) and goblet cells (MUC2+) on differentiated organoids. Counterstaining, DAPI (blue). Scale bars, 20 μm. () Schematic representation of the single-cell reseeding approach. In brief, after 6 weeks of in vitro culture, organoids derived from EPHB2high single cells were enzymatically and mechanically disaggregated. Organoid-derived single sorted cells were re-embedded in Matrigel and seeded in serial dilutions. Only Matrigel droplets in which one organoid regrew from a single cell were considered for further analysis. After 14 d, RNA was isolated from a subset of organoids cultured in stem medium. The rest of organoids were then cultured in differentiation medium and RNA was isolated after 5 d. () Intensity plot representing the ! relative gene expression of several marker genes in five single-cell–derived spheroids that were cultured in stem cell medium (1–5) and in eight single-cell–derived spheroids that underwent differentiation for 5 d (6–13). Expression was measured by qRT-PCR on mRNA extracted from each individual organoid. Numbers in color bar indicate the relative expression levels. () Confocal images showing double detection of enterocytes (FABP1+) and goblet cells (MUC2+) on differentiated organoids derived from single EPHB2high cells. Counterstaining, DAPI (blue). Scale bars, 20 μm. Change history * Change history * Author information * Supplementary informationCorrected online 20 September 2011 In the version of this article initially published online, the author list was missing the name Herbert Auer, and the name of Mercedes Gallardo was incorrectly given as María M. Gallardo. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Peter Jung & * Toshiro Sato Affiliations * Oncology program, Institute for Research in Biomedicine, Barcelona, Spain. * Peter Jung, * Anna Merlos-Suárez, * Francisco M Barriga, * Elena Sancho & * Eduard Batlle * Hubrecht Institute and University Medical Center Utrecht, Utrecht, The Netherlands. * Toshiro Sato & * Hans Clevers * Department of Gastroenterology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan. * Toshiro Sato * Department of Pathology, Hospital Universitari del Mar, Universitat Autònoma de Barcelona, Barcelona, Spain. * Mar Iglesias * Biostatistics and Bioinformatics Unit, Institute for Research in Biomedicine, Barcelona, Spain. * David Rossell * Functional Genomics Core Facility, Institute for Research in Biomedicine, Barcelona, Spain. * Herbert Auer * Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research Center, Madrid, Spain. * Mercedes Gallardo & * Maria A Blasco * Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain. * Eduard Batlle Contributions E.B. and P.J., study concept and design and writing of the manuscript. T.S. and H.C., development of original human colon culture method. P.J., acquisition, analysis and interpretation of data and modifications of the human CoSC culture protocol. A.M.-S. and F.M.B., profiling of human colon stem cells. D.R., bioinformatic analysis. E.S., conceptual and logistic support. M.M.G. and M.A.B., telomere length measurements. M.I., sample preparation and development of EphB2 sorting method. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Eduard Batlle Author Details * Peter Jung Search for this author in: * NPG journals * PubMed * Google Scholar * Toshiro Sato Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Merlos-Suárez Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco M Barriga Search for this author in: * NPG journals * PubMed * Google Scholar * Mar Iglesias Search for this author in: * NPG journals * PubMed * Google Scholar * David Rossell Search for this author in: * NPG journals * PubMed * Google Scholar * Herbert Auer Search for this author in: * NPG journals * PubMed * Google Scholar * Mercedes Gallardo Search for this author in: * NPG journals * PubMed * Google Scholar * Maria A Blasco Search for this author in: * NPG journals * PubMed * Google Scholar * Elena Sancho Search for this author in: * NPG journals * PubMed * Google Scholar * Hans Clevers Search for this author in: * NPG journals * PubMed * Google Scholar * Eduard Batlle Contact Eduard Batlle Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–11, Supplementary Data, Supplementary Table 1 and Supplementary Methods Excel files * Supplementary Table 2 (41K) List of genes whose expression is enriched in EPHB2-high cells. * Supplementary Table 3 (20M) Genome-wide expression analysis of EPHB2-high, EPHB2-medium, EPHB2-low and EPHB2-negative cells from 3 independent colon mucosa samples. Additional data
• Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors
  - Nat Med 17(10):1228-1230 (2011)
  Nature Medicine | Brief Communication Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors * Fabrizio Benedetti1 * Martina Amanzio2 * Rosalba Rosato2, 3 * Catherine Blanchard4 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1228–1230Year published:(2011)DOI:doi:10.1038/nm.2435Received09 November 2010Accepted07 July 2011Published online02 October 2011 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 Placebo analgesia is mediated by both opioid and nonopioid mechanisms, but so far nothing is known about the nonopioid component. Here we show that the specific CB1 cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (rimonabant or SR141716) blocks nonopioid placebo analgesic responses but has no effect on opioid placebo responses. These findings suggest that the endocannabinoid system has a pivotal role in placebo analgesia in some circumstances when the opioid system is not involved. View full text Author information * Author information * Supplementary information Affiliations * Department of Neuroscience, University of Turin Medical School and National Institute of Neuroscience (INN), Turin, Italy. * Fabrizio Benedetti * Department of Psychology, University of Turin, Turin, Italy. * Martina Amanzio & * Rosalba Rosato * Unit of Cancer Epidemiology, San Giovanni Battista Hospital, Preventive Oncology Center (CPO)-Piemonte, Turin, Italy. * Rosalba Rosato * Center for Endocrine and Metabolic Disorders, Aoste, Italy. * Catherine Blanchard Contributions F.B. planned and conducted the experiments, analyzed the data and wrote the manuscript. M.A. and R.R. analyzed the data and contributed to the writing of the manuscript. C.B. conducted the experiments and contributed to the writing of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Fabrizio Benedetti Author Details * Fabrizio Benedetti Contact Fabrizio Benedetti Search for this author in: * NPG journals * PubMed * Google Scholar * Martina Amanzio Search for this author in: * NPG journals * PubMed * Google Scholar * Rosalba Rosato Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Blanchard Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (221K) Supplementary Figure 1, Supplementary Tables 1–4 and Supplementary Methods Additional data
• Evidence for osteocyte regulation of bone homeostasis through RANKL expression
  - Nat Med 17(10):1231-1234 (2011)
  Nature Medicine | Brief Communication Evidence for osteocyte regulation of bone homeostasis through RANKL expression * Tomoki Nakashima1, 2, 3 * Mikihito Hayashi1, 2, 3 * Takanobu Fukunaga1, 3 * Kosaku Kurata4 * Masatsugu Oh-hora1, 3 * Jian Q Feng5 * Lynda F Bonewald6 * Tatsuhiko Kodama7 * Anton Wutz8 * Erwin F Wagner9 * Josef M Penninger10 * Hiroshi Takayanagi1, 2, 3, 11 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1231–1234Year published:(2011)DOI:doi:10.1038/nm.2452Received25 May 2011Accepted25 July 2011Published online11 September 2011 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 Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo. View full text Figures at a glance * Figure 1: New method of isolating osteocytes and RANKL expression in osteocytes. () Quantitative RT-PCR analysis of Tnfsf11 mRNA in bone tissue from which the bone marrow cells has been removed as compared to isolated bone marrow cells and T cells. (,) Separation of the EGFP-positive cell population by flow cytometry () and the morphology of the isolated cells (). DIC, differential interference contrast microscopy. () Profiling of gene expression in mouse embryonic fibroblasts, osteoblasts and osteocytes (quantitative RT-PCR analysis). Osteoblasts and osteocytes were separated by the method described above. () Cell surface expression of RANKL in isolated osteocytes. MFI, mean fluorescence intensity. () Immunohistochemical analysis of RANKL expression in femur. Bone tissue was stained with control IgG (top) or RANKL-specific (anti-RANKL) antibodies (bottom). B, bone; BM, bone marrow. () Osteoclastogenesis in coculture with osteoblasts or osteocytes. Top, TRAP staining. Scale bars: ,, 100 μm, , 40 μm. Error bars, means ± s.e.m.; *P < 0.05; **P < 0.01; *! **P < 0.005. Mouse experiments were done with the approval of the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. * Figure 2: Osteopetrotic phenotype in osteocyte-specific Tnfsf11-deficient mice. () Selective deletion of RANKL in osteocytes, but not in osteoblasts, in Tnfsf11flox/Δ; Dmp1-Cre mice with the CAG-CAT-EGFP reporter. RANKL expression was analyzed by flow cytometry in EGFP+ (osteocytes) and EGFP− (osteoblasts) populations. () Osteoclastogenesis-supporting ability of osteocytes and osteoblasts in Tnfsf11flox/Δ; Dmp1-Cre mice. () Microcomputed tomography (μCT) analysis of the femurs of Tnfsf11flox/flox and Tnfsf11flox/Δ; Dmp1-Cre littermates at 12 weeks of age (male, n = 6 or 7). Top, longitudinal view; bottom, axial view of the metaphyseal region. () Bone volume and parameters of trabecular bone in μCT analysis. () Histological analysis of the proximal tibia of Tnfsf11flox/flox and Tnfsf11flox/Δ; Dmp1-Cre littermates. () Bone morphometric analysis of Tnfsf11flox/flox and Tnfsf11flox/Δ; Dmp1-Cre littermates. () Trabecular bone mineral content per tissue volume in newborn mice in μCT analysis (postnatal day 1, male, n = 6 or 7). () Osteoclast number ! per bone surface (postnatal day 1). () Bone mineral content in Tnfsf11flox/flox and Tnfsf11flox/Δ; Dmp1-Cre littermates during growth (male, n = 5–7). Scale bars: , 1 mm; , 100 μm. Error bars, mean ± s.e.m.; ***P < 0.005; NS, not significant; ND, not detected. Mouse experiments were done with the approval of the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. Author information * Author information * Supplementary information Affiliations * Department of Cell Signaling, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan. * Tomoki Nakashima, * Mikihito Hayashi, * Takanobu Fukunaga, * Masatsugu Oh-hora & * Hiroshi Takayanagi * Japan Science and Technology Agency, Exploratory Research for Advanced Technology Program, Takayanagi Osteonetwork Project, Yushima, Bunkyo-ku, Tokyo, Japan. * Tomoki Nakashima, * Mikihito Hayashi & * Hiroshi Takayanagi * Global Center of Excellence Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Yushima, Bunkyo-ku, Tokyo, Japan. * Tomoki Nakashima, * Mikihito Hayashi, * Takanobu Fukunaga, * Masatsugu Oh-hora & * Hiroshi Takayanagi * Department of Mechanical Engineering, Faculty of Engineering, Kyushu University, Motooka, Nishi-ku, Fukuoka, Japan. * Kosaku Kurata * Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M Health Science Center, Dallas, Texas, USA. * Jian Q Feng * Department of Oral Biology, University of Missouri at Kansas City, Kansas City, Missouri, USA. * Lynda F Bonewald * Research Center for Advanced Science and Technology, Department of Molecular Biology and Medicine, University of Tokyo, Tokyo, Japan. * Tatsuhiko Kodama * Department of Biochemistry, The Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK. * Anton Wutz * Genes, Development and Disease Group, Banco Bilbao Vizcaya Argentaria Foundation, Cancer Cell Biology Program, Spanish National Cancer Center, Madrid, Spain. * Erwin F Wagner * Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria. * Josef M Penninger * Center for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia. * Hiroshi Takayanagi Contributions T.N. generated conditional knockout mice, performed most of the experiments, interpreted the results and prepared the manuscript. M.H. participated in the in vivo analyses of the mice and prepared the manuscript. T.F. and K.K. performed experiments using the three-dimensional gel-embedded cell culture system and contributed to the osteocyte isolation experiments. M.O. assisted the in vivo analyses of the mice. J.Q.F. and L.F.B. provided Dmp1-Cre deleter mice and advice on project planning and data interpretation. L.F.B. also provided the osteocyte-like cell line MLO-Y4. T.K. conducted the GeneChip analysis. A.W. and E.F.W. generated embryonic stem cells and provided technical help. J.M.P. provided advice on project planning. H.T. directed, supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hiroshi Takayanagi Author Details * Tomoki Nakashima Search for this author in: * NPG journals * PubMed * Google Scholar * Mikihito Hayashi Search for this author in: * NPG journals * PubMed * Google Scholar * Takanobu Fukunaga Search for this author in: * NPG journals * PubMed * Google Scholar * Kosaku Kurata Search for this author in: * NPG journals * PubMed * Google Scholar * Masatsugu Oh-hora Search for this author in: * NPG journals * PubMed * Google Scholar * Jian Q Feng Search for this author in: * NPG journals * PubMed * Google Scholar * Lynda F Bonewald Search for this author in: * NPG journals * PubMed * Google Scholar * Tatsuhiko Kodama Search for this author in: * NPG journals * PubMed * Google Scholar * Anton Wutz Search for this author in: * NPG journals * PubMed * Google Scholar * Erwin F Wagner Search for this author in: * NPG journals * PubMed * Google Scholar * Josef M Penninger Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroshi Takayanagi Contact Hiroshi Takayanagi Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Methods and Supplementary Figures 1–13 Additional data
• Matrix-embedded cells control osteoclast formation
  - Nat Med 17(10):1235-1241 (2011)
  Nature Medicine | Article Matrix-embedded cells control osteoclast formation * Jinhu Xiong1, 2 * Melda Onal1, 2 * Robert L Jilka1, 2 * Robert S Weinstein1, 2 * Stavros C Manolagas1, 2 * Charles A O'Brien1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1235–1241Year published:(2011)DOI:doi:10.1038/nm.2448Received03 June 2011Accepted20 July 2011Published online11 September 2011 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 Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors, thereby linking bone formation to resorption. However, RANKL is expressed by a variety of cell types, and it is unclear which of them are essential sources for osteoclast formation. Here we have used a mouse strain in which RANKL can be conditionally deleted and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to adult bone remodeling. These r! esults suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself. View full text Figures at a glance * Figure 1: Deletion of RANKL in Prx1-Cre expressing cells causes osteopetrosis. () RANKL mRNA levels in tibia, spine, calvaria (Cal.) and spleen (Spl.) of RANKLf/f (n = 9) and Prx1-Cre;RANKLf/f (n = 10) littermates (here and throughout, values are mean ± s.d.). *P < 0.05 versus RANKLf/f; Student's t test. () Femoral BMD of WT (n = 7), Prx1-Cre (n = 7), RANKLf/f (n = 9) and Prx1-Cre;RANKLf/f (n = 10) littermates. *P < 0.05 versus WT, Prx1-Cre and RANKLf/f, using two-way analysis of variance (ANOVA). () Cancellous bone volume relative to the total volume of the region of interest (BV/TV) in distal femur of WT (n = 8), Prx1-Cre (n = 8), RANKLf/f (n = 9) and Prx1-Cre;RANKLf/f (n = 10) littermates. *P < 0.05 versus WT, Prx1-Cre and RANKLf/f, using two-way ANOVA. () Representative μCT images of distal femur of 5-week-old RANKLf/f and Prx1-Cre;RANKLf/f mice. Scale bar, 1 mm. () Histological sections of distal femurs of 5-week-old RANKLf/f and Prx1-Cre;RANKLf/f mice stained with safranin O (cartilage stains red). Scale bar, 0.5 mm. () Histological sections of! distal femurs of 5-week-old RANKLf/f and Prx1-Cre;RANKLf/f mice stained for tartrate-resistant acid phosphatase (TRAP) activity (osteoclasts stain red). Scale bar, 200 μm. () Cathepsin K (CatK), TRAP and calcitonin receptor (CalR) mRNA levels in tibial RNA of RANKLf/f (n = 9) and Prx1-Cre;RANKLf/f (n = 10) mice. *P < 0.05 versus RANKLf/f; Student's t test. All values were determined in 5-week-old mice, including both sexes. Error bars represent s.d. * Figure 2: Deletion of RANKL in Osx1-Cre- and Ocn-Cre-expressing cells causes osteopetrosis. () RANKL mRNA levels in whole tibia of Osx1-Cre;RANKLf/f (n = 8), Ocn-Cre;RANKLf/f (n = 6) and Dmp1-Cre;RANKLf/f (n = 9) mice and their respective RANKLf/f littermates (n = 4–11). *P < 0.05 versus RANKLf/f littermates; Student's t test. () Quantitative PCR (qPCR) of loxP-flanked genomic DNA isolated from collagenase-digested femoral and tibial cortical bone of Dmp1-Cre;RANKLf/f (n = 9) mice and their RANKLf/f (n = 11) littermates. *P < 0.05, Student's t test. () Cancellous bone volume of the distal femurs of Osx1-Cre;RANKLf/f (n = 8), Ocn-Cre;RANKLf/f (n = 6) and Dmp1-Cre;RANKLf/f (n = 9) mice and their RANKLf/f littermates (n = 4 to 11). *P < 0.05 versus RANKLf/f littermates; Student's t test. () X-ray images of the skull, representative μCT images of the distal femur (scale bar, 1 mm), safranin O–stained histological sections of the distal femur (scale bar, 0.5 mm), TRAP-stained histological sections of the distal femur (scale bar, 200 μm) and RANKL-specific immunohi! stochemistry (RANKL IHC; scale bar, 100 μm) from Osx1-Cre;RANKLf/f, Ocn-Cre;RANKLf/f and Dmp1-Cre;RANKLf/f mice and a representative RANKLf/f littermate. Arrowheads in X-rays indicate position of erupted incisors. μCT images for each of the RANKLf/f control littermates are in Supplementary Figure 3. Region of growth plate containing hypertrophic chondrocytes in IHC images is outlined by green dashed lines and nonimmune IgG controls are in Supplementary Figure 3. All values and images are from 5-week-old mice and include both sexes. Error bars represent s.d. * Figure 3: Deletion of RANKL from Dmp1-Cre expressing cells reduces bone remodeling. () Serial BMD of Dmp1-Cre;RANKLf/f (n = 14) and RANKLf/f (n = 19) littermates. *P < 0.05 using Student's t test comparing the two genotypes at a given age. () Cancellous bone volume in distal femur or in L4 vertebra of 6-month-old Dmp1-Cre;RANKLf/f (n = 11) and RANKLf/f (n = 7) littermates. *P < 0.05, Student's t test. () Representative μCT images of distal femur and L4 vertebra of 6-month-old Dmp1-Cre;RANKLf/f and RANKLf/f littermates. Scale bar, 1 mm. () Left, qPCR of loxP-flanked RANKL genomic DNA using genomic DNA isolated from collagenase-digested femoral cortical bone of 6-month-old Dmp1-Cre;RANKLf/f (n = 11) and RANKLf/f (n = 7) littermates. Right, quantitative RT-PCR (qRT-PCR) for RANKL mRNA in tibia and L5 vertebra of same mice as at left. *P < 0.05, Student's t test. () Osteoclast (Oc) number per millimeter bone surface in cancellous bone of the distal femur of 6-month-old Dmp1-Cre;RANKLf/f (n = 4) and RANKLf/f (n = 4) littermates. *P < 0.05, Student's t test. () ! C-terminal crosslinked telopeptide of type I collagen (CTX), osteocalcin (OCN) or soluble RANKL (sRANKL) in blood plasma of 6-month-old Dmp1-Cre;RANKLf/f (n = 9) and RANKLf/f (n = 8) littermates. *P < 0.05, Student's t test. () RANKL mRNA levels in tibial cortical bone of 6-month-old Dmp1-Cre;RANKLf/f and RANKLf/f littermates, pretreated with vehicle or osteoprotegerin (OPG) and then injected with vehicle or parathyroid hormone (1–34) (PTH; n = 6–8 per group). *P < 0.05 versus RANKLf/f mice pretreated with vehicle or osteoprotegerin and then injected with vehicle using two-way ANOVA. #P < 0.05 versus RANKLf/f mice pretreated with vehicle or osteoprotegerin and then injected with parathyroid hormone using two-way ANOVA. All values include data from both sexes. Error bars represent s.d. * Figure 4: Osx1-Cre–mediated RANKL deletion in adult mice does not alter osteoclast number in cancellous bone. () X-ray images of the skull (top) and histological sections of femurs stained with safranin-O (bottom) of 6-month-old Osx1-Cre;RANKLf/f and RANKLf/f littermates exposed to doxycycline in utero and maintained on a doxycycline-containing diet until 4 months of age. Arrowheads indicate the position of erupted incisors. Scale bar, 500 μm. () qPCR of loxP-flanked genomic DNA isolated from collagenase-digested femoral cortical bone (left) and qRT-PCR of RANKL mRNA in tibia and L5 vertebra (right) of Osx1-Cre;RANKLf/f (n = 12) mice and their RANKLf/f littermates (n = 10) exposed to doxycycline as in . *P < 0.05, Student's t test. () RANKL (left) and interleukin-6 (IL-6; right) mRNA levels in bone marrow cultures from Osx1-Cre;RANKLf/f and RANKLf/f littermates exposed to doxycycline as in or from 6-month-old Dmp1-Cre;RANKLf/f and RANKLf/f littermates. Each value is mean of 3 wells. *P < 0.05 for comparisons indicated by brackets using two-way ANOVA. () Osteoclast (Oc) number per m! illimeter bone perimeter in cancellous bone of distal femur of Osx1-Cre;RANKLf/f (n = 4) and RANKLf/f (n = 4) littermates exposed to doxycycline as in . () Cathepsin K and TRAP mRNA levels in tibia and L5 vertebra of Osx1-Cre;RANKLf/f (n = 12) and RANKLf/f (n = 10) littermates exposed to doxycycline as in . () Percentage change in BMD between 4 and 6 months of age in Osx1-Cre;RANKLf/f (n = 14) and RANKLf/f (n = 8) littermates exposed to doxycycline as in . All values include data from both sexes. Error bars represent s.d. * Figure 5: Tail suspension of mice lacking RANKL in osteocytes. () Percent change of femoral BMD after 3 weeks of tail suspension or normal loading (grounded control) in RANKLf/f and Dmp1-Cre;RANKLf/f littermates. () Cancellous bone volume (left), trabecular spacing (Tb. sp.; center) and cortical thickness (Ct. th.; right), in femur of tail-suspended or grounded control RANKLf/f and Dmp1-Cre;RANKLf/f littermates. () RANKL mRNA levels in RNA prepared from collagenase-digested tibial cortical bone of tail-suspended or grounded control RANKLf/f and Dmp1-Cre;RANKLf/f littermates. () Osteoclast (Oc) number per millimeter of cancellous bone surface in distal femur of tail-suspended or grounded control RANKLf/f and Dmp1-Cre;RANKLf/f littermates. All values are from 6-month-old mice, include data from both sexes, and represent the following numbers of animals per group: grounded RANKLf/f (n = 8), suspended RANKLf/f (n = 7), grounded Dmp1-Cre;RANKLf/f (n = 8) and suspended Dmp1-Cre;RANKLf/f (n = 7), with exception of d, in which n = 5 for each gr! oup. *P < 0.05 versus grounded control of same genotype by two-way ANOVA. Error bars represent s.d. Author information * Abstract * Author information * Supplementary information Affiliations * Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences (UAMS), Little Rock, Arkansas, USA. * Jinhu Xiong, * Melda Onal, * Robert L Jilka, * Robert S Weinstein, * Stavros C Manolagas & * Charles A O'Brien * Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA. * Jinhu Xiong, * Melda Onal, * Robert L Jilka, * Robert S Weinstein, * Stavros C Manolagas & * Charles A O'Brien Contributions J.X. carried out the conditional deletion breeding, analysis of gene expression, histomorphometry, immunochemistry and tail-suspension studies. M.O. carried out the R26R breeding and histological analysis of X-gal staining. C.A.O. designed experiments, created the RANKL-flox mice and prepared the first draft of the manuscript. R.L.J., R.S.W., S.C.M. and C.A.O. provided reagents, contributed methods, discussed results and revised the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Charles A O'Brien Author Details * Jinhu Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Melda Onal Search for this author in: * NPG journals * PubMed * Google Scholar * Robert L Jilka Search for this author in: * NPG journals * PubMed * Google Scholar * Robert S Weinstein Search for this author in: * NPG journals * PubMed * Google Scholar * Stavros C Manolagas Search for this author in: * NPG journals * PubMed * Google Scholar * Charles A O'Brien Contact Charles A O'Brien Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Methods and Supplementary Figures 1–6 Additional data
• Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis
  - Nat Med 17(10):1242-1250 (2011)
  Nature Medicine | Article Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis * Guillaume Bollée1, 2, 21 * Martin Flamant3, 4, 21 * Sandra Schordan5 * Cécile Fligny1, 2 * Elisabeth Rumpel5 * Marine Milon1, 2 * Eric Schordan5 * Nathalie Sabaa3, 4 * Sophie Vandermeersch6, 7 * Ariane Galaup8, 9 * Anita Rodenas10 * Ibrahim Casal11, 12 * Susan W Sunnarborg13 * David J Salant14 * Jeffrey B Kopp15 * David W Threadgill16 * Susan E Quaggin17 * Jean-Claude Dussaule6, 7, 18 * Stéphane Germain8, 9 * Laurent Mesnard6, 7 * Karlhans Endlich5 * Claude Boucheix11, 12 * Xavier Belenfant19 * Patrice Callard7, 10 * Nicole Endlich5 * Pierre-Louis Tharaux1, 2, 20 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1242–1250Year published:(2011)DOI:doi:10.1038/nm.2491Received22 February 2011Accepted11 August 2011Published online25 September 2011 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 Rapidly progressive glomerulonephritis (RPGN) is a life-threatening clinical syndrome and a morphological manifestation of severe glomerular injury that is marked by a proliferative histological pattern ('crescents') with accumulation of T cells and macrophages and proliferation of intrinsic glomerular cells. We show de novo induction of heparin-binding epidermal growth factor–like growth factor (HB-EGF) in intrinsic glomerular epithelial cells (podocytes) from both mice and humans with RPGN. HB-EGF induction increases phosphorylation of the epidermal growth factor receptor (EGFR, also known as ErbB1) in mice with RPGN. In HB-EGF–deficient mice, EGFR activation in glomeruli is absent and the course of RPGN is improved. Autocrine HB-EGF induces a phenotypic switch in podocytes in vitro. Conditional deletion of the Egfr gene from podocytes of mice alleviates the severity of RPGN. Likewise, pharmacological blockade of EGFR also improves the course of RPGN, even when started! 4 d after the induction of experimental RPGN. This suggests that targeting the HB-EGF–EGFR pathway could also be beneficial in treatment of human RPGN. View full text Figures at a glance * Figure 1: Induction of renal HB-EGF synthesis leads to glomerular activation of EGFR during RPGN. () Representative images of in situ hybridization study in nephrotoxic serum (NTS)-injected WT animals, showing proHB-EGF expression in glomeruli (G), especially in parietal glomerular epithelial cells (Pec), in podocytes (day 4) and in crescents (Cr; day 8). White arrow, abundant proHB-EGF mRNA expression in areas where tuft-capsular podocyte bridges were present. Scale bars (orange), 50 μm. () Quantification by real-time RT-PCR of proHB-EGF mRNA in freshly isolated podocytes on day 6 after NTS injection (NTS) and in podocytes from noninjected control mice (CT; n = 3 per group). *P < 0.05 versus controls. Values are mean ± s.e.m. (n = 3 mice per group). () Western blot analysis of pEGFR and total EGFR in the renal cortex from nonchallenged controls (CT), WT mice infused with NTS ((+/+)NTS), HB-EGF–deficient mice infused with NTS ((−/−)NTS), and WT mice given intraperitoneal injections of EGFR tyrosine kinase inhibitor AG1478 ((+/+)NTS+AG1478). Values are mean ± s.e! .m. (n = 6–8 per group). *P < 0.05 versus controls at baseline; **P < 0.01 versus controls at baseline; ##P < 0.01 versus mice treated with vehicle only. () Immunofluorescence staining for pEGFR in renal cortex from CT, (+/+)NTS, (−/−)NTS and (+/+)NTS+AG1478 mice on day 8 after NTS administration. Scale bars (orange), 25 μm. * Figure 2: HB-EGF induces a migratory phenotype in podocytes in vitro. () Podocyte outgrowth over 6 d from decapsulated glomeruli of WT (+/+) or Hbegf−/− (−/−) mice (arrow). Cultures were incubated with either AG1478 (AG) or HB-EGF or vehicle. Cells are stained for WT-1 expression. () Outgrowth area from glomeruli of mice carrying functional Hbegf alleles (light blue bar) was suppressed by 500 nM of AG (dark blue bar). Sparse outgrowth from glomeruli of Hbegf−/− mice (light gray bar) was rescued by addition of 30 nM HB-EGF (black bar). () Schematic of podocyte outgrowth from isolated glomeruli, used as migration-proliferation assay to assess crescent formation in vitro. Podocytes are in a stationary state (blue) on the surface of capillary loops (gray circle) when glomeruli are plated. Subsequently, podocytes assume a migratory phenotype (orange) characterized by apical protrusions, by attachment and by migration on the substratum. Later stages of outgrowth also involve proliferation. () Representative image of F-actin reorganizatio! n and formation of RiLiSs induced by HB-EGF (30 nM for 7 min) in differentiated podocytes, with or without AG (500 nM). Induction of apical protrusions by HB-EGF is abrogated with AG (500 nM). () Quantitative analysis of RiLiS formation in differentiated podocytes. HB-EGF (30 nM) was added without (Ctl) or with inhibitors AG (500 nM), Wortmannin (Wo, 100 nM), LY294002 (LY, 30 μM), PD98059 (PD, 25 μM) and SB203580 (SB, 25 μM). () BrdU incorporation in differentiated podocytes over 48 h. () Distance of migration of differentiated podocytes within 8 h in wound assay. Data are mean ± s.e.m. (n = 3 or 4 experiments). *P < 0.05 versus untreated WT glomeruli in and versus HB-EGF alone in –. Scale bar, 300 μm in and 30 μm in . * Figure 3: Deletion of Hbegf gene prevents fatal renal destruction. () Survival curve for challenged WT and Hbegf−/− mice. () Masson's trichrome staining of kidneys and proportion of crescentic glomeruli in control mice and in NTS-injected WT and Hbegf−/− mice (day 8 after NTS; Cr, crescents; G, glomeruli; Tc, tubules with proteinaceous casts). Scale bars, 50 μm. (–) Ascites score as index of albumin plasma loss and water and sodium retention (), albuminuria () and blood urea nitrogen concentrations () in NTS-challenged WT and Hbegf−/− animals on day 8 after NTS, and in unchallenged controls (CT). Values are mean ± s.e.m. (n = 9–12 per group). *P < 0.05 versus controls at baseline; **P < 0.01 versus baseline; ***P < 0.001 versus baseline. #P < 0.05 versus NTS-treated+/+; ##P < 0.01 versus NTS-treated+/+. * Figure 4: Selective deletion of Egfr from podocytes protects from RPGN. (–) Albuminuria () blood urea nitrogen concentration () and proportion of crescentic glomeruli () in Pod-Tet on-Cre Egfrwt/wt and Pod-Tet on-Cre EgfrloxP/loxP mice at 8 d after NTS-induced RPGN (*P < 0.05 for all comparisons). () Survival curve of challenged Pod-Tet on-Cre EgfrloxP/loxP and littermate control mice in a severe model of RPGN. *P < 0.01. () Ultrastructural analysis of podocytes by transmission electron microscopy (TEM) in NTS-treated Pod-Tet on-Cre Egfrwt/wt and Pod-Tet on-Cre EgfrloxP/loxP mice. Scale bars, top, 2 μm; bottom, 1 μm. * Figure 5: Delayed EGFR tyrosine kinase inhibition stops development of crescentic RPGN. () Quantification by western blot analysis of pEGFR and total EGFR in renal cortex from nonchallenged controls (CT), NTS-injected mice treated with vehicle alone and NTS-injected mice treated with erlotinib either started 12 h before administration of NTS (days 0–14) or in a curative protocol, started 4 d later (days 4–14). Mice were killed after 14 d of RPGN. (,) Blood urea nitrogen concentration () and proportion of crescentic glomeruli in CT () in groups of mice as in . Data are mean ± s.e.m., n = 9 mice per group. **P < 0.01 versus controls at baseline (CT); ***P < 0.001 versus CT; ##P < 0.01 versus vehicle; ###P < 0.001 versus vehicle. () Ultrastructural analysis of podocytes by TEM in erlotinib-treated and vehicle-treated mice at 5 d after injection of NTS. Scale bars, 2 μm. () Masson's trichrome staining of renal cortex from a mouse treated with erlotinib (days 4–14; left) and a vehicle-treated mouse (right) on day 14. Ne, necrotic glomerular lesions; Cr, cell! ular crescents; Tc, tubular proteinaceous casts; Infilt, diffuse CD3+ cell infiltrates in vehicle-treated mice. Scale bars, 100 μm. * Figure 6: HB-EGF expression is induced in human crescentic glomerulonephritis. Representative images of immunostaining for HB-EGF using monoclonal sc-74526 antibody in sections of kidney biopsies from eight random subjects diagnosed with noncrescentic glomerulopathies (top), including diabetic nephropathy, amyloidosis, minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), IgA nephropathy (IgAN) and membranous nephropathy (MN). Bottom, immunostaining for HB-EGF in renal biopsies from eight random subjects with RPGN of various etiologies, including lupus nephritis, microscopic polyangiitis (MP), endocarditis (End), Goodpasture disease (Gp) and Wegener disease (Wg). Scale bars, 50 μm. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Guillaume Bollée & * Martin Flamant Affiliations * Unité Mixte de Recherche (UMR) 970, Paris Cardiovascular Research Centre, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France. * Guillaume Bollée, * Cécile Fligny, * Marine Milon & * Pierre-Louis Tharaux * Université Paris Descartes, Sorbonne Paris Cité, Paris, France. * Guillaume Bollée, * Cécile Fligny, * Marine Milon & * Pierre-Louis Tharaux * Université Paris Diderot, Sorbonne Paris Cité, Paris, France. * Martin Flamant & * Nathalie Sabaa * Service de Physiologie–Explorations Fonctionnelles, Hôpital Bichat, Assistance Publique–Hôpitaux de Paris (AP-HP), Paris, France. * Martin Flamant & * Nathalie Sabaa * Institut für Anatomie und Zellbiologie, Universitätsmedizin Greifswald, Greifswald, Germany. * Sandra Schordan, * Elisabeth Rumpel, * Eric Schordan, * Karlhans Endlich & * Nicole Endlich * INSERM UMR702, Hôpital Tenon, Paris, France. * Sophie Vandermeersch, * Jean-Claude Dussaule & * Laurent Mesnard * Université Pierre et Marie Curie, Sorbonne Universités, Paris, France. * Sophie Vandermeersch, * Jean-Claude Dussaule, * Laurent Mesnard & * Patrice Callard * Centre for Interdisciplinary Research in Biology, Centre National de la Recherche Scientifique UMR 7241, INSERM U1050, Paris, France. * Ariane Galaup & * Stéphane Germain * Chaire de Médecine Expérimentale, Collège de France, Paris, France. * Ariane Galaup & * Stéphane Germain * Service d'Anatomie et de Cytologie Pathologiques, AP-HP, Hôpital Tenon, Paris, France. * Anita Rodenas & * Patrice Callard * INSERM UMR 1004, Institut André Lwoff, Hôpital Paul Brousse, Villejuif, France. * Ibrahim Casal & * Claude Boucheix * Université Paris-Sud, Villejuif, France. * Ibrahim Casal & * Claude Boucheix * The Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA. * Susan W Sunnarborg * Renal Section, Boston University Medical Center, Boston, Massachusetts, USA. * David J Salant * Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA. * Jeffrey B Kopp * Department of Genetics, North Carolina State University, Raleigh, North Carolina, USA. * David W Threadgill * St. Michael's Hospital, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. * Susan E Quaggin * Service de Physiologie-Exploration Fonctionnelles, AP-HP, Hôpital Saint-Antoine, Paris, France. * Jean-Claude Dussaule * Service de Néphrologie et Dialyse, Centre Hospitalier Intercommunal André Grégoire, Montreuil, France. * Xavier Belenfant * Service de Néphrologie, Hôpital Européen Georges Pompidou, Assistance Publique–Hôpitaux de Paris, Paris, France. * Pierre-Louis Tharaux Contributions M.F., G.B. and P.-L.T. conceived the project and experiments. P.-L.T. and N.E. supervised the project. S.S., C.F., M.M., S.V. and E.S. developed methods to culture and analyze primary podocytes and conceived experiments for gene expression analysis. E.R. and M.M. carried out electron microscopy (EM) studies. S.W.S., S.E.Q., J.B.K., D.W.T., I.C. and C.B. helped generate mice with targeted deficiency of HBEGF, TGF-α, epiregulin and Egfr. A.G. and S.G. carried out in situ hybridization studies. D.J.S. and L.M. provided nephrotoxic serum and discussed data with P.-L.T. K.E., C.B. and J.-C.D. also discussed experiments with P.-L.T. and N.E. P.-L.T., G.B., M.M., C.F. and N.S. carried out all in vivo studies. M.F., A.R. and P.C. analyzed human kidney biopsies collected by X.B. G.B. and M.F. contributed equally to the study. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Pierre-Louis Tharaux Author Details * Guillaume Bollée Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Flamant Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra Schordan Search for this author in: * NPG journals * PubMed * Google Scholar * Cécile Fligny Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Rumpel Search for this author in: * NPG journals * PubMed * Google Scholar * Marine Milon Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Schordan Search for this author in: * NPG journals * PubMed * Google Scholar * Nathalie Sabaa Search for this author in: * NPG journals * PubMed * Google Scholar * Sophie Vandermeersch Search for this author in: * NPG journals * PubMed * Google Scholar * Ariane Galaup Search for this author in: * NPG journals * PubMed * Google Scholar * Anita Rodenas Search for this author in: * NPG journals * PubMed * Google Scholar * Ibrahim Casal Search for this author in: * NPG journals * PubMed * Google Scholar * Susan W Sunnarborg Search for this author in: * NPG journals * PubMed * Google Scholar * David J Salant Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey B Kopp Search for this author in: * NPG journals * PubMed * Google Scholar * David W Threadgill Search for this author in: * NPG journals * PubMed * Google Scholar * Susan E Quaggin Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Claude Dussaule Search for this author in: * NPG journals * PubMed * Google Scholar * Stéphane Germain Search for this author in: * NPG journals * PubMed * Google Scholar * Laurent Mesnard Search for this author in: * NPG journals * PubMed * Google Scholar * Karlhans Endlich Search for this author in: * NPG journals * PubMed * Google Scholar * Claude Boucheix Search for this author in: * NPG journals * PubMed * Google Scholar * Xavier Belenfant Search for this author in: * NPG journals * PubMed * Google Scholar * Patrice Callard Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Endlich Search for this author in: * NPG journals * PubMed * Google Scholar * Pierre-Louis Tharaux Contact Pierre-Louis Tharaux Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–8 and Supplementary Methods Additional data
• p38 MAPK–mediated regulation of Xbp1s is crucial for glucose homeostasis
  - Nat Med 17(10):1251-1260 (2011)
  Nature Medicine | Article p38 MAPK–mediated regulation of Xbp1s is crucial for glucose homeostasis * Jaemin Lee1, 3 * Cheng Sun1, 3 * Yingjiang Zhou1 * Justin Lee1 * Deniz Gokalp1 * Hilde Herrema1 * Sang Won Park1 * Roger J Davis2 * Umut Ozcan1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1251–1260Year published:(2011)DOI:doi:10.1038/nm.2449Received11 May 2011Accepted20 July 2011Published online04 September 2011 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 show that p38 mitogen-activated protein kinase (p38 MAPK) phosphorylates the spliced form of X-box binding protein 1 (Xbp1s) on its Thr48 and Ser61 residues and greatly enhances its nuclear migration in mice, whereas mutation of either residue to alanine substantially reduces its nuclear translocation and activity. We also show that p38 MAPK activity is markedly reduced in the livers of obese mice compared with lean mice. Further, we show that activation of p38 MAPK by expression of constitutively active MAP kinase kinase 6 (MKK6Glu) greatly enhances nuclear translocation of Xbp1s, reduces endoplasmic reticulum stress and establishes euglycemia in severely obese and diabetic mice. Hence, our results define a crucial role for phosphorylation on Thr48 and Ser61 of Xbp1s in the maintenance of glucose homeostasis in obesity, and they suggest that p38 MAPK activation in the livers of obese mice could lead to a new therapeutic approach to the treatment of type 2 diabetes. View full text Figures at a glance * Figure 1: SAPK signaling increases Xbp1s mRNA stability and nuclear translocation. () Xbp1s and actin protein levels in the MEFs infected with adenoviruses expressing LacZ (Ad-LacZ) or Xbp1s (Ad-Xbp1s) and treated with anisomycin (ANS). (,) Xbp1s and actin immunoblotting in MEFs infected with Ad-LacZ or Ad-Xbp1s and treated with TNF-α. Both a dose-response to ANS () and to TNF-α () and a time course under a constant dose of ANS () and TNF-α () were examined. () Xbp1s and actin protein levels in MEFs infected with Ad-LacZ or Ad-Xbp1s, treated with vehicle or ANS then subjected to cycloheximide (CHX). () Xbp1s protein/actin ratio before and after CHX treatment. () Xbp1s mRNA levels in MEFs after ANS stimulation. We used18S Ribosome RNA (Rn18s) for normalization of gene expression. () Xbp1s mRNA levels after addition of actinomycin D to the MEFs infected with Ad-Xbp1s and stimulated with ANS. We used Rn18s for normalization of gene expression. () Cytoplasmic and nuclear Xbp1s protein levels from MEFs infected with Ad-Xbp1s and exposed to ANS. The graph adj! acent to each blot depicts the ratio of Xbp1s in ANS- versus vehicle-treated cells. We used actin as a loading control for whole-cell or cytoplasmic immunoblots and lamin A/C as a control for nuclear lysates. Each experiment was independently reproduced three times. Error bars are ± s.e.m. Significance was determined by two-way analysis of variance (ANOVA) with Bonferroni multiple-comparison analysis (,) or Student's t test (). AU, arbitrary units. **P < 0.01, ***P < 0.001. * Figure 2: p38 MAPK increases mRNA stability of Xbp1s through activation of MK2. () Xbp1s, phospho-c-Jun (p-cJun) and actin levels from Ad-LacZ– or Ad-Xbp1s–infected MEFs that were pre-treated with JNK inhibitor VIII (JNKi) and subsequently subjected to ANS. () Xbp1s, p-cJun, phospho-p38 MAPK (p-p38), p38 MAPK and actin levels in Ad-LacZ– or Ad-Xbp1s–infected cells that were treated with ANS. The genotype of each cell type is indicated, along with its respective wild-type (WT) control. () Xbp1s, p-cJun and actin protein levels in HEK293 cells transfected with Xbp1s or co-transfected with Xbp1s and MKK7-JNK1 plasmids. () Xbp1s, phospho-ATF2 (p-ATF2), p-cJun and actin levels in Ad-LacZ– or Ad-Xbp1s–infected MEFs that were pretreated with SB203580 and then subjected to ANS. () Xbp1s, p-cJun, p-p38, p38 MAPK and tubulin levels in Ad-LacZ– or Ad-Xbp1s–infected cells that were treated with ANS. The genotype of each cell type is indicated, along with its respective wild-type (WT) control. () Xbp1s, p-ATF2, p-p38, p38 and actin levels in HEK293 c! ells transfected with Xbp1s or MKK6Glu, or co-transfected with MKK6Glu and Xbp1s together and treated with SB203580 or vehicle. () Xbp1s mRNA levels from Ad-Xbp1s–infected MEFs that were pretreated with SB203580, stimulated with ANS and subjected to actinomycin D. () Xbp1s, p-p38, p38, MK2 and tubulin levels in WT and Mapkapk2−/− cells infected with Ad-LacZ or Ad-Xbp1s and treated with ANS. () mRNA levels of Xbp1s from WT and Mapkapk2−/− cells infected with Ad-Xbp1s and treated with ANS. We used Rn18s for normalization of gene expression. () Xbp1s mRNA levels from Ad-Xbp1s–infected WT and Mapkapk2−/− MEFs stimulated either with vehicle or ANS and treated with actinomycin D. We used actin or tubulin was used as a loading control. Each experiment was independently done three times. Error bars are ± s.e.m. Significance was determined by two-way ANOVA (), three-way ANOVA () with Bonferroni multiple-comparison analysis, or Student's t test (). *P < 0.05, ***P < ! 0.001. * Figure 3: p38 MAPK phosphorylates Xbp1s at Thr48 and Ser61. () MS/MS analysis of Xbp1s after anisomycin stimulation. (,) p-Xbp1sThr48, p-Xbp1sSer61, Xbp1s and actin levels in ANS-stimulated HEK293 cells that were transfected with wild-type (WT) or mutant Xbp1s-T48A () or mutant Xbp1s-S61A (). () Coomassie blue staining (left) and western blotting (WB, right) for recombinant His-TF-Xbp1s protein. () 32P-labeled levels of phosphoryated Xbp1s, p38-α and ATF2 and total Xbp1s, p38-α and ATF2 protein levels from samples of p38 MAPK in vitro kinase assay. () p-Xbp1sThr48, p-Xbp1sSer61 and Xbp1s protein levels of His-TF-Xbp1s subjected to the p38 MAPK in vitro kinase assay. () Xbp1s, actin and lamin A/C protein levels in whole cell, cytoplasmic and nuclear extracts of HEK293 cells that we transfected with indicated Xbp1s plasmids and further stimulated with vehicle or ANS. Each experiment was independently reproduced three times. #, nonspecific band. We used actin as a loading control for whole-cell or cytoplasmic immunoblots and lamin A/C! as a control for nuclear lysates. * Figure 4: Inhibition of p38 MAPK blocks Xbp1s nuclear translocation. (,) Eight-week-old wild-type (WT) lean male mice () or age-matched ob/ob males () were fasted for 24 h and refed for 1 h. p-Xbp1sThr48, p-Xbp1sSer61, Xbp1s, p-p38, p38 and tubulin levels in total extracts and Xbp1s and lamin A/C protein levels in nuclear extracts of the liver. (–) Eight-week-old WT lean male mice were injected i.p. with SB203580 (2 mg kg−1 per day) for 3 d. Subsequently, mice were fasted for 24 h and refed for 1 h. () p-Xbp1sThr48, p-Xbp1sSer61, Xbp1s, p-ATF2 and tubulin levels in total extracts and Xbp1s and lamin A/C protein levels in nuclear extracts of the liver. () Xbp1s () and Hspa5 () mRNA levels at fasting and refed conditions in the livers of the mice either treated with vehicle or SB203580. We used Rn18s was used for normalization of gene expression. We used tubulin as a loading control for whole cell lysates and lamin A/C for nuclear lysates. Each experiment was independently reproduced three times. Error bars are ± s.e.m. **P < 0.01, ***P < ! 0.001, Student's t test. NS, Nonsignificant. #, nonspecific band. * Figure 5: Reactivation of p38 MAPK in the liver of ob/ob mice greatly enhances Xbp1s nuclear translocation. () p-p38, p38 and actin levels in the liver, muscle and WAT of 8-week-old wild-type (WT) and ob/ob mice. () MKK6 and tubulin protein levels in MEFs infected with Ad-LacZ or Ad-MKK6Glu. () (left) Blood glucose levels on day 3 and (right) circulating insulin levels on day 7 after virus injections. (,) GTT () on day 5 and ITT () on day 3 after virus injections. AUC, Area under the curve. (,) MKK6, p-p38, p38, p-ATF2 and actin levels (), total Xbp1s and tubulin as well as nuclear Xbp1s, p-Xbp1sThr48, p-Xbp1sSer61 and lamin A/C levels () in liver. () Top, mRNA levels of Xbp1s, Hspa5 and Dnajb9; bottom, p-IRE1Ser724, total IRE1 and actin protein levels. We used Rn18s for normalization of gene expression, actin or tubulin as a loading control for whole cell lysates and lamin A/C for nuclear lysates. () Insulin receptor (IR) and IRS1 tyrosine and Akt Thr308 phosphorylations, together with their total protein levels after insulin infusion. Graphs next to the blots depict the ratio be! tween phosphorylated and total protein. IP, immunoprecipitation. () mRNA levels of Gck, Ppargc1a, G6pc and Pck1 in the livers. We used actin or tubulin as a loading control for whole cell lysates and lamin A/C for nuclear lysates. We used three independent groups of mice (n = 15 in each group). Error bars are ± s.e.m. Significance was determined by two-way ANOVA with Bonferroni multiple-comparison analysis () or Student's t test . *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 6: Xbp1s–T48A-S61A cannot migrate to the nucleus in the liver and regulate glucose homeostasis. (–) Six-hour fasting blood glucose levels on day 3 (), GTT on day 5 () and ITT on day 7 () after adenovirus injections. () Xbp1s protein levels in the whole and nuclear liver extracts of the mice on day 9 after virus injections. () Xbp1s () and Hspa5 and Dnajb9 mRNA levels () in the liver. We used Rn18s for normalization of gene expression. We used tubulin as a loading control for whole cell lysates and lamin A/C for nuclear lysates. We used three independent groups of mice (n = 15 in each group). Error bars are ± s.e.m. Significance was determined by one-way ANOVA (except in ; two-way ANOVA) with Bonferroni multiple-comparison analysis. *P < 0.05, **P < 0.01, ***P < 0.001. NS, nonsignificant. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jaemin Lee & * Cheng Sun Affiliations * Division of Endocrinology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Jaemin Lee, * Cheng Sun, * Yingjiang Zhou, * Justin Lee, * Deniz Gokalp, * Hilde Herrema, * Sang Won Park & * Umut Ozcan * Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Roger J Davis Contributions Jaemin Lee and C.S. designed and carried out the experiments, analyzed the data and wrote the manuscript. Y.Z., Justin Lee, D.G., H.H., S.W.P. did the experiments. R.J.D. provided reagents and advice through out the project. U.O. developed the hypothesis, designed experiments, analyzed the data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Umut Ozcan Author Details * Jaemin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng Sun Search for this author in: * NPG journals * PubMed * Google Scholar * Yingjiang Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Justin Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Deniz Gokalp Search for this author in: * NPG journals * PubMed * Google Scholar * Hilde Herrema Search for this author in: * NPG journals * PubMed * Google Scholar * Sang Won Park Search for this author in: * NPG journals * PubMed * Google Scholar * Roger J Davis Search for this author in: * NPG journals * PubMed * Google Scholar * Umut Ozcan Contact Umut Ozcan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 and Supplementary Methods Additional data
• A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis
  - Nat Med 17(10):1261-1268 (2011)
  Nature Medicine | Article A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis * Kari A Sweeney1, 2 * Dee N Dao1, 2, 6 * Michael F Goldberg2, 6 * Tsungda Hsu1, 2 * Manjunatha M Venkataswamy2 * Marcela Henao-Tamayo3 * Diane Ordway3 * Rani S Sellers4 * Paras Jain1, 2 * Bing Chen1, 2 * Mei Chen1, 2 * John Kim1, 2 * Regy Lukose1, 2 * John Chan2, 5 * Ian M Orme3 * Steven A Porcelli2, 5 * William R Jacobs Jr1, 2, 5 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1261–1268Year published:(2011)DOI:doi:10.1038/nm.2420Received18 April 2011Accepted14 June 2011Published online04 September 2011 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 We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosisesx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4+ memory T cells and involved a di! stinct shift in the pattern of cytokine responses by CD4+ cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis. View full text Figures at a glance * Figure 1: Role of the esx-3 region in evasion of innate immunity by M. smegmatis in a high-dose infection model. () Serum concentrations of IL-12 p40, IL-6 and IFN-γ in mice (C57BL/6) infected intravenously with 5 × 107 CFU per mouse of parental Msmeg (strain mc2155), IKE (Δesx-3) or Δesx-1 strains. () Survival after intravenous inoculation of 5 × 107 CFU parental Msmeg or IKE in C57BL/6 (n = 10), Rag1−/− (n = 5) or Myd88−/− (n = 5) mice. Survival was significantly different for IKE versus parental Msmeg in C57BL/6 and Rag1−/− mice (P < 0.001, log-rank test), but not in Myd88−/− mice. () CFU in lungs and kidneys of C57BL/6 mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE infection. () CFU in lungs and kidneys of Myd88−/− mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. For , and , data are mean ± s.e.m. of three mice per group. † indicates de! ath or killing owing to extreme morbidity of all mice in a group; *P < 0.01 (analysis of variance (ANOVA)). Data for all panels are representative of six independent experiments. * Figure 2: Characterization of innate immune responses against the Msmeg IKEPLUS strain. () Serum concentrations of IL-12 p40, IL-12p70 and IFN-γ in C57BL/6 mice infected with IKE or IKEPLUS (*P < 0.01, ANOVA with Bonferroni post-test). () Growth of bacteria (CFU) in the lungs (left) and kidneys (right) of Rag1−/− mice after intravenous inoculation of Msmeg parental, IKE or IKEPLUS strains. Differences in CFU were statistically significant (P < 0.001, two-way ANOVA) for Msmeg versus either IKE or IKEPLUS at day 3. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE and IKEPLUS. () Average time to death of mice after intravenous inoculation of IKEPLUS in C57BL/6 (n = 10), Rag1−/− (n = 5) and Myd88−/− (n = 5) mice. Survival of wild-type C57BL/6 and Rag1−/− strains was significantly different than that of Myd88−/− mice (P < 0.05, log-rank test). All inoculations were done at a high intravenous dose (5 × 107 CFU per mouse). Data from and are mean ± s.e.m. of three mice per group! . Data for all panels are representative of four independent experiments. * Figure 3: Bactericidal immunity against Mtb in mice vaccinated with IKEPLUS. () Survival of C57BL/6 mice that were sham immunized (intravenous (i.v.) PBS injection; n = 21) or immunized by intravenous infection with IKEPLUS (5 × 107 CFU per mouse; n = 20) or by subcutaneous (s.c.) infection with BCG (1 × 107 CFU per mouse; n = 18) and subsequently challenged 8 weeks later with a high intravenous dose (1 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival were significant for PBS versus BCG (P = 0.0389, log-rank test), PBS versus IKEPLUS (P < 0.0001, log-rank test) or BCG versus IKEPLUS (P < 0.0001, log-rank test). () Measurement of CFU from the lungs, spleens and livers of C57BL/6 mice in a separate experiment in which mice were immunized and challenged as described in . Each symbol represents one mouse. # at base of the y axis for the liver CFU plot indicates that no colonies were obtained from IKEPLUS-vaccinated mice at day 202, consistent with clearance of Mtb infection in that tissue (entire organs were plated to give a limit of detecti! on of 1 CFU). The CFU at day 100 was not significantly different between BCG- or IKEPLUS-immunized mice in any organ (P > 0.05, ANOVA). Data shown are pooled from two independent experiments. () Survival and lung CFU of C57BL/6 mice that were sham immunized (i.v. PBS; n = 6) or immunized by intravenous infection with IKE (5 × 107 CFU per mouse; n = 6) or IKEPLUS (5 × 107 CFU per mouse; n = 6) and subsequently challenged 6 weeks later with a high intravenous dose (5 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival curves were significant for PBS versus IKEPLUS (P = 0.0007, log-rank test), PBS versus IKE (P = 0.0049, log-rank test) and IKEPLUS versus IKE (P = 0.0246, log-rank test). For lung CFU, asterisks indicate significant differences (P < 0.05, two-way ANOVA) compared to the PBS control group. Results shown are representative of four independent experiments. * Figure 4: Improvement of histopathology in IKEPLUS-immunized mice during resolution of Mtb infection. () Representative images are shown for lungs (left) and livers (right) of C57BL/6 mice immunized with IKEPLUS and challenged with Mtb as described in Figure 3. Top and middle, H&E staining; bottom, AFB staining. Scale bars correspond to 650 nm, 40 nm and 10 nm in top, middle and bottom rows, respectively. The asterisk indicates an area of residual dense granulomatous infiltrate. Filled arrowheads indicate occasional macrophages in open alveoli in the lungs of mice with resolving inflammation at day 202. Open arrowheads mark granulomatous foci in the liver, which were also substantially resolved by day 202. AFBs were visualized in macrophages in lung and liver sections at both time points. () Quantitative scoring of histopathology confirming that all three groups showed similar pathology in the lungs and liver at day 100 (P > 0.05, two-way ANOVA). However, at day 202 the survivors from the IKEPLUS-immunized group showed significantly reduced pathology scores (P < 0.05 compare! d to all other groups at day 100; one-way ANOVA with Tukey post-test). * Figure 5: Protection from Mtb challenge by subcutaneous immunization with IKEPLUS. () Survival (left) of C57BL/6 mice (n = 10–14 per group) immunized subcutaneously with PBS, IKEPLUS (1 × 108 CFU per mouse), or BCG (1 × 106 CFU per mouse) and challenged 5 weeks later with a high intravenous dose (1 × 108 CFU per mouse) of Mtb H37Rv. CFU (right) data from the same experiment show means of three mice per group. The survival curve for IKEPLUS-immunized mice was significantly different from that of the BCG-immunized mice (P < 0.001, log-rank test). The CFU levels in lungs of IKEPLUS-immunized mice were significantly different from those of surviving saline-immunized mice at days 14 and 20 (P < 0.01, two-way ANOVA) and compared to BCG-immunized mice at day 20 (P < 0.01, two-way ANOVA). () Survival (left) of mice immunized subcutaneously with PBS (n = 10), 1 × 106 CFU of BCG (n = 10) or 1 × 108 CFU of IKEPLUS (n = 9) and challenged 1 month later with ~100 CFU of aerosolized Mtb H37Rv. The survival curves for IKEPLUS- and BCG-immunized mice were significan! tly different from that of PBS-immunized mice (P < 0.05, log-rank test). The survival curve for IKEPLUS was not significantly different from that of BCG-immunized mice (P = 0.0898, log-rank test). Lung CFU (right) from three mice per group from the same experiment showed that the IKEPLUS- and BCG-vaccinated groups had significantly reduced CFU compared to saline-immunized mice at days 21, 56 and 112 (P < 0.05, two-way ANOVA). IKEPLUS-immunized mice also showed lower CFU versus saline- or BCG-immunized mice at day 175 (*P < 0.05, two-way ANOVA). Results are representative of three independent experiments. * Figure 6: Role of CD4+ T cells in IKEPLUS-induced protective immunity. () Contributions of CD4+ and CD8+ subsets, as assessed by adoptive transfer of T cells from IKEPLUS-immunized mice. Naive recipient mice (n = 5 per group) were challenged 1 d after T cell transfer with M. tuberculosis (H37Rv, 1 × 107 CFU i.v. per mouse). Left, lung CFU of Mtb for mice killed 3 weeks after Mtb challenge. The dashed line indicates mean CFU in the lungs of five mice that were directly immunized with IKEPLUS (5 × 107 CFU per mouse i.v.) at 3 weeks before challenge. A negative control group of five sham-immunized mice that did not receive T cell transfer before challenge had CFU levels that were not significantly different from those of recipients of T cells from saline-immunized donors (not shown). *P < 0.05 compared to negative control group (ANOVA). This experiment was performed three times with similar results. Right, survival curves from similarly treated groups (n = 5) of mice from one experiment. IKEPLUS CD4+, adoptive transfer of CD4+ T cells from IKEPL! US-immunized donors; BCG CD4+, transfer of CD4+ T cells from BCG-immunized donors. Mice immunized intravenously with IKEPLUS directly or injected with PBS only were included as controls. The IKEPLUS CD4+ group was significantly different from the PBS group (P = 0.035, log-rank test) but not significantly different from the directly IKEPLUS-immunized group (P = 0.3177). () Cytokine production by CD4+ T cells in the lungs of mice immunized intravenously with PBS (sham) or IKEPLUS (5 × 107 CFU per mouse) or subcutaneously with BCG (1 × 106 CFU per mouse) and challenged 8 weeks later with Mtb H37Rv (1 × 107 CFU i.v.) (n = 5 mice per time point). The graphs indicate the absolute numbers of CD4+ T cells staining positively for three, two or one of the cytokines analyzed (IFN-γ, TNF and IL-2) after re-stimulation in vitro with a peptide representing the immunodominant epitope of TB9.8. Significant differences between the IKEPLUS- and BCG-vaccinated groups are indicated (*P < 0! .05, **P < 0.01, ***P < 0.001, two-way ANOVA). Results shown a! re representative of two similar experiments. () Same as in except that the cells were re-stimulated with plate-bound monoclonal antibodies to CD3 and CD28 before analysis. () Pie charts showing the relative fractions of CD4+ T cells at 14 d after Mtb challenge producing three, two or one of the cytokines with either specific antigen re-stimulation in vitro (TB9.8 peptide) or polyclonal activation (anti-CD3). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Dee N Dao & * Michael F Goldberg Affiliations * Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA. * Kari A Sweeney, * Dee N Dao, * Tsungda Hsu, * Paras Jain, * Bing Chen, * Mei Chen, * John Kim, * Regy Lukose & * William R Jacobs Jr * Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA. * Kari A Sweeney, * Dee N Dao, * Michael F Goldberg, * Tsungda Hsu, * Manjunatha M Venkataswamy, * Paras Jain, * Bing Chen, * Mei Chen, * John Kim, * Regy Lukose, * John Chan, * Steven A Porcelli & * William R Jacobs Jr * Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA. * Marcela Henao-Tamayo, * Diane Ordway & * Ian M Orme * Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA. * Rani S Sellers * Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA. * John Chan, * Steven A Porcelli & * William R Jacobs Jr Contributions K.A.S. constructed bacterial strains, performed or contributed to the design of most experiments, and analyzed and interpreted data. D.N.D. carried out portions of the infection and challenge experiments. M.F.G. contributed to the T cell adoptive transfer studies and designed, performed and analyzed all flow cytometry analyses. T.H. participated in design and construction of bacterial strains and in the performance of mouse infection and challenge experiments. P.J. contributed to construction of bacterial strains. M.M.V. and M.H.-T. assisted with experiments analyzing responding T cell populations. R.S.S. analyzed and scored the histopathology samples. B.C., M.C., J.K. and R.L. carried out mouse infections, organ harvesting and quantification of bacilli in tissues. D.O., J.C., I.M.O., S.A.P. and W.R.J. Jr. designed and interpreted experiments. K.A.S., S.A.P. and W.R.J. Jr. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * William R Jacobs Jr Author Details * Kari A Sweeney Search for this author in: * NPG journals * PubMed * Google Scholar * Dee N Dao Search for this author in: * NPG journals * PubMed * Google Scholar * Michael F Goldberg Search for this author in: * NPG journals * PubMed * Google Scholar * Tsungda Hsu Search for this author in: * NPG journals * PubMed * Google Scholar * Manjunatha M Venkataswamy Search for this author in: * NPG journals * PubMed * Google Scholar * Marcela Henao-Tamayo Search for this author in: * NPG journals * PubMed * Google Scholar * Diane Ordway Search for this author in: * NPG journals * PubMed * Google Scholar * Rani S Sellers Search for this author in: * NPG journals * PubMed * Google Scholar * Paras Jain Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Mei Chen Search for this author in: * NPG journals * PubMed * Google Scholar * John Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Regy Lukose Search for this author in: * NPG journals * PubMed * Google Scholar * John Chan Search for this author in: * NPG journals * PubMed * Google Scholar * Ian M Orme Search for this author in: * NPG journals * PubMed * Google Scholar * Steven A Porcelli Search for this author in: * NPG journals * PubMed * Google Scholar * William R Jacobs Jr Contact William R Jacobs Jr Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9, Supplementary Tables 1–3 and Supplementary Methods Additional data
• Glutamate release by primary brain tumors induces epileptic activity
  - Nat Med 17(10):1269-1274 (2011)
  Nature Medicine | Article Glutamate release by primary brain tumors induces epileptic activity * Susan C Buckingham1 * Susan L Campbell1 * Brian R Haas1 * Vedrana Montana1 * Stefanie Robel1 * Toyin Ogunrinu1 * Harald Sontheimer1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1269–1274Year published:(2011)DOI:doi:10.1038/nm.2453Received05 May 2011Accepted27 July 2011Published online11 September 2011 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 Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into severe combined immunodeficient mice. Within 14–18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system xc− cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration–approved drug that blocks system xc−. We found that acute administration of SAS at concentrations equivalent to those used to treat Crohn's disease in humans red! uced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans. View full text Figures at a glance * Figure 1: Tumor-bearing mice show abnormal spontaneous EEG events indicative of epileptic activity. () Representative EEG recordings from three glioma-implanted mice, juxtaposing abnormal events and baselines for each. () A power spectrum from one representative event (inset) and the corresponding baseline from the same mouse. () Frequency of epileptic activity in 13 tumor-bearing mice quantified over 10 consecutive d; hourly event frequency is plotted as a function of time. () A U251-GFP tumor identified in cortical brain by EGFP fluorescence before conducting glutamate release assays. Scale bar, 1 mm. () Extracellular glutamate, released in the presence of 100 μM cystine (Cys), comparing acute cortical brain slices from sham-operated and U251-GFP-implanted mice in the presence and absence of 250 μM SAS. Error bars show means ± s.e.m. *P < 0.05, **P < 0.01. * Figure 2: Acute cortical slices from tumor-bearing mice show spontaneous epileptiform activity. (,) Cresyl violet–stained brain slices from a sham () and a glioma-implanted mouse (). Recording (Rec) and stimulating (Stim) electrodes were placed in peritumoral regions and in corresponding regions of sham slices as shown. Scale bars, 350 μm. () Higher-magnification image of the tumor shown in (, box). Scale bar, 150 μm. () Extracellular field recordings conducted in the presence of Mg2+ comparing spontaneous activity in a slice from a representative U251-GFP–bearing mouse to a slice from a sham animal. * Figure 3: Acute cortical slices from tumor-bearing mice are hyperexcitable. (,) Whole-cell recordings from representative neurons in a cortical slice from a sham-operated () versus tumor-bearing mouse () before (ACSF) and after removal of Mg2+ (Mg2+-free). Individual events are shown on an expanded time scale below each recording. () Mean latency to development of epileptiform activity comparing neurons from sham mice, to peritumoral neurons in animals implanted with U251-GFP, GBM12 and GBM22 tumors (*P < 0.05). () Mean event duration recorded in sham, U251, GBM12 and GBM22-containing brain slices. Error bars represent means ± s.e.m. * Figure 4: Cortical slices from glioma-bearing mice show increased cortical network activity and hyperexcitable layer 2 and 3 peritumoral pyramidal cells. () Representative examples of optical recordings comparing a slice from a sham and a slice from a U251-GFP–bearing mouse incubated in the voltage dye RH414 and then field stimulated with 80 μA. Each image is a pseudocolored representation of activity measured using a Neuroplex 464-diode array. Adjacent frames were recorded 1.8 ms apart. () The spread of voltage responses were measured by the number of activated diodes within the array. (,) Summary of RMP () and input resistance () recorded using whole-cell patch clamp in peritumoral neurons from U251-GFP, GBM12 and GBM22-implanted mice. () Examples of voltage responses to increasing amplitude current injections, from −100 pA to +80 pA (in 20-pA steps) in whole-cell current clamped pyramidal peritumoral neurons in glioma-bearing and in sham slices (pulse duration, 500 ms). () The average action potential number obtained in response to 20, 40, 60 and 80 pA depolarizing current pulses was plotted as a function of applied c! urrent yielding the input-output curves shown. Error bars show means ± s.e.m. *P < 0.05, ***P < 0.01. * Figure 5: SAS application reduces epileptiform activity in cortical slices from glioma-bearing mice. (,) Recordings from peritumoral neurons before (ACSF) and after removal of Mg2+ (Mg2+-free) followed by application of SAS and APV in mice bearing GBM12 () or GBM22 () tumors. () Mean response duration for peritumoral neurons recorded in U251, GBM12 and GBM22 containing brain slices comparing recordings in Mg2+-free medium to those in Mg2+-free plus SAS and Mg2+- free plus SAS and APV. Error bars show means ± s.e.m. () A cresyl violet–stained slice containing GBM22 shows the proximity of biocytin-filled recorded neuron (right inset, middle box) to the darker-stained tumor. Scale bars, 150 μm (left and middle); 20 μm (right inset). * Figure 6: Sulfasalazine reduces frequency of epileptic activity in tumor-bearing mice. () The average number of epileptic events for eight SAS-treated and six PBS-treated tumor bearing mice is shown for the 4-h period before and the 4-h period after treatment. () The average hourly event frequency is plotted for eight SAS-treated mice compared with six PBS-treated mice before and after treatment. Injection time is indicated by '0'. () The number of events that occurred for each hour during the 8-h SAS treatment block (4 h before and 4 h after) is shown for one mouse; hash marks separate 3 consecutive d and arrowheads indicate the time of SAS injection. Error bars show means ± s.e.m. **P < 0.05; ***P < 0.01. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA. * Susan C Buckingham, * Susan L Campbell, * Brian R Haas, * Vedrana Montana, * Stefanie Robel, * Toyin Ogunrinu & * Harald Sontheimer Contributions S.C.B. and S.L.C. acquired the majority of the data presented. B.R.H. was instrumental in mouse surgeries and statistical analyses of data. V.M. (supported by an American Brain Tumor Association Basic Research Fellowship) carried out glutamate release assays. S.R. assisted in electrophysiological recordings. T.O. did western blotting and glutamate uptake assays. H.S. designed experiments, supervised all research and co-wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Harald Sontheimer Author Details * Susan C Buckingham Search for this author in: * NPG journals * PubMed * Google Scholar * Susan L Campbell Search for this author in: * NPG journals * PubMed * Google Scholar * Brian R Haas Search for this author in: * NPG journals * PubMed * Google Scholar * Vedrana Montana Search for this author in: * NPG journals * PubMed * Google Scholar * Stefanie Robel Search for this author in: * NPG journals * PubMed * Google Scholar * Toyin Ogunrinu Search for this author in: * NPG journals * PubMed * Google Scholar * Harald Sontheimer Contact Harald Sontheimer Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 and Supplementary Methods Additional data
• Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155
  - Nat Med 17(10):1275-1282 (2011)
  Nature Medicine | Article Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155 * Suhwan Chang1 * Rui-Hong Wang2 * Keiko Akagi3 * Kyung-Ae Kim1 * Betty K Martin4 * Luca Cavallone5, 6 * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab)7 * Diana C Haines8 * Mark Basik5 * Phuong Mai9 * Elizabeth Poggi10 * Claudine Isaacs10 * Lai M Looi11 * Kein S Mun11 * Mark H Greene9 * Stephen W Byers10 * Soo H Teo12, 13 * Chu-Xia Deng2 * Shyam K Sharan7 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1275–1282Year published:(2011)DOI:doi:10.1038/nm.2459Received13 October 2010Accepted03 August 2011Published online25 September 2011 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 BRCA1, a well-known tumor suppressor with multiple interacting partners, is predicted to have diverse biological functions. However, so far its only well-established role is in the repair of damaged DNA and cell cycle regulation. In this regard, the etiopathological study of low-penetrant variants of BRCA1 provides an opportunity to uncover its other physiologically important functions. Using this rationale, we studied the R1699Q variant of BRCA1, a potentially moderate-risk variant, and found that it does not impair DNA damage repair but abrogates the repression of microRNA-155 (miR-155), a bona fide oncomir. Mechanistically, we found that BRCA1 epigenetically represses miR-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miR-155 promoter. We show that overexpression of miR-155 accelerates but the knockdown of miR-155 attenuates the growth of tumor cell lines in vivo. Our findings demonstrate a new mode of tumor suppression by BRC! A1 and suggest that miR-155 is a potential therapeutic target for BRCA1-deficient tumors. View full text Figures at a glance * Figure 1: R1699Q mutant ES cells show reduced survival and differentiation defects. () Schematics of generation of R1699Q ES cells using PL2F8 cells containing a null and a conditional allele of Brca1. Two halves of human HPRT1 minigenes (HP and RT) flanking the two loxP sites (shaded triangles) of the conditional allele. Cre recombinants are HAT resistant (HATR). () Southern hybridization of HATR colonies from experiments without BAC (NO BAC), wild-type (WT) BRCA1 BAC and R1699Q BRCA1 BAC. Bottom band, null allele (MT); top band, conditional allele (cko). Rescue rate, percentage Brca1ko/ko clones. Asterisk, Brca1ko/ko ES cell. () Whole mount of embryoid bodies generated from ES cells expressing wild-type (left) and R1699Q (right) BRCA1 at day 14 in culture. Bottom, higher magnification of embryoid bodies. Scale bars, 50 μm, top; 20 μm, bottom. () H&E staining of embryoid bodies generated from ES cells expressing wild-type (left) and R1699Q (right) BRCA1 at day 14 in culture. Scale bars, 100 μm, top; 50 μm, bottom. () TUNEL staining of embryoid bodies. ! Arrow, TUNEL+ cells. Scale bars, 50 μm. () Teratoma growth of one wild-type and two R1699Q clones were examined in mice (n = 5 for each group). Values are means ± s.e.m. (P = 0.007). () H&E staining of teratomas dissected 15 d after injection. Top, section of the whole teratoma; middle, magnified images of the regions indicated at top. Arrows, neurorosette structures. Bottom, neural cells immature in wild-type (left) and more differentiated in R1699Q (right) teratomas. Scale bars, 2 μm, top; 0.2 μm, middle; 50 μm, bottom. * Figure 2: Identification of miR-155 upregulation in R1699Q mutant cells and its effect on ES cell differentiation. () Quantification of miR-155 by rtPCR in wild-type (WT), R1699Q (RQ) and M1652I (MI) ES cells and embryoid bodies (EB cells) on day 7 of culture. U6 small nuclear RNA (snRNA) was used for normalization. Top, BRCA1 protein expression. () Representative pictures of miR-155 in situ hybridization in wild-type and R1699Q embryoid bodies using DIG-labeled antisense LNA of miR-155. Top, no-probe controls showing background signal (scale bar, 50 μm). Right, relative average signal. Error bars indicate s.d. () Schematic of miR-155 inducible system in ES cells. miR-155 is induced by tetracycline, which then represses the luciferase reporter (luc) containing miR-155-binding sites at the 3′ end. () miR-155 overexpression in ES cells after tetracycline (tet) induction, with U6 snRNA as control. Top, overexpression by northern hybridization; bottom, quantification by rtPCR. Error bars indicate s.d. () Representative pictures of embryoid bodies generated from wild-type ES cells with ind! uced expression of miR-155. Top, whole mount of embryoid bodies; bottom, H&E-stained sections. Scale bars, 100 μm. () Teratoma growth of wild-type ES cells with (Tet1–Tet5) and without (Con1–Con5) miR-155 induction. Left, teratoma growth. Right, average tumor volume on day 18 after injection. Minimum value of each group was excluded (n = 4; values are means ± s.e.m.). () Representative picture of teratomas in mice injected with control cells (−Tet, right) and Tet-induced cells (+Tet, left). Bottom, enlarged view. * Figure 3: BRCA1 negatively controls miR-155 expression. () Quantification of miR-155 in tumors from the Brca1ko/+;Trp53ko/+;TgR1699Q mice (RQ, n = 9). Normal liver (RQ93 nor), mammary gland (RQ515 nor) and tumors from Brca1ko/+;Trp53ko/+;TgM1652I mice (MI, n = 4) used as control. () Quantification of miR-155 in a breast tumor from an R1699Q mutation carrier. Normal, normal breast tissue. () Expression analysis of miR-155 by northern hybridization in four human breast cancer cell lines of different BRCA1 genotypes. Positive control, HEK293 cell line overexpressing miR-155; loading control, U6 snRNA; asterisk, miR-155 signal. Right, quantification of signal, normalized by U6 snRNA. () rtPCR of miR-155 in four Brca1 deficient tumors (42, 572, 907 and M161) from Brca1cko/cko;p53ko/+;MMTV Cre mice and four tumors (Con 112X, Con 172X, Con I107 and Con I224) from Her2/Neu transgenic mice. MCF7 and HCC1937 were negative and positive control, respectively. () rtPCR of miR-155 in MECs from two Brca1cko/cko;K14-Cre mice. Control, MECs from ! K14 Cre only and Brca1cko/cko. () Knockdown of BRCA1 in two clones (75 and 80) of HEK293 cells stably expressing BRCA1 short hairpin RNA (top). Loading control, β-actin (middle). Bottom, rtPCR quantification of miR-155 in the BRCA1 knockdown clones (75 and 80). Con, control. () Real-time quantification of miR-155 after ectopic expression of wild-type (WT) or R1699Q BRCA1 in BRCA1-deficient MDA-MB-436 cells. Top, expression of wild-type and R1699Q BRCA1. () miR-155 reporter assay in BRCA1-deficient HCC1937 cells using increasing amounts of wild-type BRCA1 cDNA (Con, untransfected cells and BRCA50, 50 ng; BRCA100, 100 ng). Error bars indicate s.d. * Figure 4: Mechanism of miR-155 repression by BRCA1. () Binding of BRCA1 to miR-155 promoter at two potential regions (BRCA1-1 and BRCA1-2). ES cells and embryoid bodies (EB) at day 7 in culture with wild-type and R1699Q (RQ). BRCA1 by ChIP assay. Input, 5% of total chromatin. () Effect of mutation of putative BRCA1-binding sites (Mut1, Mut2 or double mutant) on miR-155 promoter activation measured by luciferase assay (*P < 0.05, **P < 0.01, ***P < 0.005). () Effect of HDAC inhibitors on miR-155 expression measured by rtPCR in BRCA1+ MDA-MB-468 (left) and BRCA1-deficient MDA-MB-436 cells (right). Con, no treatment; Aph, apicidin. () Effect of HDAC inhibitors on wild-type miR-155 promoter (left) and mutant promoter (right) activation in MDA-MB-468 cells measured by luciferase assay (*P < 0.02, **P < 0.01, ***P < 0.005). Con, no treatment; Aph, apicidin. () ChIP assay to quantify acetylation level of histones H2A and H3 on miR-155 promoter, in wild-type and R1699Q EB cells by rtPCR. () Effect of wild-type, RQ and wild-type + RQ ! BRCA1 on acetylation of histones H2A and H3 on miR-155 promoter in MDA-MB-436 cells measured by ChIP assay and rtPCR. Con, transfection with vector only (*P < 0.02, **P < 0.01). Western blot (WB) analysis of ectopic protein expression using antibody to hemagglutinin (HA). () Association of HDAC2 on miR-155 promoter in wild-type and R1699Q EB cells analyzed by ChIP assay and rtPCR. IgG, negative control. () Co-immunoprecipitation (Co-IP) between HDAC2 and wild-type or M1652I or R1699Q BRCA1 in MECs from mice with wild-type or R1699Q BAC transgenes (left) or M1652I transgene (right). Top, IP with HDAC2 antibody; bottom, IP with human-specific BRCA1 antibody. () MDA-MB 468 cells transfected with luciferase reporter plasmid with mouse wild-type or double mutant (MT) miR-155 promoter (pro). ChIP assay was carried out with indicated antibodies (Ab). Association of BRCA1-HDAC2 complex and acetylation of histones H2A and H3 on the transfected mouse miR-155 promoter (mir155, top) an! d endogenous miR-155 promoter (MIR155). BRCA1 binding on ESRRG! , CCNB1 and STAT5A promoters with putative binding sites. Error bars indicate s.d. * Figure 5: Physiological relevance of miR-155 upregulation in BRCA1-deficient tumors. () Xenograft tumor growth of MDA-MB-468 cells with stable expression of miR-155 (P = 4.12 × 10−4, ANCOVA test). Values are mean ± s.e.m. () Growth of two clones (C9 and D6) with miR-155 stable knockdown and the parental cells (Con) orthotropically transplanted in mice (values are means ± s.e.m., *P = 0.031, **P = 0.025). () Average mass of tumors in (values are means ± s.e.m.). () Representative pictures of 66 human breast tumors in tumor tissue microarray probed with antibody to BRCA1 (Ab-1) or mouse IgG (negative control) and DIG-labeled antibody to miR-155. Scale bars, 50 μm. () rtPCR quantification of miR-155 in 28 tumors from non-BRCA1 controls and 14 tumors from BRCA1 mutation (mut) carriers (see Supplementary Table 6 for mutation description). RNU5A was used for normalization. Shaded area, low miR-155 based on twofold cut-off (dashed horizontal line). Text box, number of miR-155 high and low tumors in each group. () Schematic of role for BRCA1 in epigenetic con! trol of miR-155 promoter. In wild-type BRCA1–containing cells, miR-155 is silenced by the BRCA1-HDAC2 complex, which deacetylates H2A and H3 histones. Without functional BRCA1, the interaction of BRCA1-HDAC2 complex is disrupted, which in turn increases acetylated (Ac) H2A and H3, which activates the miR-155 promoter. Author information * Abstract * Author information * Supplementary information Affiliations * Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Suhwan Chang & * Kyung-Ae Kim * Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA. * Rui-Hong Wang & * Chu-Xia Deng * Human Cancer Genetics Program, Departments of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus, Ohio, USA. * Keiko Akagi * Science Applications International Corporation (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Betty K Martin * Lady Davis Institute, Jewish General Hospital, Montréal, Quebec, Canada. * Luca Cavallone & * Mark Basik * Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montréal, Quebec, Canada. * Luca Cavallone * The Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia. * $affiliationAuthor & * Shyam K Sharan * Pathology Histotechnology Laboratory, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, USA. * Diana C Haines * Clinical Genetics Branch, National Cancer Institute, Rockville, Maryland, USA. * Phuong Mai & * Mark H Greene * Georgetown-Lombardi Comprehensive Cancer Center Georgetown University, Washington, DC, USA. * Elizabeth Poggi, * Claudine Isaacs & * Stephen W Byers * Department of Pathology, University Malaya Medical Center, Kuala Lumpur, Malaysia. * Lai M Looi & * Kein S Mun * Department of Surgery University Malaya Cancer Research Institute, University Malaya Medical Centre, Kuala Lumpur, Malaysia. * Soo H Teo * Cancer Research Initiatives Foundation, Sime Darby Medical Centre, Kuala Lumpur, Malaysia. * Soo H Teo Consortia * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) Contributions S.C. conceived the idea, conducted all the experiments and wrote the manuscript, R.-H.W. and C.-X.D. helped with xenograft experiment, provided mouse tumor samples and cell lines, K.A. carried out bioinformatics analysis, K.-A.K. helped with experiments, B.K.M. helped with mouse work, D.C.H. performed histopathological analysis, L.C. analyzed human tumor samples, M.B., P.M., M.H.G., KConFab, L.M.L., K.S.M., S.H.T., E.P., C.I. and S.W.B. provided human tumor samples, S.K.S. conceived the idea, supervised the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Shyam K Sharan Author Details * Suhwan Chang Search for this author in: * NPG journals * PubMed * Google Scholar * Rui-Hong Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Keiko Akagi Search for this author in: * NPG journals * PubMed * Google Scholar * Kyung-Ae Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Betty K Martin Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Cavallone Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) * Diana C Haines Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Basik Search for this author in: * NPG journals * PubMed * Google Scholar * Phuong Mai Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Poggi Search for this author in: * NPG journals * PubMed * Google Scholar * Claudine Isaacs Search for this author in: * NPG journals * PubMed * Google Scholar * Lai M Looi Search for this author in: * NPG journals * PubMed * Google Scholar * Kein S Mun Search for this author in: * NPG journals * PubMed * Google Scholar * Mark H Greene Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen W Byers Search for this author in: * NPG journals * PubMed * Google Scholar * Soo H Teo Search for this author in: * NPG journals * PubMed * Google Scholar * Chu-Xia Deng Search for this author in: * NPG journals * PubMed * Google Scholar * Shyam K Sharan Contact Shyam K Sharan Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10, Supplementary Tables 1–6, Supplementary Methods and Supplementary Discussion Additional data
• Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis
  - Nat Med 17(10):1283-1289 (2011)
  Nature Medicine | Article Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis * Coralie Dorard1, 2 * Aurélie de Thonel3, 4 * Ada Collura1, 2 * Laetitia Marisa5 * Magali Svrcek1, 2, 6 * Anaïs Lagrange1, 2 * Gaetan Jego3, 4 * Kristell Wanherdrick1, 2 * Anne Laure Joly3, 4 * Olivier Buhard1, 2 * Jessica Gobbo3, 4 * Virginie Penard-Lacronique7 * Habib Zouali8 * Emmanuel Tubacher8 * Sylvain Kirzin9 * Janick Selves9 * Gérard Milano10 * Marie-Christine Etienne-Grimaldi10 * Leila Bengrine-Lefèvre11 * Christophe Louvet11 * Christophe Tournigand11 * Jérémie H Lefèvre2, 12 * Yann Parc2, 12 * Emmanuel Tiret2, 12 * Jean-François Fléjou1, 2, 6, 13 * Marie-Pierre Gaub14 * Carmen Garrido3, 4, 15, 16 * Alex Duval1, 2, 16 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:1283–1289Year published:(2011)DOI:doi:10.1038/nm.2457Received23 February 2011Accepted01 August 2011Published online25 September 2011 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 Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and tre! atment response in CRC. View full text Figures at a glance * Figure 1: Identification of HSP110 as a new target gene for frequent mutation in MSI CRC cell lines and primary tumors. () Principle of the study we performed using RT-PCR to find exon-skipping events due to MSI in CRC cell lines. n = the number of analyzed exons. IR, intronic repeat. () BET staining of RT-PCR product in agarose gel detecting the presence of a specific, additional HSP110 band in MSI CRC cell lines. Its size implied exon 9 skipping in HSP110 mRNA. We did not detect its expression in all MSS cell lines tested. The results were confirmed in a series of primary CRCs (T) that were MSI or MSS, respectively, and their matching normal mucosa (N). () Allelic profiles for several MSI CRC cell lines and primary tumors and controls (MSS CRC and LBL cell lines) at the intronic HSP110 T17. MSS samples were weakly polymorphic, whereas MSI CRC cell lines and primary tumors always showed aberrant alleles that fell outside the polymorphic zone (in orange). Numbers indicate the size of the HSP110 T17 deletion in MSI tumor samples (in base pairs). () Mutation of the intronic HSP110 T17 repeat wa! s more frequent in MSI primary CRCs (100%) and in premalignant adenomas (53%) than that of any known coding microsatellite alteration. Numbers indicate the size of the HSP110 T17 deletion in MSI tumor or adenoma samples. * Figure 2: Expression of HSP110ΔE9 relatively to mutational status of HSP110 T17 sequence in MSI CRC. () Amplification plots corresponding to HSP110wt and HSP110ΔE9 RT-PCR products. Results are expressed (E) as n-fold difference in HSP110ΔE9 relative to HSP110wt expression (ΔCt), where ΔCt was determined in each case by subtracting the average Ct value of the HSP110ΔE9 mRNA from the average Ct value of the HSP110wt mRNA. () Values of E ratio are shown for the 13 MSI CRC cell lines and 43 primary tumor samples. MSS LBLs (n = 20), MSS CRC cell lines (n = 7; ALA, COLO320, SW480, FET, GLY, FRI, EB) and MSS primary CRCs (n = 20) were also tested as controls. A significant difference was observed between MSI and MSS primary CRCs with respect to HSP110ΔE9 expression (P = 8.5 × 10–5; Student's t test). Medium E values are indicated by red bars in each case. () Black and red bars correspond to CRCs in which HSP110ΔE9 mRNA expression was low or high, respectively (below or above the median value calculated in our tumor series; see also Supplementary Table 2). The size of the! T17 deletion was significantly different between these two groups of tumors (P = 1.1 × 10–13; Student's t test). () Two different HSP110 antibodies (Abs) were used in western blots—one recognizing the C-terminal part of the protein and detecting the α and β HSP110 isoforms (upper blot) and the other targeting the N-terminal part and detecting HSP110ΔE9 (this Ab detects HSP110βE9 only weakly). Error bars correspond to the s.d. of measured densitometric values. *P < 0.05. a.u., arbitrary units. * Figure 3: HSP110ΔE9 is a dominant-negative mutant that binds to HSP110 and blocks its chaperone function. () Protein aggregation was evaluated in heated extracts from MEF HSF1− cells transfected with HSP70, HSP110wt, HSP110ΔE9, and HSP110wt and HSP110ΔE9 combined, as reported26. Each bar is the mean value of four different experiments. *P < 0.05. () Immunoprecipitation (IP) with HA antibody (HSPs) was followed by immunoblotting with GFP antibody (HSP110 proteins) in lysates from HCT116 cells co-transfected with a GFP-tagged HSP110wt or HSP110ΔE9 (left) or an empty GFP vector (right) together with the indicated hemagglutinin (HA)-tagged HSP. () Immunoprecipitation of HSP110wt, using an antibody that recognizes the C terminus of HSP110, was followed by immunoblotting with GFP antibody in lysates from HCT116 cells transfected with GFP-tagged HSP110wt and/or HSP110ΔE9. Inputs, protein level in total cell lysates. () Top, fluorescence microscopic analysis in HCT116 cells of GFP-tagged HSP110wt or HSP110ΔE9 (green) and nuclei (blue). Scale bars, 0.5 μm. Bottom, cell fractionat! ion studies in HCT116 cells transfected with GFP-tagged HSP110wt or HSP110ΔE9. HSP90 serves as cytosolic marker. () We did cell fractionation studies on HCT116 cells transfected with GFP-tagged HSP110wt, HSP110ΔE9 or both (HSP11wt + HSP110ΔE9). Exp1 and Exp2 constitute two independent transfection experiments. HSP90 serves as a cytosolic marker and histone H3 as a nuclear marker. * Figure 4: HSP110ΔE9 has an antitumor effect in xenografts, blocks the HSP110 antiapoptotic effect and sensitizes cancer cells to die. () Antitumor effect of HSP110ΔE9 in xenografts. Mean tumor volumes were measured in nude mice (± s.d., six mice per group. *P < 0.05) injected with HCT116 cells transfected either with a GFP vector (open circles) or GFP-tagged HSP110ΔE9 (filled circles). Inset, expression of the transfected proteins. HSP70 serves as a loading control. () Apoptosis was measured by immunodetection of caspase 8 (CASP8) and PARP cleavage (left), or by fluorescence-activated cell sorting (FACS) analysis of caspase 3 activity (right), in HCT116 cells transfected with GFP-tagged HSP110wt, HSP110ΔE9 or an empty vector (GFP) and treated with recombinant TRAIL ligand. () FACS analysis of apoptosis in GFP-positive (GFPpos, transfected with a GFP-empty vector or HSP110ΔE9) and GFP-negative (GFPneg, non transfected) HCT116 cells treated with TRAIL. () The percentage of apoptosis (chromatin condensation) induced by TRAIL was determined in HCT116 cells transfected with GFP-tagged HSP110wt and increase! d doses of HSP110ΔE9 or an empty vector (GFP). () Immunoblot of caspase 8 (CASP8) and PARP cleavage in LoVo cells transfected as described in and treated, when indicated, with TRAIL ligand. () HCT116 (MSI) or SW480 (MSS) cells transfected with HSP110wt (1.5 μg) were co-transfected with two different doses (c1, 0.5 μg and c2, 1 μg) of either GFP-empty vector or HSP110ΔE9. After treatment with oxaliplatin (Oxa, 40 μM, 48 h), apoptosis was assessed by FACS analysis as in . *P < 0.05. a.u., arbitrary units. * Figure 5: Clinical impact of HSP110ΔE9 expression in people with MSI CRC. Kaplan-Meier univariate analyses of disease-free survival (DFS) in persons with stage 2 or stage 3 MSI CRC are shown according to their HSP110ΔE9 expression. For statistical analyses, only the first 5 years are shown. Subjects from both series were combined using series-specific cutoff points to define HSP110ΔE9 expression classes (75% and 50% for first and second series, respectively). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Carmen Garrido & * Alex Duval Affiliations * Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche Saint-Antoine, Equipe 'Instabilité des Microsatellites et Cancers', Paris, France. * Coralie Dorard, * Ada Collura, * Magali Svrcek, * Anaïs Lagrange, * Kristell Wanherdrick, * Olivier Buhard, * Jean-François Fléjou & * Alex Duval * Université Pierre et Marie Curie Paris, Paris, France. * Coralie Dorard, * Ada Collura, * Magali Svrcek, * Anaïs Lagrange, * Kristell Wanherdrick, * Olivier Buhard, * Jérémie H Lefèvre, * Yann Parc, * Emmanuel Tiret, * Jean-François Fléjou & * Alex Duval * INSERM, Dijon, France. * Aurélie de Thonel, * Gaetan Jego, * Anne Laure Joly, * Jessica Gobbo & * Carmen Garrido * University of Burgundy, Dijon, France. * Aurélie de Thonel, * Gaetan Jego, * Anne Laure Joly, * Jessica Gobbo & * Carmen Garrido * Programme 'Cartes d'Identité des Tumeurs', Ligue Nationale Contre le Cancer, Paris, France. * Laetitia Marisa * Assistance Publique–Hôpitaux de Paris (AP-HP), Hôpital Saint-Antoine, Service d'Anatomie et Cytologie Pathologiques, Paris, France. * Magali Svrcek & * Jean-François Fléjou * INSERM, Institute Gustave Roussy, Villejuif, France. * Virginie Penard-Lacronique * Centre d'Etude du Polymorphisme Humain, Fondation Jean Dausset, Institut de Génétique Moléculaire, Paris, France. * Habib Zouali & * Emmanuel Tubacher * INSERM, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France. * Sylvain Kirzin & * Janick Selves * Laboratoire d'Oncopharmacologie, Centre Antoine Lacassagne, Nice, France. * Gérard Milano & * Marie-Christine Etienne-Grimaldi * Service d'Oncologie Médicale, Hôpital Saint-Antoine, AP-HP, Paris, France. * Leila Bengrine-Lefèvre, * Christophe Louvet & * Christophe Tournigand * AP-HP, Service de Chirurgie Générale et Digestive, Hôpital Saint-Antoine, Paris, France. * Jérémie H Lefèvre, * Yann Parc & * Emmanuel Tiret * AP-HP, Hôpital Saint-Antoine, Tumorothèque Cancer Est, Paris, France. * Jean-François Fléjou * INSERM, Développement et Physiopathologie de l'Intestin et du Pancréas, Strasbourg, France. * Marie-Pierre Gaub * Centre Hospitalier Universitaire Dijon, Dijon, France. * Carmen Garrido Contributions C.D. carried out analyses of aberrant splicing events due to MSI in CRC and genetic study of HSP110 in CRC cells and primary tumors. A.d.T. carried out analyses of wild-type and mutated HSP110 chaperone functions in CRC cells. A.C., M.S., A.L., K.W. and O.B. assisted with the mutational screening of primary CRC. J.G. (with G.J. and A.L.J.) carried out mouse work. L.M. carried out the clinical study and survival analyses. H.Z. and E. Tubacher assisted with the in silico search of candidate genes containing intronic microsatellite sequences. V.P.-L., S.K., J.S., G.M., M.-C.E.-G., L.B.-L., C.L., C.T., J.H.L., Y.P., E. Tiret, J.-F.F. and M.-P.G. provided CRC samples and clinical data. A.D. and C.G. conceived the project, coordinated and directed the study, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Carmen Garrido or * Alex Duval Author Details * Coralie Dorard Search for this author in: * NPG journals * PubMed * Google Scholar * Aurélie de Thonel Search for this author in: * NPG journals * PubMed * Google Scholar * Ada Collura Search for this author in: * NPG journals * PubMed * Google Scholar * Laetitia Marisa Search for this author in: * NPG journals * PubMed * Google Scholar * Magali Svrcek Search for this author in: * NPG journals * PubMed * Google Scholar * Anaïs Lagrange Search for this author in: * NPG journals * PubMed * Google Scholar * Gaetan Jego Search for this author in: * NPG journals * PubMed * Google Scholar * Kristell Wanherdrick Search for this author in: * NPG journals * PubMed * Google Scholar * Anne Laure Joly Search for this author in: * NPG journals * PubMed * Google Scholar * Olivier Buhard Search for this author in: * NPG journals * PubMed * Google Scholar * Jessica Gobbo Search for this author in: * NPG journals * PubMed * Google Scholar * Virginie Penard-Lacronique Search for this author in: * NPG journals * PubMed * Google Scholar * Habib Zouali Search for this author in: * NPG journals * PubMed * Google Scholar * Emmanuel Tubacher Search for this author in: * NPG journals * PubMed * Google Scholar * Sylvain Kirzin Search for this author in: * NPG journals * PubMed * Google Scholar * Janick Selves Search for this author in: * NPG journals * PubMed * Google Scholar * Gérard Milano Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Christine Etienne-Grimaldi Search for this author in: * NPG journals * PubMed * Google Scholar * Leila Bengrine-Lefèvre Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Louvet Search for this author in: * NPG journals * PubMed * Google Scholar * Christophe Tournigand Search for this author in: * NPG journals * PubMed * Google Scholar * Jérémie H Lefèvre Search for this author in: * NPG journals * PubMed * Google Scholar * Yann Parc Search for this author in: * NPG journals * PubMed * Google Scholar * Emmanuel Tiret Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-François Fléjou Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Pierre Gaub Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen Garrido Contact Carmen Garrido Search for this author in: * NPG journals * PubMed * Google Scholar * Alex Duval Contact Alex Duval Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Information (3M) Supplementary Figures 1–5 and Supplementary Tables 1–4 Additional data
• A human memory T cell subset with stem cell–like properties
  - Nat Med 17(10):1290-1297 (2011)
  Nature Medicine | Article A human memory T cell subset with stem cell–like properties * Luca Gattinoni1, 9 * Enrico Lugli2, 9 * Yun Ji1 * Zoltan Pos3, 4 * Chrystal M Paulos5, 6 * Máire F Quigley7, 8 * Jorge R Almeida8 * Emma Gostick7 * Zhiya Yu1 * Carmine Carpenito5, 6 * Ena Wang3, 4 * Daniel C Douek8 * David A Price7, 8 * Carl H June5, 6 * Francesco M Marincola3, 4 * Mario Roederer2, 9 * Nicholas P Restifo1, 9 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:1290–1297Year published:(2011)DOI:doi:10.1038/nm.2446Received29 April 2011Accepted19 July 2011Published online18 September 2011 Abstract * Abstract * Accession codes * 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 Immunological memory is thought to depend on a stem cell–like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human st! em cell–like memory T cell population is of direct relevance to the design of vaccines and T cell therapies. View full text Figures at a glance * Figure 1: Identification of TSCM cells in human blood. () Flow cytometry analysis of sorted human CD45RO−CD62L+ naive CD8+ T cells before and 14 d after stimulation with α-CD3/CD28-coated beads and IL-2 in the presence or absence of 5 μM TWS119. Numbers indicate the percentage of cells in the CD45RO−CD62L+ gate. () Flow cytometry analysis of TWS119-generated CD45RO−CD62L+ naive-like CD8+ T cells overlaid with CD45RO−CD62L+ naive and memory (non-CD45RO−CD62L+) cells from a healthy donor (HD). () Flow cytometry analysis of PBMC from a healthy donor. Dot plots show the gating strategy to identify CD95+, IL-2Rβ+ TSCM cells. () Percentages of circulating CD8+ T cell subsets in 29 healthy donors. () Flow cytometry analysis of PBMC from a representative healthy donor. Overlaid histogram plots show expression levels of a given molecule in different CD8+ T cell subsets. CD8+ T cell subsets were defined as follows: TN cells, CD3+CD8+CD45RO−CCR7+CD45RA+CD62L+CD27+CD28+IL7Rα+CD95−; TSCM cells, CD3+CD8+CD45RO–CCR7+CD45RA+! CD62L+CD27+CD28+IL7Rα+CD95+; TCM cells, CD3+CD8+CD45RO+CD45RA−CCR7+CD62L+; TEM cells, CD3+CD8+CD45RO+CD45RA−CCR7−CD62L−. * Figure 2: TSCM cells possess attributes of conventional memory T cells. () TREC copy number in sorted CD8+ T cell subsets relative to TN cells. Data are represented as means ± s.e.m. of four donors. () Intracellular cytokine staining of PBMCs from a representative healthy donor after stimulation with SEB. Graphs show naive-like (NL) gated T cells. NL, CD45RO−CCR7+CD45RA+CD27+CD28+. Numbers represent the percentage of CD95+ (TSCM) and CD95− (TN) cells producing a single cytokine. () Percentages of CD8+ T cell subsets producing IFN-γ, IL-2 and TNF-α in six healthy donors (obtained as described in ). () Pie charts depicting the quality of the cytokine response in CD8+ T cell subsets in six healthy donors as determined by the Boolean combination of gates identifying IFN-γ+, IL-2+ and TNF-α+ cells. () Carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution in sorted CD8+ T cell subsets after stimulation with 25 ng ml−1 of IL-15 for 10 d. Data are shown after gating on CD8+ cells. PD, percentage divided; PI, proliferation index. SSC! , side scatter. (,) Percentage of divided cells () and proliferation index of different CD8+ T cell subsets (), after stimulation as in panel . Data are represented as means ± s.e.m. of 9 donors. () Flow cytometry analysis of PBMC from HLA-A2+ donors. Graphs show tetramer-binding cells versus CD95 expression in the NL (CD45RO−CCR7+CD45RA+CD27+IL7Rα+) gate. () Percentage of tetramer-binding cells expressing CD95 in the NL gate, determined as in panel . Data represent the donors tested for tetramer specificity. HD, healthy donor; MP, melanoma patient. () Frequency of two immunodominant CMV-specific TCRβ clonotypes relative to all CMV-specific TCRβ clonotypes in pp65-specific T cell subsets isolated over a period of 22 months from a representative donor. The CDR3β amino acid sequences are shown. Changes in the frequencies of immunodominant clonotypes are depicted as the thickness of the bars, with the magnitude scale shown on the right. *P < 0.05; **P < 0.01; ***P < 0.0! 01; NS, not significant (t test, ,,, and χ2 permutation test,! ). * Figure 3: TSCM cells represent a distinct, less-differentiated T cell memory subset. () Heat map of differentially expressed genes (P < 0.01; one-way repeated measures analysis of variance (ANOVA), false discovery rate <5%, Benjamini-Hochberg's method) among CD8+ T cell subsets. Red and blue colors indicate increased and decreased expression, respectively. () Robust multichip analysis (RMA)-normalized intensity of selected genes progressively downregulated (naive-associated genes) or upregulated (effector-associated genes) from TN cells TSCM cells TCM cells TEM cells. Data are represented as means ± s.e.m. of three donors. () MDS analysis of differentially expressed genes (P < 0.01, false discovery rate <5%). Numbers represent the differentially regulated genes among each CD8+ T cell subset (P < 0.01 (t test) and > twofold change in expression). () Heat map of differentially expressed genes among TSCM and TCM cells (P < 0.01 (t test) and > twofold change in expression). Red and blue colors indicate increased and decreased expression, respectively. Full gene! names are listed in the Supplementary Methods. * Figure 4: Enhanced self-renewal and multipotency of TSCM cells. () Percentage of CD8+ T cells expressing CCR7 and CD62L (right) and CD45RA (left) relative to cell division after exposure to 25 ng ml−1 of IL-15 for 10 d. Slopes were compared using a Wilcoxon rank test, *P = 0.0391. Pre, the phenotype of sorted CD8+ T cell subsets before stimulation. () Percentage of CFSE-diluted CD8+ T cells that retained the parental phenotype after stimulation with 25 ng ml−1 of IL-15 for 10 d. *P < 0.05; **P < 0.01 (t test). () Percentage of CD8+ T cells expressing CCR7 and CD62L (right) and CD45RA (left) relative to cell division after stimulation with α-CD3/CD2/CD28-coated beads for 6 d. Pre, the phenotype of sorted CD8+ T cell subsets before stimulation. () Percentage of CFSE-diluted CD8+ T cells with a given phenotype after stimulation with α-CD3/CD2/CD28-coated beads for 6 d. () Stemness index of CD8+ memory T cell subsets. *P < 0.05 (t test). Data are represented as means ± s.e.m. of eight (,), six (,) or four () donors. * Figure 5: Increased proliferative capacity, survival and antitumor activity of TSCM cells. () 3H-thymidine incorporation by sorted CD8+ T cell subsets after stimulation with α-CD3/CD2/CD28-coated beads. Data are represented as means ± s.e.m. of ten donors. Results are normalized to the number of seeded cells, as different cell numbers were obtained from different sorts. c.p.m., counts per min. *P < 0.05; **P < 0.01; ***P < 0.001 (t test). () Flow cytometry analysis of human T cells in the spleen, lymph nodes (LN) and liver of a representative NSG mouse at 4 weeks after adoptive transfer of CD4+ T cells (5 × 106) with or without sorted CD8+ T cell subsets (106). Graphs show T cells after gating on human CD45+ cells. Numbers indicate the percentage of cells in the CD4+CD8− or CD4−CD8+ gates. () Total human CD8+ T cell recovery in the spleens, LN and livers from six NSG mice 4 weeks after adoptive transfer of CD4+ T cells with or without sorted CD8+ T cell subsets. A total of six mice per T cell subset from two independent experiments (three replicate mice per! T cell subset per experiment) are shown. Horizontal lines indicate median values. *P < 0.05; **P < 0.01 (t test). (–) In vivo bioluminescent imaging (), percentage change of body weight (), and survival of NSG mice () bearing M108-luciferase mesothelioma after adoptive transfer of CD4+ T cells (106) with or without sorted CD8+ T cell subsets (3 × 106) expressing a mesothelin-specific chimeric antigen receptor. ***P < 0.001, one-way repeated measures ANOVA () and log-rank (Mantel-Cox) test (). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE23321 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Luca Gattinoni, * Enrico Lugli, * Mario Roederer & * Nicholas P Restifo Affiliations * Center for Cancer Research, National Cancer Institute, US National Institutes of Health (NIH), Bethesda, Maryland, USA. * Luca Gattinoni, * Yun Ji, * Zhiya Yu & * Nicholas P Restifo * ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Enrico Lugli & * Mario Roederer * Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, US National Institutes of Health, Bethesda, Maryland, USA. * Zoltan Pos, * Ena Wang & * Francesco M Marincola * Center for Human Immunology, US National Institutes of Health, Bethesda, Maryland, USA. * Zoltan Pos, * Ena Wang & * Francesco M Marincola * Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Chrystal M Paulos, * Carmine Carpenito & * Carl H June * Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Chrystal M Paulos, * Carmine Carpenito & * Carl H June * Department of Infection, Immunity and Biochemistry, Cardiff University School of Medicine, Heath Park, Cardiff, UK. * Máire F Quigley, * Emma Gostick & * David A Price * Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, US National Institutes of Health, Bethesda, Maryland, USA. * Máire F Quigley, * Jorge R Almeida, * Daniel C Douek & * David A Price Contributions L.G., E.L., Y.J., Z.P., C.M.P., J.R.A., Z.Y. and C.C. carried out experiments; L.G., E.L., Y.J., Z.P., C.M.P. and J.R.A. analyzed experiments; L.G., E.L., C.M.P., E.W., D.C.D., D.A.P., C.H.J., F.M.M., M.R. and N.P.R. designed experiments; E.G., M.F.Q. and D.A.P. contributed reagents; E.L. and M.R. edited the manuscript; and L.G. and N.P.R. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Luca Gattinoni or * Nicholas P Restifo Author Details * Luca Gattinoni Contact Luca Gattinoni Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico Lugli Search for this author in: * NPG journals * PubMed * Google Scholar * Yun Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Zoltan Pos Search for this author in: * NPG journals * PubMed * Google Scholar * Chrystal M Paulos Search for this author in: * NPG journals * PubMed * Google Scholar * Máire F Quigley Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge R Almeida Search for this author in: * NPG journals * PubMed * Google Scholar * Emma Gostick Search for this author in: * NPG journals * PubMed * Google Scholar * Zhiya Yu Search for this author in: * NPG journals * PubMed * Google Scholar * Carmine Carpenito Search for this author in: * NPG journals * PubMed * Google Scholar * Ena Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel C Douek Search for this author in: * NPG journals * PubMed * Google Scholar * David A Price Search for this author in: * NPG journals * PubMed * Google Scholar * Carl H June Search for this author in: * NPG journals * PubMed * Google Scholar * Francesco M Marincola Search for this author in: * NPG journals * PubMed * Google Scholar * Mario Roederer Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas P Restifo Contact Nicholas P Restifo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–15, Supplementary Tables 1–9 and Supplementary Methods Additional data
• Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells
  - Nat Med 17(10):1298-1303 (2011)
  Nature Medicine | Letter Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells * Barthelemy Diouf1, 2 * Qing Cheng1, 2 * Natalia F Krynetskaia1, 2 * Wenjian Yang1, 2 * Meyling Cheok1, 2 * Deqing Pei1, 3 * Yiping Fan4 * Cheng Cheng1, 3 * Evgeny Y Krynetskiy1, 2 * Hui Geng5 * Siying Chen5 * William E Thierfelder1, 2 * Charles G Mullighan1, 6 * James R Downing1, 6 * Peggy Hsieh5 * Ching-Hon Pui1, 6, 7 * Mary V Relling1, 2 * William E Evans1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1298–1303Year published:(2011)DOI:doi:10.1038/nm.2430Received02 November 2010Accepted05 July 2011Published online25 September 2011 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 DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (~11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic ! deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance. View full text Figures at a glance * Figure 1: Gene copy number loss and MSH2 protein expression in primary human leukemia cells. (,) MSH2 protein () and mRNA () expression measured in primary ALL cells isolated from bone marrow aspirates of 90 individuals newly diagnosed with ALL, constituting the discovery cohort. The filled circles indicate values in individuals. () We determined MSH2 protein levels by western blot analysis and normalized them to the GAPDH signal. We defined MSH2-L ALL cells as those with low MSH2 protein signals on a western blot of 1 million ALL cells (<8% RU). () Pathway of proteins upstream of PKCζ and MSH2 degradation. Depicted in color are four proteins corresponding to the genes that were deleted more frequently in MSH2-L leukemias. () dChip SNP array data analysis revealed overrepresentation of monoallelic copy number loss (yellow) in MSH2-L leukemia cells. () MSH2 protein levels in leukemia cells from the validation cohort (all individuals with deletions of one or more of these genes with sufficient cells for western analysis and a 2:1 matched cohort of individuals with AL! L but without these deletions). The horizontal lines in , and depict the median values for each cohort. * Figure 2: Protein expression in primary leukemia cells with hemizygous deletions, treatment outcome and drug sensitivity according to leukemia cell MSH2 phenotype. () Western blot analysis of two individuals with hemizygous deletions of FRAP1 and two individuals with hemizygous deletions of PRKCZ shows ~30–60% lower amounts of the corresponding protein compared to the matched individuals that did not have these deletions. (,) Kaplan-Meier analysis of overall survival () and cumulative incidence of hematological relapse () in children whose ALL cells had the MSH2-L ALL phenotype compared to those with the MSH2-H phenotype. The difference in overall 10-year survival is statistically significant (P = 0.009, log-rank test), whereas the difference in cumulative incidence of a hematological relapse approached the conventional level of statistical significance (P = 0.06, Gray's test). () CCRF-CEM cells in which FRAP1, HERC1, PIK3C2B or PRKCZ were knocked down (KD) had significantly greater resistance to the thiopurines antileukemic agents 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP) (P < 0.02). In contrast, these cells had increased sen! sitivity to the alkylating agent melphalan. * Figure 3: PRKCZ, PIK3C2B, HERC1 and FRAP1 inhibition and MSH2 stability. We transduced human leukemia cells (CCRF-CEM) with shRNA against PRKCZ, PIK3C2B, HERC1 or FRAP1 or with the non-target control. (–) Western blot analysis of the effect of these knockdowns on each protein knocked down and on MSH2 protein levels after HERC1 (), PRKCZ (), PIK3C2B () and FRAP1 () stable knockdown. () We measured phosphorylation of AKT (Ser473) and P70S6K1 (Thr389) by phospho-specific antibodies in ,, and . () Depicted is the decrease in MSH2 protein levels following treatment with increasing concentrations of rapamycin to inhibit FRAP1. () MSH2 protein levels after PRKCZ, PIK3C2B, HERC1 or FRAP1 inhibition as quantified by densitometry, normalized to GAPDH signal and expressed as a percent of the control. Values are means ± s.d. of three independent experiments. () To determine the half-life of MSH2 protein, we treated CCRF-CEM cells with 5 μg ml−1 cycloheximide to inhibit protein synthesis. () The results of Western blotting of MSH2 quantified by densitom! etry and normalized to GAPDH, expressed as a percent of the time zero value, showed a shorter half-life of MSH2 after PIK3C2B, PRKCZ, HERC1 and FRAP1 inhibition.() Inhibition of FRAP1 by rapamycin (rap) (300 nM) resulted in accumulation of ubiquitinated (ub) MSH2 protein in CEM cells after 48 h (); the lower MSH2 protein levels were reversed by the proteasome inhibitor MG132 (10 nM). Ab, antibody. * Figure 4: Increase in PP2A activity through inhibition or knockdown of FRAP1, HERC1 or PIK3C2B with rescue by okadaic acid (OA) and the effects on MMR activity. () Phosphatase activity as measured colorimetrically using threonine phosphopeptide, K-R-pT-I-R-R, as a substrate. Error bars represent the s.d. of three replicate experiments. () Increase of PP2A activity by FRAP1, HERC1 or PIK3C2B knockdown decreases PKCζ activation (phosphorylation) and MSH2 protein levels, both of which are rescued by okadaic acid. OA+ indicates incubation of the cells for 24 h with 10 nM of okadaic acid. () The phospho PKCζ signal quantified by densitometry is shown. Values are means ± s.d. of three independent experiments. () Using an M13mp2 DNA substrate containing a two-base loop, the repair efficiency is expressed as a percentage: 100× (the ratio of mixed colonies in the control group – the ratio of mixed colonies in repaired group)/(the ratio of mixed colonies in the control group). We did each experiment in triplicate; data represent the means ± s.d. of three separate determinations. () We depleted cytosolic extracts of MSH2 with antibody o! r mock-depleted them with mouse IgG. The data are averages from three (mock depleted and MSH2 depleted) experiments. The error bars represent the means ± s.d. of three independent experiments. () Graph showing DNA repair efficiency after immunodepletion of MSH2 or mock depletion with MSH2- and IgG-specific antibodies. Author information * Author information * Supplementary information Affiliations * Hematological Malignancies Program, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Barthelemy Diouf, * Qing Cheng, * Natalia F Krynetskaia, * Wenjian Yang, * Meyling Cheok, * Deqing Pei, * Cheng Cheng, * Evgeny Y Krynetskiy, * William E Thierfelder, * Charles G Mullighan, * James R Downing, * Ching-Hon Pui, * Mary V Relling & * William E Evans * Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Barthelemy Diouf, * Qing Cheng, * Natalia F Krynetskaia, * Wenjian Yang, * Meyling Cheok, * Evgeny Y Krynetskiy, * William E Thierfelder, * Mary V Relling & * William E Evans * Department of Biostatistics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Deqing Pei & * Cheng Cheng * Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Yiping Fan * Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health (NIH), Bethesda, Maryland, USA. * Hui Geng, * Siying Chen & * Peggy Hsieh * Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Charles G Mullighan, * James R Downing & * Ching-Hon Pui * Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. * Ching-Hon Pui Contributions W.E.E. designed and supervised experiments and their analyses and wrote the manuscript with B.D. B.D., Q.C., N.F.K., M.C., E.Y.K., H.G., S.C., P.H., W.E.T. and C.G.M. performed experiments and participated in their analyses. J.R.D., C.G.M. and M.V.R. directed experiments and contributed to the genomic analyses. D.P., Y.F. and C.C. performed the statistical analyses. W.Y. led the genomic analyses in collaboration with other authors. C.-H.P. led the clinical trials and provided the ALL samples. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * William E Evans Author Details * Barthelemy Diouf Search for this author in: * NPG journals * PubMed * Google Scholar * Qing Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Natalia F Krynetskaia Search for this author in: * NPG journals * PubMed * Google Scholar * Wenjian Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Meyling Cheok Search for this author in: * NPG journals * PubMed * Google Scholar * Deqing Pei Search for this author in: * NPG journals * PubMed * Google Scholar * Yiping Fan Search for this author in: * NPG journals * PubMed * Google Scholar * Cheng Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Evgeny Y Krynetskiy Search for this author in: * NPG journals * PubMed * Google Scholar * Hui Geng Search for this author in: * NPG journals * PubMed * Google Scholar * Siying Chen Search for this author in: * NPG journals * PubMed * Google Scholar * William E Thierfelder Search for this author in: * NPG journals * PubMed * Google Scholar * Charles G Mullighan Search for this author in: * NPG journals * PubMed * Google Scholar * James R Downing Search for this author in: * NPG journals * PubMed * Google Scholar * Peggy Hsieh Search for this author in: * NPG journals * PubMed * Google Scholar * Ching-Hon Pui Search for this author in: * NPG journals * PubMed * Google Scholar * Mary V Relling Search for this author in: * NPG journals * PubMed * Google Scholar * William E Evans Contact William E Evans Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10, Supplementary Tables 1–8 and Supplementary Methods Additional data
• The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension
  - Nat Med 17(10):1304-1309 (2011)
  Nature Medicine | Letter The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension * Ewout J Hoorn1, 6 * Stephen B Walsh2, 6 * James A McCormick1 * Antje Fürstenberg2 * Chao-Ling Yang1 * Tom Roeschel3 * Alexander Paliege3 * Alexander J Howie4 * James Conley1 * Sebastian Bachmann3 * Robert J Unwin2, 6 * David H Ellison1, 5, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1304–1309Year published:(2011)DOI:doi:10.1038/nm.2497Received01 April 2011Accepted15 July 2011Published online02 October 2011 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 Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use1, 2. These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, h! ydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment. View full text Figures at a glance * Figure 1: Effects of tacrolimus on arterial pressure and electrolyte handling in mice. () Effects of tacrolimus on systolic blood pressure (SBP) over a period of 14 days (d). We determined significant differences by two-way analysis of variance (ANOVA). () Effects of tacrolimus on 24-h urine sodium excretion. *P < 0.05 by ANOVA. () Effects of tacrolimus on 24-h urine potassium excretion. Significance was determined by unpaired t test. () Correlation between urine sodium excretion and SBP rise after tacrolimus treatment, calculated as the change from the average baseline value to the final-day SBP. Significance was determined using linear regression. () Comparison of the rise in SBP caused by tacrolimus (Tac) treatment during high-salt (HS) and normal-salt (NS) diets. During the baseline (BL) SBP measurements, both groups were fed the NS diet. The P value was obtained using two-way ANOVA to analyze whether the change in SBP in one group was significantly different from the change in SBP in the other group. () Effect of tacrolimus (Tac, n = 13) on plasma aldoste! rone concentrations (PAldo), compared with vehicle (Veh) (n = 5), during NS diet. HS suppressed plasma aldosterone despite Tac (n = 5). To illustrate an activated renin angiotensin system, plasma aldosterone concentrations are also shown in untreated wild-type mice fed a low-sodium diet (LS, n = 5). *P < 0.05, compared with NS + Veh. Significance was determined by unpaired t test. BW, body weight. Error bars are means ± s.e.m. * Figure 2: Effects of tacrolimus on transport proteins and kinases in kidney and in vitro. () Localization of calcineurin A-α in kidney; left images show immunohistochemical detection of calcineurin; right images show NCC in the same field delineating distal convoluted tubules. A control without primary antibody is shown for comparison. Dots (•) indicate distal convoluted tubules. Scale bars, 50 μm (top and bottom); 20 μm (middle). () Immunoblots showing effects of tacrolimus on the sodium chloride cotransporters (NCC and pNCC at ~130 kDa, and NKCC2 and pNKCC2 at ~140 kDa) and on the calcium channel TRPV5 (at ~90 kDa). Densitometry analysis (fold change compared to vehicle, normalized for actin) for each are shown at the right. Significance was determined by unpaired t test. () Immunoblots showing effects of tacrolimus on WNK3 (at ~200 kDa), WNK4 (at ~150 kDa) and SPAK. Densitometry analysis (fold change compared to vehicle, normalized for actin) is shown at the right; densitometry of SPAK was done by averaging all isoforms. Significance was determined by unp! aired t test. () Immunoblots showing effects of tacrolimus on HEK293 cells. NCC expression was undetectable when cells were not induced with tetracycline (Un). Induced cells were either untreated (0), or treated with vehicle (Veh) or with tacrolimus (Tac). Bands corresponding to total NCC were detected at ~130 kDa (*) and 110 kDa (#), indicating mature and immature forms, respectively, whereas the band corresponding to pNCC was detected only at 130 kDa. Representative immunoblots are shown. Densitometry was normalized for actin. Significance was determined by unpaired t test. NS, not significant. Error bars are means ± s.e.m. * Figure 3: Effects of tacrolimus on systolic blood pressure (SBP), sodium handling and NCC abundance in mice in which NCC was deleted, inhibited or overexpressed. () Effects of tacrolimus on SBP of NCC-knockout mice and littermates. Significance was determined by ANOVA. () Effect of treatment with hydrochlorothiazide (HCTZ) or vehicle (Veh) on established tacrolimus (Tac)-induced hypertension in wild-type mice. Statistical analysis was performed by unpaired t tests. () Effects of HCTZ on urine sodium to creatinine ratio (UNa/UCreat) in tacrolimus-treated animals (Tac + HCTZ) and in untreated mice (HCTZ only). For comparison, UNa/UCreat in tacrolimus-treated animals given vehicle is also shown. Each triangle, dot or circle indicates a single mouse. () Comparison of effects of tacrolimus treatment (TAC) on SBP in wild-type (WT) and transgenic mice overexpressing NCC (Tg(NCC)). Baseline (BL) and final-day SBPs are shown. The P value was obtained using two-way ANOVA to analyze whether the change in SBP from BL to the final day in one group was significantly different from the change in SBP in the other group. () Immunoblots comparing tota! l NCC and pNCC abundances from kidneys of vehicle (Veh)-treated WT mice and tacrolimus (Tac)-treated WT and Tg(NCC) mice. () Quantification of the effects of tacrolimus (Tac) on NCC and pNCC abundance in WT and TgNCC mice (n = 5 in each group). Significance was determined by ANOVA. NS, not significant. Error bars are means ± s.e.m. * Figure 4: Functional data and immunostaining in human subjects with CNI-induced hypertension compared with controls. () Comparison of effects of 10 mg bendroflumethiazide on fractional chloride excretion (ΔFECl) in kidney transplant recipients with tacrolimus-induced hypertension (Tacrolimus), healthy volunteers (Control) and kidney transplant recipients receiving sirolimus (Sirolimus). Significance was determined by ANOVA. () Comparison of extracellular fluid volume versus total body fluid volume (ECF/TBF) in kidney transplant patients with tacrolimus-induced hypertension compared with controls, measured by bioimpedance. Significance was determined by unpaired t test. () Comparison of the ratio of extracellular to total body water (ECW/TBW) in kidney transplant recipients with tacrolimus-induced hypertension compared with controls, measured by bioimpedance. Significance was determined by unpaired t test. () Comparison of plasma renin activity (PRA) in kidney transplant recipients with tacrolimus-induced hypertension compared with controls. Significance was determined by unpaired t test. ! () Comparison of plasma aldosterone concentration (PAldo) in kidney transplant recipients with tacrolimus-induced hypertension compared with controls. Significance was determined by unpaired t test. () Representative confocal immunofluorescence images of renal tissue showing NCC and pNCC in kidney transplant recipients with tacrolimus-induced hypertension compared with azathioprine-treated kidney transplant recipients and with healthy controls. Scale bars, 100 μm. Images from additional biopsy samples and clinical characteristics are provided in Supplementary Tables 4–6 and Supplementary Figure 3. Error bars are means ± s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ewout J Hoorn, * Stephen B Walsh, * Robert J Unwin & * David H Ellison Affiliations * Division of Nephrology & Hypertension, Oregon Health & Science University, Portland, Oregon, USA. * Ewout J Hoorn, * James A McCormick, * Chao-Ling Yang, * James Conley & * David H Ellison * UCL Centre for Nephrology, University College London, London, UK. * Stephen B Walsh, * Antje Fürstenberg & * Robert J Unwin * Department of Anatomy, Charité, Berlin, Germany. * Tom Roeschel, * Alexander Paliege & * Sebastian Bachmann * Department of Pathology, University College London, London, UK. * Alexander J Howie * Veterans Affairs Medical Center, Portland, Oregon, USA. * David H Ellison Contributions E.J.H. and S.B.W. carried out most of the experiments, analyzed the data and wrote the initial manuscript. J.A.M. generated the mice overexpressing the NCC and participated in animal experiments and analyses. J.C. did the aldosterone infusion experiments. A.F. contributed to the human experiments and, together with A.J.H., to the kidney biopsy tissue staining. C.-L.Y. conducted the cell studies. T.R., A.P. and S.B. carried out the calcineurin immunohistochemistry. R.J.U. and D.H.E. conceived of the study, supervised the work and edited the manuscript. All authors reviewed the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * David H Ellison Author Details * Ewout J Hoorn Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen B Walsh Search for this author in: * NPG journals * PubMed * Google Scholar * James A McCormick Search for this author in: * NPG journals * PubMed * Google Scholar * Antje Fürstenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Chao-Ling Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Tom Roeschel Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Paliege Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander J Howie Search for this author in: * NPG journals * PubMed * Google Scholar * James Conley Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastian Bachmann Search for this author in: * NPG journals * PubMed * Google Scholar * Robert J Unwin Search for this author in: * NPG journals * PubMed * Google Scholar * David H Ellison Contact David H Ellison Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (389K) Supplementary Tables 1–6 and Supplementary Figures 1–4 Additional data
• A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice
  - Nat Med 17(10):1310-1314 (2011)
  Nature Medicine | Letter A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice * Ichiro Inoshima1, 4 * Naoko Inoshima1, 4 * Georgia A Wilke1 * Michael E Powers2 * Karen M Frank3 * Yang Wang1 * Juliane Bubeck Wardenburg1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:1310–1314Year published:(2011)DOI:doi:10.1038/nm.2451Received17 June 2011Accepted22 July 2011Published online18 September 2011 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 Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone1, 2. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia3, 4, 5. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleava! ge in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease. View full text Figures at a glance * Figure 1: ADAM10 contributes to lethal S. aureus pneumonia. () Survival curves for Adam10−/− mice relative to nondeleted littermate controls after infection with S. aureus strain Newman. n = 14 control mice and 15 Adam10−/− mice. (,) H&E-stained lung tissues derived from control and Adam10−/− mice 18–24 h after infection with a sublethal () or lethal () inoculum of S. aureus. Scale bars, 40 μm () and 1 mm (). * Figure 2: Hla induces ADAM10-dependent epithelial barrier disruption and E-cadherin cleavage. () Cell-associated metalloprotease activity measured in A549 cells transfected with irrelevant or ADAM10-specific siRNA following treatment for the time periods indicated with 10 μg ml−1 (300 nM) active Hla or the nontoxigenic mutant HlaH35L. Activity was quantified by detection of a fluorescent substrate product. *P ≤ 0.05. () ECIS recordings of A549 monolayers treated with PBS, the HlaH35L mutant (50 μg ml−1), or irrelevant (Irr) or ADAM10-specific (A10) siRNA transfectants treated with 50 μg ml−1 Hla. () Immunoblot analysis of full-length E-cadherin (FL) and accumulation of the CTF after treatment of A549 cells with controls DMSO and ionomycin compared to 20 μg ml−1 HlaH35L or Hla over the indicated time course. () Concentration dependence of E-cadherin cleavage in A549 cells exposed to 1–50 μg ml−1 (30 nM–1.5 μM) Hla for 1 h. () Immunofluorescence microscopy images showing surface expression of E-cadherin (green) after treatment with Hla for 2 h. Nu! clei (blue) are stained with DAPI. Mean pixel intensity scored for ≥75 cells, 20.5 ± 0.87 (PBS) and 16.5 ± 1.72 (Hla), P = 0.02. Scale bar, 20 μm. (,) Cellular metalloprotease activity and E-cadherin cleavage induced by treatment of A549 cells for 2 h with 10 μg ml−1 Hla, the monomeric HlaH35L mutant, a pre-pore locked mutant (HlaPPL) that is reverted to the wild-type toxin in the presence of dithiothreitol (HlaPPL + DTT), or Hla in the presence of the pore-blocking methyl-β-cyclodextrin (MβCD) () or by Hla in the presence of medium (F12K), PBS, Dulbecco's PBS (DPBS) or PBS supplemented with 0.9 mM Ca2+, 0.493 mM Mg2+ or 2.67 mM K+ (). Error bars represent s.e.m. * Figure 3: Hla is required for E-cadherin cleavage and disruption of epithelial barrier function in S. aureus pneumonia. () BAL fluid analysis 6, 12 and 24 h after infection of C57BL/6J mice with S. aureus USA300 or its isogenic mutant harboring a disruption of the hla locus (Hla−) to assess E-cadherin cleavage, measured by immunoblotting for the released NTF. (,) Simultaneous evaluations of barrier disruption assessing BAL for cell count () and protein concentration () in groups of seven mice. () BAL fluid analysis from C57BL/6J mice that received an intranasal dose of 0.4 μg purified Hla or HlaH35L or control PBS, assessed as described in . () Histopathology of mouse lung tissues 4 h after treatment with PBS or Hla. Tissues were analyzed by H&E staining (top) or E-cadherin immunohistochemistry (bottom). Scale bars, 40 μm. (,) Immunoblot analysis of cleaved E-cadherin NTF () and quantification of cell and protein content () present in BAL fluid from Adam10−/− mice relative to control littermates after treatment with 0.4 μg purified Hla delivered by intranasal route. (,) E-cadherin cl! eavage () and cell and protein recovery () from BAL samples of C57BL/6J mice infected with 3 × 108 WT or Hla−S. aureus as compared to infection with 7 × 108 Hla−S. aureus. Statistical analysis for panels ,, and was performed using a two-tailed Student's t-test; *P < 0.05 and **P < 0.02. * Figure 4: An ADAM10-specific metalloprotease inhibitor prevents Hla-mediated injury. () Toxin-induced (20 μg ml−1, time periods as indicated) E-cadherin cleavage detected by immunoblot analysis of A549 cells that were pretreated with the metalloprotease inhibitor GI254023X (20 μM) or DMSO vehicle. () ECIS-based monitoring of A549 monolayer resistance after Hla treatment (20 μg ml−1) of cells exposed to GI254023X (20 μM) or DMSO vehicle control. () Binding and oligomerization of radiolabeled, active Hla to A549 cells after treatment with GI254023X (Hla7, oligomeric Hla; Hla, monomeric Hla). () Immunoblot analysis of cleaved E-cadherin NTF present in BAL fluid from GI254023X-treated mice relative to DMSO-treated mice after intranasal instillation of purified Hla. () Mortality curves in mice treated with DMSO vehicle or GI254023X upon challenge with lethal inocula of strain Newman (top, n = 14 mice, 5 × 108S. aureus per mouse; bottom, n = 8 mice, 6.3 × 108S. aureus per mouse). Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Ichiro Inoshima & * Naoko Inoshima Affiliations * Department of Pediatrics, University of Chicago, Chicago, Illinois, USA. * Ichiro Inoshima, * Naoko Inoshima, * Georgia A Wilke, * Yang Wang & * Juliane Bubeck Wardenburg * Department of Microbiology, University of Chicago, Chicago, Illinois, USA. * Michael E Powers & * Juliane Bubeck Wardenburg * Department of Pathology, University of Chicago, Chicago, Illinois, USA. * Karen M Frank Contributions I.I. performed mouse infection modeling, in vivo E-cadherin cleavage studies and ECIS studies. N.I. performed mouse breeding and genetic analysis and assisted with infection modeling. G.A.W. performed siRNA transfections, analyzed ADAM10 expression on 16HBE14o- cells and performed cellular assays of metalloprotease activity. M.E.P. examined the effects of GI254023X on toxin binding and performed ECIS experiments. K.M.F. generated the HlaPPL mutant. Y.W. performed ECIS experiments. J.B.W. performed cellular assays of metalloprotease activity, E-cadherin cleavage and immunofluorescence microscopy and wrote the paper. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Juliane Bubeck Wardenburg Author Details * Ichiro Inoshima Search for this author in: * NPG journals * PubMed * Google Scholar * Naoko Inoshima Search for this author in: * NPG journals * PubMed * Google Scholar * Georgia A Wilke Search for this author in: * NPG journals * PubMed * Google Scholar * Michael E Powers Search for this author in: * NPG journals * PubMed * Google Scholar * Karen M Frank Search for this author in: * NPG journals * PubMed * Google Scholar * Yang Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Juliane Bubeck Wardenburg Contact Juliane Bubeck Wardenburg Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–6 and Supplementary Methods Additional data
• Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results
  - Nat Med 17(10):1315-1319 (2011)
  Nature Medicine | Technical Report Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results * Gooitzen M van Dam1 * George Themelis2 * Lucia M A Crane1 * Niels J Harlaar1, 2 * Rick G Pleijhuis1 * Wendy Kelder1 * Athanasios Sarantopoulos2 * Johannes S de Jong1 * Henriette J G Arts3 * Ate G J van der Zee3 * Joost Bart4 * Philip S Low5 * Vasilis Ntziachristos2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:1315–1319Year published:(2011)DOI:doi:10.1038/nm.2472Received15 October 2010Accepted11 March 2011Published online18 September 2011 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 The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90–95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α–targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α–targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery. View full text Figures at a glance * Figure 1: The tumor-specific fluorescent agent folate-FITC. () Folate is conjugated through an ethylenediamine spacer to fluorescein isothiocyanate (FITC), resulting in folate-FITC, with a molecular weight of 917 kDa. () A schematic presentation of the targeting of ovarian cancer. Folate-FITC is targeted toward FR-α and internalized upon binding, shuttling folate-FITC into the cytoplasm. * Figure 2: Intra-operative multispectral imaging system. () Multispectral fluorescence camera system. () Positioning before draping. () Intraoperative application including draping. (–) Intraoperative screenshots of simultaneously detected and depicted images in color (,) and corresponding fluorescence during surgery (,) in a patient with high-grade serous ovarian carcinoma and extensive peritoneal carcinomatosis (stage III, FR-α positive). * Figure 3: Quantification of tumor deposits ex vivo. (,) Color image () with the corresponding tumor-specific fluorescence image () of a representative area in the abdominal cavity. () Scoring was based on three different color images (median 7, range 4–22) and their corresponding fluorescence images (FLI) (median 34, range 8–81); P < 0.001 by five independent surgeons. * Figure 4: Microphotographs of different ovarian tumors. (–) Three different tumor types are shown: fibrothecoma (), borderline serous tumor () and high-grade serous carcinoma (). Top row, routine staining with H&E; middle row, immunohistochemical staining (IHC) for FR-α; lower row, unstained slides observed with fluorescent microscopy (FM) to detect the intravenously administered folate-FITC. The fibrothecoma () shows no expression of FR-α nor binding of folate-FITC, which corresponds with the absence of lesions visualized intraoperatively using the fluorescence camera system. Both the borderline serous tumor () and the high-grade serous tumor () show epithelial expression of FR-α and binding of folate-FITC, which corresponds with the presence of visible lesions intraoperatively. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Surgery, Division of Surgical Oncology, BioOptical Imaging Center, University of Groningen, Groningen, The Netherlands. * Gooitzen M van Dam, * Lucia M A Crane, * Niels J Harlaar, * Rick G Pleijhuis, * Wendy Kelder & * Johannes S de Jong * Technische Universität München & Helmholtz Zentrum, München, Germany. * George Themelis, * Niels J Harlaar, * Athanasios Sarantopoulos & * Vasilis Ntziachristos * Department of Gynaecology, Division of Gynaecological Oncology, University of Groningen, Groningen, The Netherlands. * Henriette J G Arts & * Ate G J van der Zee * Department of Pathology and Molecular Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. * Joost Bart * Department of Chemistry, Purdue University, West Lafayette, Indiana, USA. * Philip S Low Contributions G.M.v.D. supervised the project, designed and performed the study, interpreted data and wrote the manuscript. G.T. designed the camera system, performed the study, interpreted data with technical and computer analyses support and wrote the manuscript. L.M.A.C. designed and performed the study, interpreted data and wrote the manuscript. N.J.H. performed the study and interpreted data. R.G.P. performed the study and interpreted data. W.K. performed the study and interpreted data. A.S. designed the camera system, performed the study and interpreted data with technical and computer analysis support. J.S.d.J. performed the study and interpreted data. H.J.G.A. designed and performed the study, interpreted data and wrote the manuscript. A.G.J.v.d.Z. designed and performed the study, interpreted data and wrote the manuscript. J.B. designed and performed the study, interpreted data, performed the pathology analyses and wrote the manuscript. P.S.L. designed and performed the study, an! d interpreted data. V.N. supervised the project, designed and performed the study, designed and provided the camera system and interpreted data, and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Gooitzen M van Dam or * Vasilis Ntziachristos Author Details * Gooitzen M van Dam Contact Gooitzen M van Dam Search for this author in: * NPG journals * PubMed * Google Scholar * George Themelis Search for this author in: * NPG journals * PubMed * Google Scholar * Lucia M A Crane Search for this author in: * NPG journals * PubMed * Google Scholar * Niels J Harlaar Search for this author in: * NPG journals * PubMed * Google Scholar * Rick G Pleijhuis Search for this author in: * NPG journals * PubMed * Google Scholar * Wendy Kelder Search for this author in: * NPG journals * PubMed * Google Scholar * Athanasios Sarantopoulos Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes S de Jong Search for this author in: * NPG journals * PubMed * Google Scholar * Henriette J G Arts Search for this author in: * NPG journals * PubMed * Google Scholar * Ate G J van der Zee Search for this author in: * NPG journals * PubMed * Google Scholar * Joost Bart Search for this author in: * NPG journals * PubMed * Google Scholar * Philip S Low Search for this author in: * NPG journals * PubMed * Google Scholar * Vasilis Ntziachristos Contact Vasilis Ntziachristos Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (229K) Supplementary Figure 1 and Supplementary Methods Movies * Supplementary Video 1 (37M) Intraoperative application of the multispectral intraoperative fluorescence camera system Additional data
• Corrigendum: Peroxisome proliferation–associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity
  - Nat Med 17(10):1320 (2011)
  Nature Medicine | Corrigendum Corrigendum: Peroxisome proliferation–associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity * Sabrina Diano * Zhong-Wu Liu * Jin Kwon Jeong * Marcelo O Dietrich * Hai-Bin Ruan * Esther Kim * Shigetomo Suyama * Kaitlin Kelly * Erika Gyengesi * Jack L Arbiser * Denise D Belsham * David A Sarruf * Michael W Schwartz * Anton M Bennett * Marya Shanabrough * Charles V Mobbs * Xiaoyong Yang * Xiao-Bing Gao * Tamas L HorvathJournal name:Nature MedicineVolume: 17,Page:1320Year published:(2011)DOI:doi:10.1038/nm1011-1320aPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.17, 1121–1127 (2011); published online 28 August 2011; corrected after print 16 September 2011 In the version of this article initially published, the top electrophysiological trace of Figure 4a was inadvertently repeated as the bottom electrophysiological trace of Figure 4b. The scientific conclusions of the paper were not affected by the error. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Sabrina Diano Search for this author in: * NPG journals * PubMed * Google Scholar * Zhong-Wu Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Jin Kwon Jeong Search for this author in: * NPG journals * PubMed * Google Scholar * Marcelo O Dietrich Search for this author in: * NPG journals * PubMed * Google Scholar * Hai-Bin Ruan Search for this author in: * NPG journals * PubMed * Google Scholar * Esther Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Shigetomo Suyama Search for this author in: * NPG journals * PubMed * Google Scholar * Kaitlin Kelly Search for this author in: * NPG journals * PubMed * Google Scholar * Erika Gyengesi Search for this author in: * NPG journals * PubMed * Google Scholar * Jack L Arbiser Search for this author in: * NPG journals * PubMed * Google Scholar * Denise D Belsham Search for this author in: * NPG journals * PubMed * Google Scholar * David A Sarruf Search for this author in: * NPG journals * PubMed * Google Scholar * Michael W Schwartz Search for this author in: * NPG journals * PubMed * Google Scholar * Anton M Bennett Search for this author in: * NPG journals * PubMed * Google Scholar * Marya Shanabrough Search for this author in: * NPG journals * PubMed * Google Scholar * Charles V Mobbs Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoyong Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Xiao-Bing Gao Search for this author in: * NPG journals * PubMed * Google Scholar * Tamas L Horvath Search for this author in: * NPG journals * PubMed * Google Scholar
• Corrigendum: Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras-mediated pancreatic cancer
  - Nat Med 17(10):1320 (2011)
  Nature Medicine | Corrigendum Corrigendum: Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras-mediated pancreatic cancer * H Rosie Xing * Carlos Cordon-Cardo * Xinzhu Deng * William Tong * Luis Campodonico * Zvi Fuks * Richard KolesnickJournal name:Nature MedicineVolume: 17,Page:1320Year published:(2011)DOI:doi:10.1038/nm1011-1320bPublished online11 October 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.9, 1267–1268 (2003); published online 07 September 2003; corrected after print 11 October 2011 In the version of this article initially published, there are irregularities with the tubulin loading controls in lanes 1 through 4 and with the KSR1 bands in lanes 7 and 8 of Figure 2f. The authors have repeated the experiment and have provided a new figure panel that is now published as part of the correction notices linked to the HTML version and attached to the PDF version of the article. The original figures remain in both online versions of the article. The authors have also made a correction to Supplementary Figure 6b, which has been added to the supplementary file online. H. Rosie Xing does not agree to this correction. Figure 1: Figure 2f * Full size image (39 KB) Additional data Author Details * H Rosie Xing Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Cordon-Cardo Search for this author in: * NPG journals * PubMed * Google Scholar * Xinzhu Deng Search for this author in: * NPG journals * PubMed * Google Scholar * William Tong Search for this author in: * NPG journals * PubMed * Google Scholar * Luis Campodonico Search for this author in: * NPG journals * PubMed * Google Scholar * Zvi Fuks Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Kolesnick Search for this author in: * NPG journals * PubMed * Google Scholar