Latest Articles Include:
- The dangers of the fast track
- Nat Med 17(4):389 (2011)
Nature Medicine | Editorial The dangers of the fast track Journal name:Nature MedicineVolume: 17,Page:389Year published:(2011)DOI:doi:10.1038/nm0411-389Published online07 April 2011 The US Food and Drug Administration (FDA) wants to create a way to fast-track approval of new medical devices, but they must first address the problems of the existing accelerated process for bringing devices to market that are substantially equivalent to ones already in commercial distribution. View full text Additional data - Nuclear leak reinforces need for drugs to combat radiation
- Nat Med 17(4):391 (2011)
Nature Medicine | News Nuclear leak reinforces need for drugs to combat radiation * Cassandra WillyardJournal name:Nature MedicineVolume: 17,Page:391Year published:(2011)DOI:doi:10.1038/nm0411-391Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Last month, in the aftermath of Japan's 9.0-magnitude earthquake and subsequent tsunami, evacuation centers surrounding the crippled Fukushima Daiichi Nuclear Power Station stockpiled nearly a quarter-million doses of potassium iodide as a preventative measure against radiation poisoning. These pills protect people from the long-term risks of thyroid cancer associated with chronic radiation exposure, but they do little to guard against the ill effects of high-dose radiation toxicity. Unfortunately, no drugs are currently approved to treat the extreme radiation sickness that plant workers or emergency personnel may experience. Yet, thanks to investment from the US government, several candidate compounds might soon be available in the event of another nuclear catastrophe. The Project BioShield Act, passed by Congress in 2004, and the Pandemic and All-Hazard Preparedness Act, signed into law two years later, allotted billions of dollars in funding for research into medical countermeasures to be used in the case of nuclear, chemical and biological attacks. These government awards include more than $500 million for the treatment and prevention of so-called acute radiation syndrome (ARS), the extreme radiation sickness associated with exposure to high doses of ionizing radiation over a short period of time. In addition to terrorism, nuclear plant disasters are a leading cause of ARS. The 1986 explosion at Ukraine's Chernobyl plant, for example, caused 134 confirmed cases of ARS, accounting for almost one third of the reported incidences of the disease worldwide (Health Phys., 462–469, 2007). As Nature Medicine went to press, engineers in Japan were making headway in containing leaks at the Fukushima site and seemed to have averted a meltdown. But radiation at the plant had already spiked to dangerous levels, forcing hundreds of exposed emergency workers to temporarily evacuate. If any of these workers are diagnosed with ARS, their treatment options currently are limited to antibiotics, blood transfusions and fluid supplements that deal with the symptoms of the disease. Physicians also sometimes administer cancer drugs that help the immune system rebound, but these drugs must be given in medical facilities. Now, however, researchers are developing biologics and small molecule drugs that be used in the field to stem radiation's ill effects. One of the lead candidates—a drug called CBLB502 being developed by Cleveland BioLabs of Buffalo, New York—binds an immune protein called Toll-like receptor 5 to activate a cell survival pathway. In unpublished data from rodent and monkey models, the drug, which is derived from a protein found in the flagella of Salmonella bacteria, remained "very efficacious" up to 48 hours after exposure, according to Cleveland BioLabs' chief scientific officer Andrei Gudkov. The US Food and Drug Administration (FDA) granted the compound fast-track status in July 2010, and, backed by a $15.6 million award from the US government, the company has already tested CBLB502 in 150 healthy volunteers in two phase 1 safety studies. According to spokesperson Rachel Levine, the company plans to submit an approval application by the end of next year. Meanwhile, the Pennsylvania-based biotech Onconova is advancing its own compound, Ex-RAD, which works by inhibiting proapoptosis proteins such as p53 as well as downstream regulators of cell death. According to Ramesh Kumar, Onconova's president and CEO, the small molecule has been tested for safety in more than 50 people, with few adverse effects reported. Fresh blood Asahi Shimbun via Getty Images Radiation drugs needed. Instead of trying to block cell death, some companies are developing treatments that simply replace the cells lost to radiation. For example, the California-based biotech Cellerant Therapeutics has a system based on blood progenitor cells that can form mature infection-fighting and clotting blood cells upon infusion by intravenous drip. Importantly, these cells—dubbed CLT-008—do not produce the mature T cells that cause immune reactions, so just one product can be stored to serve as a temporary therapy for all ARS-affected individuals. What's more, unlike many other ARS drug candidates, CLT-008 seems to work up to a week after exposure. "By that time, you'd actually have a chance to evacuate people out of the city," notes Mark Whitnall, head of the radiation countermeasures program at the Armed Forces Radiobiology Research Institute in Bethesda, Maryland. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Cassandra Willyard Search for this author in: * NPG journals * PubMed * Google Scholar - Trial success spurs planning for rollout of HIV prevention pills
- Nat Med 17(4):392 (2011)
Nature Medicine | News Trial success spurs planning for rollout of HIV prevention pills * Elie DolginJournal name:Nature MedicineVolume: 17,Page:392Year published:(2011)DOI:doi:10.1038/nm0411-392Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. BOSTON — The antiretroviral pill Truvada topped Time magazine's list of medical breakthroughs last year after a study showed that taking the pill daily can prevent HIV infection. The drug, made by California's Gilead Sciences, could provide a powerful new tool to curb the worldwide AIDS epidemic. But, as HIV experts discussed here at the annual Conference on Retroviruses and Opportunistic Infections last month, questions of cost effectiveness and drug resistance, among others, still need to be answered before the product is ready for primetime. In November of last year, an international team led by Robert Grant, a virologist at the University of California–San Francisco (UCSF), reported that the strategy, called preexposure prophylaxis (PrEP), led to a 44% reduction in HIV acquisition in a trial of nearly 2,500 sexually active gay men in six countries (N. Engl. J. Med.363, 2587–2599, 2010). But that finding was based on data collected over only 16 months on average. "After the publication, there were questions about how durable the results would be," says Grant. So the researchers tracked the study subjects in the so-called iPrEx study for three more months, crunched the numbers and found that the efficacy of the preventative pill was still 42% on average, as Grant reported here. The protection rate seen in the iPrEx study was much higher among people who regularly took their Truvada pills. But monitoring adherence remains a major challenge in trials of self-administered medicines. This became even clearer when Rivet Amico, a public health researcher at the University of Connecticut–Storrs, and her colleagues analyzed drug concentrations in the blood of around 180 HIV-negative study participants. The researchers found that none of the trial's indicators of pill use—including investigator-initiated counts of pills or bottles and self-reported data—fully matched direct measurements of drug concentration in the blood. Further probing the same data set, Peter Anderson, a clinical pharmacologist at the University of Colorado–Denver, found that older people, people in the US and trial participants who engaged in the riskiest form of sex were the most likely to have detectable amounts of antiretroviral drugs in their blood, perhaps the most accurate indicator of pill use. "Using drug levels really helped us understand the iPrEx results and the need for understanding drug-taking behavior better," says Anderson. Given the patchy adherence rates, many researchers were concerned about the possibility of drug resistance evolving. But, according to data presented by UCSF's Teri Liegler, none of the 36 men who were taking Truvada and became infected with the virus showed any signs of resistance to either tenofovir or emtricitabine, the two drugs that are combined in the Truvada pill. A separate mathematical model of the South African HIV epidemic, presented by Ume Abbas from the Cleveland Clinic Foundation in Ohio, showed that wide PrEP rollout is expected to contribute far less to drug resistance than standard antiretroviral therapy. Beyond Truvada Truvada is not the only possible preemptive weapon against infection. Currently, researchers are testing either Truvada or tenofovir alone in large-scale PrEP efficacy trials in 4700 Kenyan and Ugandan heterosexual couples in which one partner is HIV-positive, 2,400 injecting drug users in Thailand and 8,900 heterosexual women in sub-Saharan Africa. The results are expected over the next two years. Other PrEP candidates at earlier stages in the pipeline include Tibotec Pharmaceuticals' dapivirine, a reverse transcriptase inhibitor, and Pfizer's maraviroc, an entry inhibitor. "As a field, we need those data," says Connie Celum of the University of Washington in Seattle, who is heading the heterosexual couple trial in East Africa. Currently, she notes, researchers only know that PrEP works in one population—gay men—so "those data are going to be critical to informing implementation." The iPrEx trial, meanwhile, is now moving into an open-label phase where study subjects in the control arm will be offered Truvada instead of placebo. Albert Liu, a trial investigator at the San Francisco Department of Public Health, hopes that adherence rates will go up now that participants know that the drug works. The trial extension will also provide additional insights into sexual practices and drug safety, he notes. Getty Images Doses of Truvada. In the initial November 2010 publication, the iPrEx investigators reported that nausea was the only serious side effect associated with the drug. However, at the meeting, UCSF metabolism researcher Kathleen Mulligan presented data showing that subjects taking Truvada had 0.5–1% lower bone mineral density compared with those receiving placebo. The two groups, however, experienced similar rates of bone fractures. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Trial in youngest group points to HIV treatment overhaul
- Nat Med 17(4):393 (2011)
Nature Medicine | News Trial in youngest group points to HIV treatment overhaul * Elie DolginJournal name:Nature MedicineVolume: 17,Page:393Year published:(2011)DOI:doi:10.1038/nm0411-393Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. BOSTON — By some estimates, around 1,800 children, mostly newborns, become infected with HIV each day. But even though the stakes are high, the most commonly used strategy to combat the deadly virus among infected children in resource-limited countries may need a massive overhaul. According to data presented here last month at the Conference on Retroviruses and Opportunistic Infections, babies born with HIV should immediately start receiving antiretroviral drugs known as protease inhibitors, not the reverse transcriptase inhibitor widely used throughout the developing world. "We have created a bit of a stir at the guideline level moving forward," says Paul Palumbo, a pediatrician at Dartmouth Medical School in Hanover, New Hampshire. "We need to move into considerations of using [protease inhibitors] as a first-line therapy." The reverse transcriptase–blocking drug nevirapine, marketed as Viramune by the German company Boehringer-Ingelheim, is the cornerstone of both preventing mother-to-child transmission of HIV and treating infections in affected infants in the third world, where the vast majority of the world's 2.1 million HIV-positive children live. But in children who become infected despite receiving nevirapine as prophylaxis, the drug often selects for resistant viruses. To avoid using the same medicine for prophylaxis and treatment, researchers have sought a different drug regimen for kids once they become infected, and a protease inhibitor called Kaletra—a formulation of ritonavir-boosted lopinavir developed by Chicago-based Abbott Laboratories—has emerged as the front-runner. Last year, the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) group, for which Palumbo serves as a vice chair, showed that Kaletra repressed viral loads better than nevirapine in HIV-positive youngsters who had previously been exposed to the latter drug (N. Engl. J. Med., 1510–1520, 2010). In response, the World Health Organization (WHO) recommended that Kaletra should be used among nevirapine-exposed kids. But, owing to logistical concerns and a lack of data demonstrating Kaletra's general superiority, the agency stopped short of advocating Kaletra as a first-line treatment for all HIV-infected children. Design Pics/Newscom Experts are rethinking HIV treatments for infants. Now, in the first head-to-head trial of the two drugs conducted anywhere in the world for young children not previously exposed to nevirapine, the IMPAACT team found that Kaletra led to half as many drug failures as nevirapine among close to 300 infants under the age of three at ten study sites across sub-Saharan Africa and India. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - New organization pledges scientific expertise for viral outbreaks
- Nat Med 17(4):394 (2011)
Nature Medicine | News New organization pledges scientific expertise for viral outbreaks * Adam MannJournal name:Nature MedicineVolume: 17,Page:394Year published:(2011)DOI:doi:10.1038/nm0411-394aPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. WASHINGTON, DC — The next time a viral outbreak like H1N1 influenza or the SARS virus threatens the world, Robert Gallo wants the scientific community to be ready. To make that happen, last month Gallo launched the Global Virus Network (GVN), an international group of leading virologists and medical researchers tasked with providing scientific expertise to government agencies in the face of emerging infectious viral agents. "An independent voice of medical laboratory scientists would be a great addition to the surveillance world," says Gallo, who co-discovered the HIV retrovirus in 1984 and is currently director of the Institute of Human Virology at the University of Maryland School of Medicine in Baltimore. At a meeting here in early March, representatives from 16 countries gathered to determine the network's mandate and mission. As envisioned by its members, the GVN will serve as a clearinghouse for rapid data collection and disease containment. In response to an outbreak, the organization would send researchers from one of its many international academic centers into the field to collect samples and help local officials diagnose and treat infected people. http://www.sardari.com Global Virus Network co-founders Reinhard Kurth (left), Robert Gallo (center) and William Hall (right). The GVN, which hopes to secure a budget of at least $25 million per year with donations from governments, research centers, charitable organizations and private companies, would engage with—yet remain autonomous of—other agencies such as the World Health Organization (WHO) and the US Centers for Disease Control and Prevention (CDC). Both of these agencies, which have ongoing discussions with GVN officials about how the groups can best work together, are also involved in controlling viral pathogens but are often more constrained by political considerations and region-specific funding support. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Adam Mann Search for this author in: * NPG journals * PubMed * Google Scholar - UK plans 30% funding bump for translational research
- Nat Med 17(4):394 (2011)
Nature Medicine | News UK plans 30% funding bump for translational research * Bea PerksJournal name:Nature MedicineVolume: 17,Page:394Year published:(2011)DOI:doi:10.1038/nm0411-394bPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. LONDON — The push for translational medicine got a boost in the UK last month when the government announced that it would devote £775 million ($1.2 billion) over five years for translational research focusing on high-priority disease areas such as cancer, heart disease and dementia. This funding cycle represents a 30% increase on the first round of such grants, which began in 2007. "It's not a massive amount of money, but it's a realignment of priorities," says Chris Torrance, chief executive of the translational genomics company Horizon Discovery based in Cambridge, UK. "In a period where other people are taking cuts, it's significant," he adds. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Bea Perks Search for this author in: * NPG journals * PubMed * Google Scholar - Speedy sequencing technologies help track food-borne illness
- Nat Med 17(4):395 (2011)
Nature Medicine | News Speedy sequencing technologies help track food-borne illness * Michelle PflummJournal name:Nature MedicineVolume: 17,Page:395Year published:(2011)DOI:doi:10.1038/nm0411-395aPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. The monumental Food Safety Modernization Act, signed into law by US President Barack Obama in January, promises to add much-needed improvements to the security of the US food supply. Yet a technological advance, rather than a major legislative overhaul, could have the largest impact on the government's ability to identify contaminated foods and rid them from store shelves. In a proof-of-principle study published last month, scientists from the US Food and Drug Administration (FDA) reported that, compared with traditional DNA fingerprinting, next-generation sequencing more precisely identified the bacterial strain and the food product responsible for a 2009–2010 outbreak of Salmonella that sickened more than 250 people across the US. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Michelle Pflumm Search for this author in: * NPG journals * PubMed * Google Scholar - Drugs development is cheaper than widely claimed, experts say
- Nat Med 17(4):395 (2011)
Nature Medicine | News Drugs development is cheaper than widely claimed, experts say * Michelle PflummJournal name:Nature MedicineVolume: 17,Page:395Year published:(2011)DOI:doi:10.1038/nm0411-395bPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. The sky-high costs of research and development are often cited by the pharmaceutical industry to justify the steep prices charged for prescription medicines. But the widely touted sticker price of bringing a new drug to market might be radically inflated, new research shows. In a study published in February, two health policy experts argue that companies spend around $60 million after discovery costs to test a new biologic or small-molecule drug—a far cry from the $1.3 billion estimate normally bandied about by the drug industry. If confirmed, the markedly lower price of drug development could undermine big pharma's claims that generous tax breaks and high drug prices are needed to spur medical innovation. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Michelle Pflumm Search for this author in: * NPG journals * PubMed * Google Scholar - Mutation-specific cystic fibrosis treatments on verge of approval
- Nat Med 17(4):396-397 (2011)
Nature Medicine | News Mutation-specific cystic fibrosis treatments on verge of approval * Elie DolginJournal name:Nature MedicineVolume: 17,Pages:396–397Year published:(2011)DOI:doi:10.1038/nm0411-396Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. More than two decades after scientists discovered the mutated gene responsible for cystic fibrosis, the first drugs that target the defective protein involved in the disease may be on the cusp of market approval. In February, researchers announced preliminary results from a phase 3 trial showing that a pill called VX-770 led to substantial improvements in lung capacity in people with cystic fibrosis. Similar drugs in development could also reach patients within the next few years. "This is one of the most exciting developments in [cystic fibrosis] therapies since I've been practicing," says Paula Anderson, a pulmonary physician at the University of Arkansas for Medical Sciences in Little Rock. "We've never had any other treatment that was really specifically targeted at mutations." In 1989, a team that included Francis Collins, now director of the US National Institutes of Health, showed that mutations in a gene encoding a chloride channel protein called cystic fibrosis transmembrane conductance regulator, or CFTR, are always responsible for cystic fibrosis, an inherited disorder that slows mucus clearing from the airways and thereby makes individuals susceptible to deadly lung infections1, 2, 3. After the landmark genetic discovery, researchers widely assumed that cures would be just around the corner. But no approved therapies to date target the underlying genetic defect; instead, existing therapies such as antibiotics and mucus-thinning drugs can only treat the symptoms of the disease. Creative Commons The CFTR protein. VX-770 is different. This small-molecule drug from Vertex Pharmaceuticals, a Cambridge, Massachusetts–based company, interacts directly with CFTR, propping open the defective protein to allow a more normal flow of chloride ions across the cell membrane, thereby restoring the function of airway cells. In the recently announced trial, which enrolled 161 participants who carried at least one copy of a CFTR mutation called G551D, the people taking VX-770 showed improved lung function and reported fewer respiratory problems than those on placebo. The drug also restored chloride levels in patients' sweat to near-normal levels, indicating that the chloride pumps throughout the body were back up and running. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Orphan cystic fibrosis drugs find sister diseases
- Nat Med 17(4):397 (2011)
Nature Medicine | News Orphan cystic fibrosis drugs find sister diseases * Elie DolginJournal name:Nature MedicineVolume: 17,Page:397Year published:(2011)DOI:doi:10.1038/nm0411-397Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. With fewer than 80,000 people in the world diagnosed with cystic fibrosis, the disease hardly presents itself as a lucrative market for drug development. But it's not just people with cystic fibrosis who harbor mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. "There are other diseases that CFTR mutations are associated with," notes Melissa Ashlock, former vice president of drug discovery for Cystic Fibrosis Foundation Therapeutics. As such, CFTR modulators designed for one ailment—be it cystic fibrosis or otherwise—could have broader market potential beyond the single orphan disease. Although most people with mutations in CFTR develop cystic fibrosis, some individuals experience less severe disorders, including chronic pancreatitis, male infertility, sinusitis and airway abnormalities. These people are also likely to benefit from CFTR-targeted agents such as Vertex Pharmaceutical's VX-770 and VX-809. But, given that many of these more mild diseases are more sporadic than cystic fibrosis, "whether they would be good candidates for being treated with a chronic therapy that's going to be quite expensive is unclear," says Sam Moskowitz, director of the cystic fibrosis basic science program at MassGeneral Hospital for Children in Boston. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Genetic insights beginning to divide autism diagnosis
- Nat Med 17(4):398 (2011)
Nature Medicine | News Genetic insights beginning to divide autism diagnosis * Monica HegerJournal name:Nature MedicineVolume: 17,Page:398Year published:(2011)DOI:doi:10.1038/nm0411-398aPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. News has already emerged that the American Psychiatric Association is considering expanding its definition of autism to include a broader range of developmental and mental syndromes in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders. But under the radar, geneticists are moving in the opposite direction, parsing the disease into more specific subtypes on the basis of its molecular etiology. Autism is not a biological disorder, but rather a behavioral description, with the underlying disease "a constellation of rare genetic disorders," says John Spiro, the senior associate director for research at the New York–based Simons Foundation Autism Research Initiative. Last month, Spiro spoke at the first international symposium on a genetic subtype of autism known as Phelan-McDermid syndrome, an event that took place at the New York Academy of Medicine. Stephen Scherer, director of the Centre for Applied Genomics at The Hospital for Sick Children in Toronto echoes Spiro's view, saying that autism may in fact be a "bunch of different genetic disorders that have a common clinical outcome." For example, Rett syndrome, a neurodevelopmental disorder that affects primarily girls and falls under the broad 'autism spectrum disorder' umbrella, has a distinct genetic cause—a mutation typically in the MECP2 gene on the X chromosome, enabling diagnosis of affected individuals. The genetic subtype was first described in 1999 (Nat. Genet.23, 185–188, 1999). Genotyping autism subtypes. Researchers are just beginning to parse other subtypes on the basis of genetics and, in particular, copy number variants, where entire portions of a chromosome are either deleted or duplicated. A tiny deletion on chromosome 22 is behind Phelan-McDermid syndrome and is estimated to occur in fewer than 1 in 10,000 pregnancies. Deletions and duplications on chromosomes 16, 15 and 1, meanwhile, have also been identified and are being considered as separate subtypes of autism by geneticists. These subtypes do not have lay names and are simply referred to by their chromosomal aberration. For instance, autism caused by mutations to chromosome 16 is now often being called 16p11.2 (or 16p for short) and has been estimated to occur in about 1% of all autism cases (N. Engl. J. Med.358, 667–675, 2008). View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Monica Heger Search for this author in: * NPG journals * PubMed * Google Scholar - France promotes research into rare diseases with new plan
- Nat Med 17(4):398-399 (2011)
Nature Medicine | News France promotes research into rare diseases with new plan * Barbara CasassusJournal name:Nature MedicineVolume: 17,Pages:398–399Year published:(2011)DOI:doi:10.1038/nm0411-398bPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. PARIS — On 28 February, international Rare Disease Day, the French government finally unveiled a four-year plan to bolster research and treatment into this group of ailments, which include sickle anemia and amyotrophic lateral sclerosis. The plan, which spans from this year to 2014, aims to improve the quality of care for people with rare diseases, bolster research and expand European and international cooperation in this area. It is a continuation of France's first National Plan for Rare Diseases, which ran from 2005 to 2008 and was claimed to be a world first. (A second iteration of the plan was due to be launched in 2010, but it was delayed in part because of a government reshuffle in November.) View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Barbara Casassus Search for this author in: * NPG journals * PubMed * Google Scholar - Sequencing reveals suite of commensal and pathogenic viruses
- Nat Med 17(4):399 (2011)
Nature Medicine | News Sequencing reveals suite of commensal and pathogenic viruses * Elie DolginJournal name:Nature MedicineVolume: 17,Page:399Year published:(2011)DOI:doi:10.1038/nm0411-399Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. VANCOUVER, CANADA — After coming to realize that symbiotic bacteria play a large part in running our bodies, scientists are slowly beginning to appreciate the importance of our viral communities, too. As researchers discussed here last month at the International Human Microbiome Congress, new sequencing techniques are revealing that these viruses—collectively called the 'virome'—often differ significantly between healthy and diseased individuals. "There's no question that these viral populations are affecting human health," says Frederic Bushman, a molecular virologist at the University of Pennsylvania School of Medicine in Philadelphia. "But we're just at an early stage in figuring out who's there and what they're doing. Well down the road we'll be asking how to engineer [the virome] to affect health outcomes." Metagenomic studies of viral communities trace their roots back to 2003 when Forest Rohwer and his colleagues at San Diego State University first sequenced the bacteriophage viruses living in a single human fecal sample (J. Bacteriol.185, 6220–6223, 2003). Since then, newer high-throughput sequencing methods have started to produce vastly more data on many more samples, yet the applications of virome studies to disease are only just starting to be worked out. Equinox Graphics / Photo Researchers, Inc. Researchers make headway sequencing the human virome. To better understand the links between viruses and diet, for example, Bushman and his colleagues placed five people on either a high-fat or a low-fat diet and then sequenced their stool samples over a ten-day period. Reporting at the March meeting, Bushman showed that the bacteriophage populations of people on the same diets grew more similar as the experiment proceeded, raising the possibility that viral communities could be engineered to combat obesity. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - After half-century's wait, approval paves path for new lupus drugs
- Nat Med 17(4):400 (2011)
Nature Medicine | News After half-century's wait, approval paves path for new lupus drugs * Heidi LedfordJournal name:Nature MedicineVolume: 17,Page:400Year published:(2011)DOI:doi:10.1038/nm0411-400aPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. On 9 March, Benlysta (belimumab) became the first lupus drug approved by US regulators in over half a century. The event was cause for celebration, not only for Human Genome Sciences, the Rockville, Maryland biotechnology company that originally developed the drug, but also for its many competitors racing to be the next to bring a lupus therapy to market. Clinical trials in lupus are notoriously difficult because the disease is so variable. For decades, companies have tried to bring a lupus drugs to market only to see their most promising candidates fail. One challenge is that patients frequently take two or three drugs, including steroids and other immunosuppressants, to tame their overactive immune systems, and this can mask the effects of an experimental treatment. Benlysta's approval, however, has given the community hope that such hurdles can be overcome. "This is the path that will give industry the confidence it needs to move forward with other therapies," says Margaret Dowd, president of the Lupus Research Institute in New York. Benlysta, a human monoclonal antibody drug, is now approved by the US Food and Drug Administration to treat systemic lupus erythematosus, a painful autoimmune disorder that can damage the joints, heart, kidneys and lungs. In many instances of lupus, unusually high amounts of a protein known as B lymphocyte stimulator (BLyS, pronounced 'bliss') allow overactive immune cells to slip past the body's defenses and into the circulation. Benlysta works by blocking BLyS and thereby allowing B cells to undergo regular programmed cell death instead of going rogue. Table 1: Lupus drugs in late-stage clinical trials Full table At least three other companies are developing drugs that target BLyS. One of these experimental medicines, atacicept, developed by Merck KGaA in Darmstadt, Germany and a Seattle biotechnology company called ZymoGenetics, targets both BLyS and a closely related protein called 'a proliferation-inducing ligand' (APRIL). Atacicept is currently in simultaneous phase 2 and phase 3 testing for systemic lupus, but the companies halted trials of the drug in patients with a severe form of the disease owing to an increase in infections. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Heidi Ledford Search for this author in: * NPG journals * PubMed * Google Scholar - Pooled trials drowning in conflict-of-interest oversights
- Nat Med 17(4):400-401 (2011)
Nature Medicine | News Pooled trials drowning in conflict-of-interest oversights * Nazlie LatefiJournal name:Nature MedicineVolume: 17,Pages:400–401Year published:(2011)DOI:doi:10.1038/nm0411-400bPublished online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Many influential meta-analyses of clinical trial data may be riddled with buried conflicts of interest. According to a report published last month, even when potential conflicts are disclosed in primary studies, they are almost never included in subsequent pooled analyses. The authors of the report say that more transparency is needed in meta-analyses because clinicians and medical organizations regularly rely on such reviews to inform their decisions. Clinical trial reporting guidelines have changed tremendously over the last decade, with strict protocols now in place for disclosing potential financial conflicts. Yet the same guidelines do not exist for meta-analyses. For example, the Cochrane Handbook for Systematic Reviews of Interventions used to guide the drafting of meta-analyses does not explicitly ask authors to list financial conflicts of interest found in the primary studies used. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Nazlie Latefi Search for this author in: * NPG journals * PubMed * Google Scholar - African genomics project takes shape at Cape Town meeting
- Nat Med 17(4):401 (2011)
Nature Medicine | News African genomics project takes shape at Cape Town meeting * Linda NordlingJournal name:Nature MedicineVolume: 17,Page:401Year published:(2011)DOI:doi:10.1038/nm0411-401Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. CAPE TOWN — More than 200 medical researchers met under sunny skies here on 4 and 5 March to discuss practical ways for the African continent to start benefiting from advances in genomics. The meeting's aim was to inform the design of the $37 million, five-year Human Heredity and Health in Africa (H3Africa) initiative. The initiative, funded by the Wellcome Trust, a UK medical research charity, and the US National Institutes of Health (NIH), aims to bring modern medical technology to bear on Africa's heavy disease burden. Africans are extremely genetically diverse—yet little is known about this variance and its health impact. To date, three quarters of the thousands of genetics studies completed worldwide have been conducted on populations of European descent. Africans are also poorly represented in international genetics projects such as the HapMap and 1,000 Genomes projects. This gap presents both an opportunity and a challenge for Africa, NIH director Francis Collins said at the meeting. The rapidly falling cost of sequencing and genetic analysis has put the technology within reach for cash-strapped African researchers. Moreover, the importance of Africa as the birthplace of humanity makes African genetics an important and intriguing area of study, he said. But the continent's poor health systems—including scant medical data, low research capability and a lack of trained health professionals—means this research effort risks being managed outside the continent, he continued. "We need capacity building in areas where disease occurs." Irene Abdou/Newscom Sudanese children. The H3Africa initiative plans to help plug Africa's genomics knowledge gap by addressing shortfalls in equipment, training and regulations. Although the exact remit for H3Africa remains flexible, a white paper presented at the Cape Town meeting set out a number of possible activities for the initiative. These include the development of regional centers of excellence in genotyping and sequencing and a continent-wide bioinformatics network. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Linda Nordling Search for this author in: * NPG journals * PubMed * Google Scholar - News in brief: Biomedical briefing
- Nat Med 17(4):402-403 (2011)
Nature Medicine | News News in brief: Biomedical briefing Journal name:Nature MedicineVolume: 17,Pages:402–403Year published:(2011)DOI:doi:10.1038/nm0411-402Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Wanted: science Criticizing the UK government's response to the swine flu pandemic and other emergency situations, the House of Commons Science and Technology Committee published a report last month urging the government to more closely involve scientists in planning ahead for crises, rather than using them to contain the chaos and panic that often result after a disaster. "There is insufficient effort to sit down with scientists and discuss issues of concern," Andrew Miller, a Labour Party parliamentarian and chair of the committee, told Nature Medicine. Booster shot US officials rolled out a new National Vaccine Plan in February to ensure that all Americans have access to preventative vaccines. The ten-year strategy—the first such update since 1994—aims to modernize technology for immunization campaigns and sets new benchmarks for vaccine safety and surveillance. "The updated plan... provides a roadmap to vaccine priorities for the next decade," says Bruce Gellin, director of the National Vaccine Program Office. Brazilian blueprint In the wake of a 964-million-reais ($579 million) cut to science spending in Brazil's 2011 budget, government officials last month established a new panel to build a roadmap for research and development in the country. Led by Brazilian-born neuroscientist Miguel Nicolelis of Duke University School of Medicine in Durham, North Carolina, the 21-member 'Commission of the Future' will identify barriers to scientific advancement and create a long-term business plan for the nation. Vaccine victory Vaccine manufacturers breathed a huge sigh of relief in February after the US Supreme Court declared that they cannot be sued outside of the special Vaccine Court established by Congress in 1986. In a 6-2 decision, the justices ruled that a lawsuit alleging a link between vaccines and seizures could not be filed in the Pennsylvania courts. The ruling "is a victory for parents and children," says Paul Offit, a pediatric vaccine expert at the Children's Hospital of Philadelphia. "It continues to allow for a stable industry and encourages vaccine makers to stay in place." Jacking up GINA Three US states introduced legislation over the past two months to further safeguard consumers against discrimination on the basis of their genetic makeup. Extending protections granted under the federal Genetic Information Nondiscrimination Act (GINA), the new legal protections from Massachusetts, California and Vermont prohibit insurance companies from using genetic test results to deny people of life, disability or long-term care coverage. "There are many weaknesses and gaps in GINA," says Mark Rothstein, founding director of the University of Louisville's Institute of Bioethics, Health Policy and Law in Kentucky. "The states are going to have to fill in the blanks." People Out of the Gates Creative Commons The former pharma executive who ran the global health program at the Bill & Melinda Gates Foundation will step down this summer to move back to his native Japan. In his five years at the Seattle-based foundation, Tachi Yamada, an accomplished gastroenterologist and former chairman of research and development at GlaxoSmithKline, led efforts to develop and deliver low-cost vaccines for the developing world, and oversaw the Grand Challenges Explorations program. "We have helped establish a sense of urgency about the big problems out there," Yamada says. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Straight talk with...Seth Berkley
- Nat Med 17(4):404 (2011)
Nature Medicine | News Straight talk with...Seth Berkley * Roxanne KhamsiJournal name:Nature MedicineVolume: 17,Page:404Year published:(2011)DOI:doi:10.1038/nm0411-404Published online07 April 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 On 13 June, donors to the GAVI Alliance will gather in London to affirm their commitment to fund immunizations in the developing world. At the meeting, participants will address the estimated $3.7 billion financing gap needed over the next four years to scale up childhood vaccination efforts to meet the demand forecasts for those countries that receive assistance from the Geneva-based organization. But attendees of the pledging conference will also be discussing something not on the formal agenda: the announcement last month that Seth Berkley, who founded and heads the International AIDS Vaccine Initiative (IAVI), will take over the helm of the alliance in August. Berkley will lead a unique chapter in GAVI's development as the organization narrows in on the looming deadline set by Millennium Development Goal 4, which aims to reduce child mortality by two thirds by 2015. Yet, in a sense, these efforts will be a continuation of the work Berkley has fostered at IAVI since he formally launched the New York–based nonprofit in 1996. Berkley, an epidemiologist who previously held jobs with the Rockefeller Foundation, the Carter Center and the US Centers for Disease Control and Prevention, has witnessed ups and downs in the vaccination field, from the disappointing STEP trial in 2007 to the more recent good news from the 2009 Thai study, which reported as much as 31% protection against HIV. spoke with Berkley about what he has learned in his quest for a preventative shot against AIDS. View full text Additional data Author Details * Roxanne Khamsi Search for this author in: * NPG journals * PubMed * Google Scholar - Mutations to the rescue
- Nat Med 17(4):405-407 (2011)
Nature Medicine | News Mutations to the rescue * Mike May1Journal name:Nature MedicineVolume: 17,Pages:405–407Year published:(2011)DOI:doi:10.1038/nm0411-405Published online07 April 2011 Genetic mutations are usually the source of debilitating disease. But, for a number of rare inherited blood and skin disorders, spontaneous DNA changes can repair and even reverse disease symptoms. explores the therapeutic potential of this 'natural gene therapy'. View full text Additional data Affiliations * Mike May is a freelance science writer living in Austin, Texas. Author Details * Mike May Search for this author in: * NPG journals * PubMed * Google Scholar - Missed connections
- Nat Med 17(4):408-410 (2011)
Nature Medicine | News Missed connections * Marissa Miley1Journal name:Nature MedicineVolume: 17,Pages:408–410Year published:(2011)DOI:doi:10.1038/nm0411-408Published online07 April 2011 A surprising percentage of people with autism also suffer from seizures, but doctors have been baffled by this overlap for decades. Now, various groups of scientists have begun exploring how the same genetic risk factors and aberrations in nerve signaling in early brain development might underlie both these disorders. reports on how solving this riddle could point to better treatments for epilepsy and autism. View full text Additional data Affiliations * Marissa Miley is an author and science writer based in New York. Author Details * Marissa Miley Search for this author in: * NPG journals * PubMed * Google Scholar - Engage with, don't fear, community labs
- Nat Med 17(4):411 (2011)
Nature Medicine | News | Opinion Engage with, don't fear, community labs * Ellen D Jorgensen1 * Daniel Grushkin2 * AffiliationsJournal name:Nature MedicineVolume: 17,Page:411Year published:(2011)DOI:doi:10.1038/nm0411-411Published online07 April 2011 The do-it-yourself biology movement has exploded in recent years, culminating in the formation of the world's first community laboratory, opened late last year. As this grassroots effort continues to grow, professional biomedical researchers stand to benefit from partnering with the legions of garage biotechnology enthusiasts. 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 * Molecular biologist Ellen D. Jorgensen is an adjunct assistant professor at the New York Medical College in Valhalla and the president of Genspace. * Daniel Grushkin is a science writer in Brooklyn, New York and vice president of Genspace. Author Details * Ellen D Jorgensen Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Grushkin Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - That obscure object of desire
- Nat Med 17(4):412 (2011)
Nature Medicine | Book Review That obscure object of desire * Jan Vijg1Journal name:Nature MedicineVolume: 17,Page:412Year published:(2011)DOI:doi:10.1038/nm0411-412Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. The Youth Pill: Scientists at the Brink of an Anti-Aging Revolution David Stipp Current, 2010 320 pp., hardcover, $26.95 ISBN: 1617230006 Buy this book: USUKJapan Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Demographic shifts will soon have a major impact on the world economy. Fifteen years from now, there will be almost 70 million Americans over the age of 65. In China, home to the most extraordinary economic growth spurt in history, there will soon be a ratio of only one working-age adult to every six people over age 65. In Europe the situation is grimly similar. As the incidence of virtually all major chronic diseases increases with age, lower birth rates and the ever-decreasing mortality rate among the elderly will require a steep boost in healthcare expenditures. View full text Author information Affiliations * Jan Vijg is at the Albert Einstein College of Medicine, Bronx, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Jan Vijg Author Details * Jan Vijg Contact Jan Vijg Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - The flip side of cardiolipin import
- Nat Med 17(4):413 (2011)
Nature Medicine | Correspondence The flip side of cardiolipin import * Coen C Paulusma1 * Roderick H J Houwen2 * Patrick L Williamson3 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Page:413Year published:(2011)DOI:doi:10.1038/nm0411-413aPublished online07 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. To the Editor: We read with interest the paper by Ray and colleagues1 reporting that elevated cardiolipin levels during pulmonary infection impair lung and lung surfactant function. They suggest that Atp8B1 is a cardiolipin importer that helps clear this lipid from lung surfactant and that it contains a 40-amino-acid cardiolipin-binding domain that reduces lung injury when introduced into infected lungs. We do appreciate the inference that elevated cardiolipin contributes to the etiology of pneumonia and the observation that Atp8B1 overexpression improves lung function after experimental pneumonia. However, the mechanism of pulmonary cardiolipin accumulation and the role of Atp8B1 therein is not illuminated by the experiments of Ray and colleagues1. 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 * Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands. * Coen C Paulusma * Department of Pediatrics, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands. * Roderick H J Houwen * Department of Biology, Amherst College, Amherst, Massachusetts, USA. * Patrick L Williamson Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Coen C Paulusma Author Details * Coen C Paulusma Contact Coen C Paulusma Search for this author in: * NPG journals * PubMed * Google Scholar * Roderick H J Houwen Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick L Williamson Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Reply to "The flip side of cardiolipin import"
- Nat Med 17(4):413-414 (2011)
Nature Medicine | Correspondence Reply to "The flip side of cardiolipin import" * Bill B Chen1 * Jian Fei Jiang2 * Nancy B Ray3 * Valerian E Kagan2 * Rama K Mallampalli1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:413–414Year published:(2011)DOI:doi:10.1038/nm0411-413bPublished online07 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Ray et al. reply: In their correspondence, Paulusma and colleagues1 question whether Atp8B1 functions in cardiolipin import in lung epithelia. Of note, the natural substrate for Atp8b1 has not been conclusively identified. Unlike many studies using functional assays showing phosphatidylserine flippase (out-to-in) activity of Atp8b1 and its yeast homologs (for example, Dnf2p; ref. 2), we have used both functional and biochemical approaches to map a cardiolipin binding domain (CBD). With regard to nitrobenzoxadiazole (NBD)-cardiolipin used in our studies3, we optimized solubility of this probe in medium by including apoprotein-containing Infasurf (5%). We show that NBD-cardiolipin is internalized in mouse embryonic cells4 and in lung epithelia where it is detected within lysosomes but not mitochondria (Fig. 1). We also observe that ectopically expressed Atp8b1 increases NBD-cardiolipin uptake in lung epithelia (Fig. 2). Figure 1: Cardiolipin within mouse lung epithelia. Lung epithelial cells (2 × 106 per dish) were labeled with fluorescent NBD-labeled cardiolipin (CL) for 15 min and co-stained with the lysosomal marker Lysotracker (Invitrogen, 1:10,000) for 30 min before confocal imaging. Shown are fluorescence uptake images in cells indicative of colocalization of CL with Lysotracker (arrows). * Full size image (56 KB) * Figures index * Next figure Figure 2: Cardiolipin import by Atp8b1 in mouse lung epithelia. Cells transduced with a control lentivirus or with a lentivirus encoding Atp8b1 were incubated with 25 μM dipalmitoylphosphatidylcholine-cardiolipin liposomes containing 1.25 μM NBD-cardiolipin (yellow) at 37 °C for 30 min. At the end of incubation, cells were washed with 2% fatty acid–free BSA in PBS and then fixed in 4% paraformaldehyde with 2 μg ml−1 Hoechst 33342 (blue) as a counterstain. Shown are images acquired with a Nikon Eclipse TE200 inverted scope demonstrating internalized NBD-CL within cells transduced with Atp8b1. View full text Figures at a glance * Figure 1: Cardiolipin within mouse lung epithelia. Lung epithelial cells (2 × 106 per dish) were labeled with fluorescent NBD-labeled cardiolipin (CL) for 15 min and co-stained with the lysosomal marker Lysotracker (Invitrogen, 1:10,000) for 30 min before confocal imaging. Shown are fluorescence uptake images in cells indicative of colocalization of CL with Lysotracker (arrows). * Figure 2: Cardiolipin import by Atp8b1 in mouse lung epithelia. Cells transduced with a control lentivirus or with a lentivirus encoding Atp8b1 were incubated with 25 μM dipalmitoylphosphatidylcholine-cardiolipin liposomes containing 1.25 μM NBD-cardiolipin (yellow) at 37 °C for 30 min. At the end of incubation, cells were washed with 2% fatty acid–free BSA in PBS and then fixed in 4% paraformaldehyde with 2 μg ml−1 Hoechst 33342 (blue) as a counterstain. Shown are images acquired with a Nikon Eclipse TE200 inverted scope demonstrating internalized NBD-CL within cells transduced with Atp8b1. 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 * Department of Internal Medicine, Acute Lung Injury Center of Excellence, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. * Bill B Chen & * Rama K Mallampalli * Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. * Jian Fei Jiang & * Valerian E Kagan * Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA. * Nancy B Ray Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rama K Mallampalli Author Details * Bill B Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Jian Fei Jiang Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy B Ray Search for this author in: * NPG journals * PubMed * Google Scholar * Valerian E Kagan Search for this author in: * NPG journals * PubMed * Google Scholar * Rama K Mallampalli Contact Rama K Mallampalli Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Journal home * Current issue * For authors * Subscribe * E-alert sign up * RSS feed Correspondence To the Editor: We read with interest the paper by Ray and colleagues reporting that elevated cardiolipin levels during pulmonary infection impair lung and lung surfactant function. 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- Nat Med 17(4):415-416 (2011)
Nature Medicine | Article Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity * Tracy L Flach1, 7 * Gilbert Ng1, 7 * Aswin Hari1 * Melanie D Desrosiers1 * Ping Zhang2 * Sandra M Ward2 * Mark E Seamone3 * Akosua Vilaysane3 * Ashley D Mucsi1 * Yin Fong1 * Elmar Prenner4 * Chang Chun Ling2 * Jurg Tschopp5 * Daniel A Muruve3 * Matthias W Amrein6 * Yan Shi1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:479–487Year published:(2011)DOI:doi:10.1038/nm.2306Received26 July 2010Accepted18 January 2011Published online13 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Abstract * Abstract * 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 As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4+ T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function–associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behav! e as a general sensor for solid structures. View full text Figures at a glance * Figure 1: Alum shows affinity for DC surface without inducing phagocytosis. () Left, serum IgG1 OVA-specific antibody titers 2 weeks after immunization with the indicated type of alum as adjuvant. Right, similar to the left except that clinical alum AlOH and AlPO4 were tested. () An SEM image of CsAl mounted on an AFM tip with epoxy. () Typical binding forces between a DC2.4 cell and four forms of alum. Blank refers to a tip with no alum attached. The number in parentheses indicates number of independent repeats of that operation (a representative curve for each is shown). At top right is a schematic depiction of the assay. The bottom right graph is a comparison of force curves between an MSU tip and an alum tip in their interaction with a DC2.4 cell. DC2.4 cell interaction with an MSU (or latex, not shown) tip is characterized by a gradual increase in binding strength and by a point of loss of retraction force curve indicated by the arrow (point of irreversible binding: phagocytosis). () SEM of DC2.4 cells cultured for 2 h with either CsAl (top) or! MSU (bottom). * Figure 2: Alum targets only DCs. () The binding forces of the indicated cells with a CsAl tip as compared to a blank tip. B cells (A20, Bjab, Raj and Ramos), macrophages (RAW 264.7, Ana Ry 1 and J744) and DCs (DC2.4 and PMA-transformed THP-1) were cultured on glass disks by direct adhesion or via a poly-D-lysine substrate coating. () Similar to except that GM-CSF plus IL-4 and DAP supernatant were used to differentiate BMDCs and macrophages, respectively, and magnetic cell sorting beads were used to purify splenic B cells. All examined with an Imject tip. () Environmental SEM image and EDS analysis of alum (CsAl precipitate) content in DC2.4 cells treated with alum for 2 h. Cells were washed three times without further treatment or gold sputter coating before data collection. In areas of high carbon reading, the aluminum (natural contaminant) in the glass is shielded from the electron beams due to the cell mass. () Similar to except that BMDCs from C57BL/6 or TLR4-knockout mice were contacted by AlOH. () Si! milar to except that DC2.4 cells were pretreated with 10 μg ml−1 of LPS for 30 min before the assay. * Figure 3: Alum's direct affinity for membrane lipids. () CsAl precipitate crystals were stained with equal molar concentrations of either phosphatidylcholine (PC), nitro-2-1,3-benzoxadiazol-4-yl (NBD), phosphatidylethanolamine (PE, NBD), sphingomyelin (Sph, BODIPY), or cholesterol (Chol, BODIPY). The bound fluorescence intensity was analyzed by FACS. () A synthetic cholesterol, SW.I.30, was attached to a gold-coated AFM tip via the thiol group at the end of the aliphatic chain extension. Top, a schematic depiction of the experiments. Left graph, the reading of the maximal attraction forces for SW.I.30. n represents the number of independent repeats. Control is a similar synthesis with the aliphatic extension from the hydroxyl group on the head of cholesterol. Right graph, a direct comparison between both cholesterol (SW.I.30) and synthetic sphingomyelin (PZ-3019) in their biological orientation in the membrane with reference to alum crystal contact. Blank, an unmodified tip. PZ-3019 is depicted at the bottom. () Synthetic bilay! er membranes from defined membrane lipids were laid on a glass surface for contact by an Imject tip. The red indicates the upper leaflet for contact. Average binding forces were recorded; the number on the top indicates the number of repeats. Error bars are s.e.m. * Figure 4: Alum triggers membrane lipid sorting and the activation of the Src-ITAM-Syk-PI3K pathway for DC activation. () Left, similar to Figure 1c except the CsAl binding with DC2.4 cell was recorded in the presence of the indicated inhibitors. Blank is a tip without alum. All other readings were recorded with an alum functionalized tip. Right, similar to the left except that an AlOH tip was used. () Similar to except that BMDCs cultured from wild-type (C57BL/6) or the indicated knockouts; Syk, Src (Hck, Fgr and Lyn triple-deficient) or ITAM (FcRγ and DAP12 double-deficient (Fcer1g−/− Tyrobp−/−)) were used in place of wild-type DCs without inhibitors. () Western blot analysis of total and phosphorylated Erk1/2 in DC2.4 and RAW 264.7 cells treated with alum. () Left, binding forces between DC2.4 or RAW 264.7 cells and AlOH-coated tips in the presence of the ndicated Erk inhibitor or activator. Right, similar to except DC2.4 and RAW 264.7 cells were pretreated with ferrocene and PD098059 before alum stimulation. () The draining LN CD11c+ cells were analyzed for Alexa levels 36 h aft! er subcutaneous injection of Alexa-OVA and alum mixture as described in the Online Methods. The numbers indicate the CD11c+Alexa+ cells as a percentage of the total cells. * Figure 5: Alum-mediated DC binding and activation requires the ITAM-Syk pathway but not Nlrp3-ASC. () Left, similar to Figure 4a except BMDCs from wild-type C57BL/6, Pycard−/− or Nlrp3−/− mice were used. Right, similar to graph on left except that an AlOH tip was used. () FACS analysis of CD40, CD80 and CD86 expression on BMDCs from wild-type, Nlrp3−/− and Pycard−/− mice left untreated or activated with MSU, bacteria (BAC, killed DH5αEscherichia coli) or CsAl precipitate for 24 h. () ELISA results of TNF-α (left) and IL-1β (right) production from DCs left untreated or activated with CpG, E. coli or CsAl precipitate for 6h. The loss of IL-1β production in Pycard−/− and Nlrp3−/− DCs confirms their deficiencies. () Similar to except BMDCs from wild-type, Syk-, Src- or ITAM-deficient Fcer1g−/− Tyrobp−/− DCs were used. MSU, known to trigger very little or no TNF-α production from BMDCs (our own observation), was used as a baseline control. * Figure 6: DCs following alum contact gain strong adhesion to CD4 T cells. () Alexa levels in untreated or BMDCs incubated with CsAl precipitate, Alexa-OVA and conjugated CsAl precipitate–Alexa-OVA as measured by FACS after 2 h. Piceatannol or cytochalasin B was also incubated with CsAl–Alexa-OVA conjugates. () Top left, splenic B or CD4+ cell binding by DC2.4 cells treated with CsAl precipitate for 4 h. Excess alum was washed away before the binding assay. The top right graph is similar to the left except that BMDCs from TLR4-knockout mice were treated with AlOH. Bottom right, identical data to those at top left depicted in a bar graph for better statistical representation; one-way analysis of variance (ANOVA) for all four is 0.014. () Similar to bottom right panel except that DCs were pretreated with soluble OVA for 2 h before the reading and that the approaching cell was a magnetic cell sorting–purified splenic OT-II T cell. One-way ANOVA for all four is 0.022. () Left, BMDC surface expression of ICAM-1 after treatment with AlOH, CsAl or C! pG for 24 h was analyzed by FACS with YN1/1.7 antibody. Right, similar to except that untreated, alum-treated wild-type or alum-treated Icam1−/− BMDCs were used to make contact with either wild-type CD4+ T cells or LFA-1–knockout CD4+ T cells. () SEM image of a magnetic cell sorting–purified untreated splenic DC (left) and a DC treated with alum with subsequent Histopaque gradient purification (top right). Bottom right, a DC recovered from the pellet. () Serum OVA-specific IgG1 titers measured by ELISA 2 weeks after C57BL/6 mice were intravenously immunized with OVA and DC treated with CsAl precipitate and purified with a Histopaque gradient. Standard immunization control is CsAl-based standard OVA immunization. Control is an untreated mouse serum. () Similar to , except that Nlrp3−/− DCs were used. () A proposed mechanism of how alum invokes the humoral immune response in vaccination. In response to the alum-antigen mixture, DC membrane lipids serve as a surrog! ate receptor to the crystal surface. The ensuing lipid sorting! triggers the aggregation of ITAM containing molecules, Syk recruitment and PI3K activation. However, alum does not enter DCs; it instead delivers the antigen into the DCs via endocytic uptake. DCs process the antigen in their MHC class II compartment and at the same time become activated as a consequence of the inflammatory phagocytosis. The activated DCs strongly engage CD4+ cells via ICAM-1 and LFA-1 binding, leading to the subsequent cognate B cell activation. Here cross-presentation of MHC class I antigens is absent, resulting in no CTL induction. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Tracy L Flach & * Gilbert Ng Affiliations * Immunology Research Group, Department of Microbiology & Infectious Diseases, and Snyder Institute, University of Calgary, Calgary, Alberta, Canada. * Tracy L Flach, * Gilbert Ng, * Aswin Hari, * Melanie D Desrosiers, * Ashley D Mucsi, * Yin Fong & * Yan Shi * Department of Chemistry and Alberta Ingenuity Center for Carbohydrate Science, University of Calgary, Calgary, Alberta, Canada. * Ping Zhang, * Sandra M Ward & * Chang Chun Ling * Department of Medicine, University of Calgary, Calgary, Alberta, Canada. * Mark E Seamone, * Akosua Vilaysane & * Daniel A Muruve * Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. * Elmar Prenner * Department of Biochemistry, University of Lausanne, Epalinges, Switzerland. * Jurg Tschopp * Department of Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada. * Matthias W Amrein Contributions Y.S. designed experiments with input from M.W.A. and wrote the manuscript with assistance from T.L.F. and D.A.M. T.L.F. performed the experiments unless indicated otherwise below. G.N. performed EDS and SEM assays and developed methods for lipid-crystal binding analysis. A.H. and A.D.M. performed CD4-DC binding analysis and EDS and SEM work and contributed to bilayer lipid synthesis. M.D.D. performed antibody induction and cytokine studies with assistance from Y.F. S.M.W., P.Z. and C.C.L. performed aliphatic chain extension on cholesterol and sphingomyelin. M.E.S. and A.V. performed western blotting. E.P. provided Langmuir trough and technical assistance. J.T. and D.A.M. provided inflammasome-deficient mice and technical assistance and consultation. M.W.A. supervised all aspects of work involving AFM. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yan Shi Author Details * Tracy L Flach Search for this author in: * NPG journals * PubMed * Google Scholar * Gilbert Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Aswin Hari Search for this author in: * NPG journals * PubMed * Google Scholar * Melanie D Desrosiers Search for this author in: * NPG journals * PubMed * Google Scholar * Ping Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra M Ward Search for this author in: * NPG journals * PubMed * Google Scholar * Mark E Seamone Search for this author in: * NPG journals * PubMed * Google Scholar * Akosua Vilaysane Search for this author in: * NPG journals * PubMed * Google Scholar * Ashley D Mucsi Search for this author in: * NPG journals * PubMed * Google Scholar * Yin Fong Search for this author in: * NPG journals * PubMed * Google Scholar * Elmar Prenner Search for this author in: * NPG journals * PubMed * Google Scholar * Chang Chun Ling Search for this author in: * NPG journals * PubMed * Google Scholar * Jurg Tschopp Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel A Muruve Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias W Amrein Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Shi Contact Yan Shi Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7, Supplementary Methods and Supplementary Data Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Trastuzumab resistance: all roads lead to SRC
- Nat Med 17(4):416-418 (2011)
Nature Medicine | Article Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways * Siyuan Zhang1 * Wen-Chien Huang1 * Ping Li1 * Hua Guo1 * Say-Bee Poh1 * Samuel W Brady1, 2 * Yan Xiong1 * Ling-Ming Tseng1 * Shau-Hsuan Li1 * Zhaoxi Ding1 * Aysegul A Sahin3 * Francisco J Esteva1, 2, 4 * Gabriel N Hortobagyi4 * Dihua Yu1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:461–469Year published:(2011)DOI:doi:10.1038/nm.2309Received03 June 2010Accepted21 January 2011Published online13 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Abstract * Abstract * 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 Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzuma! b-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance. View full text Figures at a glance * Figure 1: SRC hyperactivation is a key signaling alteration in acquired trastuzumab-resistant cells. () MTS assay comparing cell proliferation of indicated parental breast cancer cell lines and their corresponding acquired TtzmR sublines upon treatment with freshly added trastuzumab (Ttzm, 2 μg ml−1) for 4 d. () Tumor volume of mammary fat pad xenografts derived from either BT474 parental (P) or TtzmR subline upon treatment of IgG control or Ttzm (10 mg per kg body weight, intraperitoneally) weekly. Tumor volume at various times of treatment is presented as percentage of original tumor size at day 0 of treatment. () Representative histograms from flow cytometric analysis of EGFR and ERBB2 abundance in BT474 parental and TtzmR cells. () Relative amounts of EGFR, ERBB2, HER3 and IGF-1R in the indicated parental and TtzmR cells analyzed by flow cytometry. () Immunoblots comparing major cell signaling changes between the indicated parental and TtzmR sublines. P indicates phosphorylation; for example, P-EGFR-Y1068 is EFGR phosphorylated at Tyr1068 () Immunoblots assessing the! impact of overexpression of EGFR and IGF-1R in BT474 parental (BT474.P) cells on signaling. () MTS assay evaluating trastuzumab resistance of BT474 cells overexpressing EGFR or IGF-1R, treated as in . () Immunoblots of EGFR and phosphorylated SRC after shRNA-mediated EGFR knockdown in BT474.TtzmR cells. () MTS assay evaluating sensitivity of TtzmR to trastuzumab after EGFR knockdown. Cells were treated as in . () Left, immunoblots of knock-down of SRC in BT474.TtzmR cells by SRC shRNA. Right, MTS assay assessing trastuzumab sensitivity of TtzmR cells after SRC knockdown. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 2: SRC is activated in PTEN-deficient de novo trastuzumab-resistant cells. () MTS assay comparing cell proliferation of the indicated parental cell lines and their corresponding PTEN knockdown sublines upon trastuzumab treatment (Ttzm, 2 μg ml−1, 4 d). PTEN knockdown by either PTEN antisense (PTEN.as) oligonucleotides or PTEN shRNA is described in Online Methods. PTEN nonsense oligonucleotides, PTEN.ns. () Immunoblots assessing the effects of PTEN knockdown on phosphorylation of SRC Tyr416 (P-SRC-Y416) and other signals. () Immunoblots examining effects of inhibition of PI3K-AKT pathway on P-SRC-Y416 in PTEN knockdown cells. () Immunoblots assessing P-SRC-Y416 after reconstitution of wild-type (WT) PTEN, PTEN C124S or PTEN G129E mutants in PTEN-deficient MDA-MB-468 cells. () Coimmunoprecipitation assay to test for interactions between PTEN and SRC in BT474.P cells. () In vitro PTEN phosphatase assay detecting capability of purified GST-PTEN proteins (WT, C124S or G129E) to directly dephosphorylate the P-SRC-Y416 and P-SRC-Y527 phosphopeptides. (! ) Immunoblots evaluating the efficiency of SRC knockdown by SRC shRNA in BT474 PTEN knockdown cells. () MTS assay assessing trastuzumab sensitivity of PTEN knockdown cells with or without SRC knockdown. Cells were treated as in . All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 3: SRC is a key modulator of trastuzumab response. () Immunoblots comparing SRC phosphorylation status in the indicated cells expressing a constitutively active SRC mutant (Y527F) or a kinase-dead SRC mutant (K295R). () MTS assay assessing trastuzumab sensitivity of cells transfected with SRC Y527F or SRC K295R mutant. Cells were treated as in Figure 1a. Error bars, s.e.m. () 3D tumor spheroid assay comparing response to trastuzumab treatment of BT474.GFP and BT474.SRC Y527F. 3D tumor spheroid assay was carried out as described in Online Methods. Scale bar, 100 μm. () Top, representative BT474 orthotopic xenograft tumors. Scale bar, 1 cm. Bottom, volume of mammary fat pad xenograft tumors derived from either GFP-labeled BT474 parental (GFP) or SRC Y527F–expressing cells upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is percentage of original tumor size at day zero of treatment. Error bars, s.e.m. Ttzm-treated GFP group versus SRC Y527F group (ANOVA, P < 0.001, two-sided). () Correlation betwee! n clinical response rate and amount of tumor phospho-SRC-Y416 (pSRC) in patients who received first-line trastuzumab-based therapy. Complete response (CR), partial response (PR) and stable disease (SD) were grouped together and compared with SD. Patient response was compared by Fisher's exact test (P = 0.011, two-sided). () Low versus high tumor phospho-SRC-Y416 abundance and overall survival of patients who received first-line trastuzumab-based therapy. Difference of overall survival was analyzed by Kaplan-Meier survival model with log-rank test (P = 0.044, two-sided). * Figure 4: SRC inhibition induced signaling alterations in multiple trastuzumab-resistant models. () Immunoblots detecting EGF-induced EGFR dimerization in BT474.TtzmR cells stably infected with control shRNA or SRC shRNA. Cross-linking of cell membrane EGFR is described in Online Methods. MW, molecular weight. () Immunoblots comparing EGFR phosphorylation upon EGF treatment in BT474.TtzmR cells with or without SRC knockdown. () Immunoblots assessing EGFR signaling in BT474.TtzmR cells upon treatment with the SRC inhibitor saracatinib with or without EGF treatment. Cells were pretreated with saracatinib or vehicle 1 h before EGF stimulation. () Immunoblots assessing abundance of HER3 phosphorylation and other signaling events upon treatment with saracatinib alone, trastuzumab alone or combination treatment. () Immunoblots evaluating HER3 activation in response to trastuzumab treatment after stable knockdown of SRC in TtzmR cells by shRNA. (–) Immunoblots showing the impact of trastuzumab plus saracatinib on AKT signaling in multiple trastuzumab-resistant models: BT474.! TtzmR (), PTEN knockdown (), or constitutively active SRC mutant (SRC Y527F) expressing (). * Figure 5: Trastuzumab treatment plus SRC inhibition overcomes multiple resistance mechanisms in vitro. () MTS assay examining the effect of SRC inhibition in combination with trastuzumab treatment in the indicated four trastuzumab-resistant models. BT474.TtzmR cells, cells overexpressing IGF-1R and EGFR, and PTEN.shRNA cells were treated as described in Online Methods. () MTS assay evaluating the effects of trastuzumab, saracatinib or combination treatment in control BT474-GFP and trastuzumab-resistant cells overexpressing SRC Y527F. () 3D tumor spheroid assay comparing the cell proliferation of BT474.TtzmR cells upon treatment with trastuzumab alone, saracatinib alone or combination treatment. Tumor spheroid assay was carried out as described in Online Methods. Scale bar, 100 μm. () MTS assay comparing SRC inhibition by saracatinib and AKT inhibition by triciribine on overcoming trastuzumab resistance. () TUNEL assay examining induction of apoptosis by saracatinib and trastuzumab combined treatment in control (Con.shRNA) and PTEN knockdown (PTEN.shRNA) cells. TUNEL-positive! cells were detected by flow cytometry. () The induction of DNA fragmentation (indicated by sub-G1 population detected by flow cytometry) by combined treatment with saracatinib and trastuzumab in BT474.PTEN knockdown cells (PTEN.as) and BT474.SRC constitutively active (Y527F) cells. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 6: Trastuzumab plus saracatinib combinatorial treatment overcomes trastuzumab resistance in vivo. () Top, representative immunofluorescence images of SRC knockdown in PTEN.shRNA xenografts using intratumoral injection of SRC.shRNA-containing virus. Scale bar, 100 μm. Bottom, volume of trastuzumab-resistant PTEN-deficient tumors with or without SRC knockdown upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is presented as percentage of original tumor size at day zero of treatment. () Top, representative immunohistochemistry (IHC) images of in vivo inhibition of SRC-Y416 phosphorylation by saracatinib or AKT-S473 phosphorylation by triciribine in BT474.TtzmR xenograft tumors. Scale bar, 100 μm. Bottom, TtzmR xenograft tumor volume in response to different treatments. () Left, representative in vivo luciferase images of mice at day 0 and 21 days after indicated treatment. Left side of animal, BT474 control.shRNA tumors; right side, BT474 PTEN.shRNA tumors. Right, tumor volume in response to different treatments. () Left, representative IHC stain! ing of AKT-S473 phosphorylation after different treatments (vehicle or trastuzumab plus saracatinib) in BT474.PTEN.shRNA xenograft tumors. Scale bar, 50 μm. Right, overall AKT-S473 phosphorylation IHC staining intensity between trastuzumab-alone group and combination-treatment group. Phospho-AKT (pAKT) staining was compared between each group by Fisher's exact test (P = 0.049, two-sided). () Top, representative tumor sections with TUNEL staining. Scale bar, 50 μm. Bottom, in situ TUNEL staining of apoptotic cells in tumors treated as indicated. All error bars, s.e.m. All in vivo data were generated from a minimum of five replicates. () Model of SRC as a common node downstream of multiple resistance pathways and conquering trastuzumab resistance by targeting SRC. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Siyuan Zhang, * Wen-Chien Huang, * Ping Li, * Hua Guo, * Say-Bee Poh, * Samuel W Brady, * Yan Xiong, * Ling-Ming Tseng, * Shau-Hsuan Li, * Zhaoxi Ding, * Francisco J Esteva & * Dihua Yu * Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, USA. * Samuel W Brady, * Francisco J Esteva & * Dihua Yu * Department of Pathology, MD Anderson Cancer Center, Houston, Texas, USA. * Aysegul A Sahin * Department of Breast Medical Oncology, MD Anderson Cancer Center, Houston, Texas, USA. * Francisco J Esteva & * Gabriel N Hortobagyi Contributions S.Z., W.-C.H. and D.Y. designed experiments and analyzed data; S.Z., W.-C.H., H.G., P.L., S.-B.P., S.W.B., Y.X., L.-M.T. and Z.D. carried out experiments; S.Z., H.G. and S.-H.L. did statistical analysis of clinical data; A.A.S. collected tumor samples and evaluated immunohistochemistry staining with H.G.; G.N.H. and F.J.E. collected clinical patient information and analyzed patient response data; S.Z., S.W.B. and D.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dihua Yu Author Details * Siyuan Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Wen-Chien Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Ping Li Search for this author in: * NPG journals * PubMed * Google Scholar * Hua Guo Search for this author in: * NPG journals * PubMed * Google Scholar * Say-Bee Poh Search for this author in: * NPG journals * PubMed * Google Scholar * Samuel W Brady Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Ling-Ming Tseng Search for this author in: * NPG journals * PubMed * Google Scholar * Shau-Hsuan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaoxi Ding Search for this author in: * NPG journals * PubMed * Google Scholar * Aysegul A Sahin Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco J Esteva Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriel N Hortobagyi Search for this author in: * NPG journals * PubMed * Google Scholar * Dihua Yu Contact Dihua Yu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (524K) Supplementary Figures 1–22, Supplementary Table 1 and Supplementary Methods Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - An amino acid profile to predict diabetes?
- Nat Med 17(4):418-420 (2011)
Nature Medicine | Article Metabolite profiles and the risk of developing diabetes * Thomas J Wang1, 2, 3 * Martin G Larson3, 4 * Ramachandran S Vasan3, 5 * Susan Cheng2, 3, 6 * Eugene P Rhee1, 7, 8 * Elizabeth McCabe2, 3 * Gregory D Lewis1, 2, 8 * Caroline S Fox3, 9, 10 * Paul F Jacques11 * Céline Fernandez12 * Christopher J O'Donnell2, 3, 8 * Stephen A Carr8 * Vamsi K Mootha8, 13, 14 * Jose C Florez8, 13 * Amanda Souza8 * Olle Melander15 * Clary B Clish8 * Robert E Gerszten1, 2, 8 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:448–453Year published:(2011)DOI:doi:10.1038/nm.2307Received07 April 2010Accepted19 January 2011Published online20 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Abstract * Abstract * 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 Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines and other polar metabolites were profiled in baseline specimens by liquid chromatography–tandem mass spectrometry (LC-MS). Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly significant associations with future diabetes: isoleucine, leucine, valine, tyrosine and phenylalanine. A combination of three amino acids predicted future diabetes (with a more than fivefold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential key role of amino acid metabolism early in the pathogenesis of diabetes and s! uggest that amino acid profiles could aid in diabetes risk assessment. View full text Author information * Abstract * Author information * Supplementary information Affiliations * Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Thomas J Wang, * Eugene P Rhee, * Gregory D Lewis & * Robert E Gerszten * Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Thomas J Wang, * Susan Cheng, * Elizabeth McCabe, * Gregory D Lewis, * Christopher J O'Donnell & * Robert E Gerszten * Framingham Heart Study of the National Heart, Lung, and Blood Institute and Boston University School of Medicine, Framingham, Massachusetts, USA. * Thomas J Wang, * Martin G Larson, * Ramachandran S Vasan, * Susan Cheng, * Elizabeth McCabe, * Caroline S Fox & * Christopher J O'Donnell * Department of Mathematics and Statistics, Boston University, Boston, Massachusetts, USA. * Martin G Larson * Cardiology Section, Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts, USA. * Ramachandran S Vasan * Division of Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Susan Cheng * Renal Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Eugene P Rhee * Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. * Eugene P Rhee, * Gregory D Lewis, * Christopher J O'Donnell, * Stephen A Carr, * Vamsi K Mootha, * Jose C Florez, * Amanda Souza, * Clary B Clish & * Robert E Gerszten * Division of Endocrinology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Caroline S Fox * National Heart, Lung, and Blood Institute, Division of Intramural Research, Bethesda, Maryland, USA. * Caroline S Fox * Jean Mayer US Department of Agriculture Human Nutrition Research Center, Tufts University, Boston, Massachusetts, USA. * Paul F Jacques * Department of Experimental Medical Science, Lund University, Malmö, Sweden. * Céline Fernandez * Diabetes Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Vamsi K Mootha & * Jose C Florez * Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Vamsi K Mootha * Department of Clinical Sciences, Lund University, Malmö, Sweden. * Olle Melander Contributions T.J.W. conceived of the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. A.S. and E.P.R., under the direction of C.B.C., developed the metabolic profiling platform, performed mass spectrometry experiments and analyzed the data. S.A.C. and V.K.M. helped in the establishment of the metabolite profiling platform and manuscript revision. G.D.L. contributed to data analysis and manuscript generation. M.G.L., R.S.V., S.C. and E.M. helped in experimental design, performed statistical analyses and assisted in manuscript generation. C.J.O. and C.S.F. helped in experimental design and manuscript revision. P.F.J. directed the dietary analyses in the Framingham Heart Study and contributed to manuscript revision. J.C.F. assisted in the interpretation of the data and contributed to manuscript revision. O.M. and C.F. performed the replication analyses in the Malmö Diet and Cancer cohort and contributed to manuscript revision. R.E.G. conceived of! the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. Competing financial interests T.J.W., R.S.V., M.G.L., V.K.M. and R.E.G. are named as co-inventors on a patent application to the US Patent Office pertaining to metabolite predictors of diabetes. J.C.F. has received consulting honoraria from Publicis Healthcare, Merck, bioStrategies, XOMA and Daiichi-Sankyo and has been a paid invited speaker at internal scientific seminars hosted by Pfizer and Alnylam Pharmaceuticals. Corresponding authors Correspondence to: * Thomas J Wang or * Robert E Gerszten Author Details * Thomas J Wang Contact Thomas J Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Martin G Larson Search for this author in: * NPG journals * PubMed * Google Scholar * Ramachandran S Vasan Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Eugene P Rhee Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth McCabe Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory D Lewis Search for this author in: * NPG journals * PubMed * Google Scholar * Caroline S Fox Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F Jacques Search for this author in: * NPG journals * PubMed * Google Scholar * Céline Fernandez Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J O'Donnell Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen A Carr Search for this author in: * NPG journals * PubMed * Google Scholar * Vamsi K Mootha Search for this author in: * NPG journals * PubMed * Google Scholar * Jose C Florez Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Souza Search for this author in: * NPG journals * PubMed * Google Scholar * Olle Melander Search for this author in: * NPG journals * PubMed * Google Scholar * Clary B Clish Search for this author in: * NPG journals * PubMed * Google Scholar * Robert E Gerszten Contact Robert E Gerszten Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (127K) Supplementary Figure 1, Supplementary Tables 1–4 and Supplementary Methods Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Derailing heterotopic ossification and RARing to go
- Nat Med 17(4):420-421 (2011)
Nature Medicine | Article Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists * Kengo Shimono1, 4 * Wei-en Tung1, 4 * Christine Macolino1 * Amber Hsu-Tsai Chi1 * Johanna H Didizian1, 4 * Christina Mundy1 * Roshantha A Chandraratna2 * Yuji Mishina3 * Motomi Enomoto-Iwamoto1, 4 * Maurizio Pacifici1, 4 * Masahiro Iwamoto1, 4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:454–460Year published:(2011)DOI:doi:10.1038/nm.2334Received15 September 2010Accepted18 February 2011Published online03 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Abstract * Abstract * 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 Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-li! ke kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans. View full text Figures at a glance * Figure 1: RAR agonists block chondrogenesis and intramuscular rBMP-2-driven heterotopic ossification. () Micromass cultures of mesenchymal cells from E11.5 mouse limb treated with increasing doses of all-trans-retinoic acid (RA) or the RAR-γ agonist NRX204647 and stained with alcian blue on day 8. () Similar micromass cultures prepared from E11.5 RAR-γ–null or wild-type (WT) limb mesenchymal cells and treated with all-trans-retinoic acid (100 nM) for 8 d. () Micromass cultures prepared from dual RAR-α– and RAR-β–deficient (αβ-def) or wild-type limb mesenchymal cells and treated with all-trans-retinoic acid as above. () Heterotopic ossification masses induced by implantation of rBMP-2-filled collagen sponge into a microsurgically created pocket in the calf muscles. The mice were treated with vehicle, all-trans-retinoic acid (12 mg per kg body weight per day) or NRX204647 (1.2 mg per kg body weight per day) by gavage. Large round mineralized heterotopic ossification masses were visible by μCT in mice that received vehicle, markedly decreased in mice treated with r! etinoic acid and absent in NRX204647-treated mice. Ectopic tissues were sectioned and examined by Masson trichrome (MT) and alcian blue (AB) staining, immunofluorescence for myosin heavy chain (MHC: red) and osteocalcin (OC: green) and TRAP staining. Scale bar for μCT, 5 mm; for histology, 200 μm. () Heterotopic ossification tissue induced by subcutaneous implantation of rBMP-2/Matrigel mixture. Treatment and analysis of ectopic tissue were as above. Scale bar for μCT, 5 mm; for alcian blue, 2.5 mm. * Figure 2: Effectiveness of different retinoids against heterotopic ossification. () Chemical structures of synthetic RAR-γ agonists and natural 13-cis-retinoic acid. () Suppression of heterotopic ossification by retinoids evaluated by measuring BV/TV in ectopic masses in control versus retinoid-treated mice by μCT on day 12. Values from control groups were set at 100% and used to calculate relative values in experimental groups. Each group had ≥ 8 samples; data shown as means ± s.e.m. (*P < 0.01 versus control). () Macro views (top), soft X-ray radiograms (middle) and histological sections stained with H&E (bottom) of ectopic masses collected from mice treated with vehicle or CD1530. Scale bar in top and middle images, 1 cm; bottom images, 100 μm. () Selectivity of RAR-γ agonist action. Subcutaneous heterotopic ossification was triggered in wild-type, heterozygous RAR-γ (HT) and RAR-γ–null (KO) mice, and mice were treated with RAR-γ agonist CD1530 (4 mg per kg body weight per day) or vehicle for 12 d. *P < 0.01 versus control. () Evaluation o! f rebound effects. Mice implanted with rBMP-2–Matrigel mixture subcutaneously were treated with vehicle, CD1530 or RAR-α agonist NRX195183 for 10 days. Treatment was stopped, and heterotopic ossification was evaluated by μCT at later time points. () Window of opportunity tests. Mice implanted as above were left untreated up to day 6 and were then treated with CD1530 or NRX195183 until day 12. * Figure 3: RAR-γ agonists block FOP-like heterotopic ossification. () ADTC5 cells expressing the strong constitutively active ALK2 Q207D or control empty vector were transfected with the BMP signaling reporter Id1-luc and then treated with 0, 10, 30 or 100 nM CD1530. Reporter activity was normalized to phRG-TK encoding Renilla luciferase. Error bars represent s.d. for triplicate samples. () ALK2 Q207D–transgenic mouse model of FOP-like heterotopic ossification. Consecutive soft X-ray images of the same mice, injected with Ad-Cre and cardiotoxin, taken at P7, P21 and P35 showing massive heterotopic ossification in vehicle-treated mice (double arrowheads), but markedly less in companion mice treated with CD1530 (4.0 mg per kg body weight per day). Scale bar, 1 cm. () μCT images showing massive heterotopic ossification in vehicle-treated ALK2 Q207D–transgenic mice injected with Ad-Cre and cardiotoxin (double arrowheads) but not in companion CD1530-treated mice. Scale bar, 1 cm. () Bright-field (BF) and fluorescent images showing ectopic s! keletal tissue (positive for alizarin complexon, AR) in vehicle-treated ALK2 Q207D–transgenic mice injected with Ad-Cre and cardiotoxin but not in those treated with CD1530. Positive GFP fluorescence confirmed that Ad-Cre had activated transgene expression in all mice. There was minimal GFP and AR fluorescence in mice that had not been injected with Ad-Cre and cardiotoxin. Scale bar, 5 mm. * Figure 4: Mechanisms of RAR-γ agonist action. () Id1-luc reporter activity in ATDC5 cells. Error bars represent s.d. for triplicate samples. () Immunoblots showing phosphorylated Smad1, Smad5 and Smad8 (p-Smad1,5,8) protein amounts in control (DMSO) ATDC5 cells treated with rBMP-2 and in cells treated with CD1530 (top) and overall Smad1 amounts in control cells and in CD1530-treated cells (bottom). Membranes were reblotted with α-tubulin–specific antibodies for normalization. () Dual Smad1 and β-actin immunofluorescence staining and nuclear DAPI staining showing that Smad1 was largely cytoplasmic in control cells and relocated to the nucleus during rBMP-2 treatment, but there was minimal detectable Smad1 signal in cytoplasm or nucleus of cells concurrently treated with CD1530. Red, Smad1; green, β-actin; blue, nuclei. () Immunoblots similar to those in showing that the overall amounts of Smad1, Smad4 and Smad5 with or without CD1530 and rBMP-2 treatment. () Immunoblots showing that the decreases in Smad1 levels eli! cited by CD1530 treatment were counteracted by co-treatment with the proteasome inhibitors AW9155 or PI-108. * Figure 5: RAR-γ agonists reprogram the differentiation potentials of skeletal progenitor cells. (,) ATDC5 cells were grown in the presence of vehicle (control) or 1 μM CD1530 for 2 d, rinsed, replated and then grown for an additional 7 d with or without 100 ng ml−1 rBMP-2. Control cells responded well to rBMP-2 and underwent chondrogenic differentiation revealed by strong alcian blue () and alkaline phosphatase () staining, but the cells pretreated with CD1530 did not and failed to stain. () Similar experiments with GFP-expressing MSCs derived from mouse bone marrow show that the cells underwent differentiation upon rBMP-2 treatment (top) but CD1530 pretreated MSCs did not and failed to stain with alizarin red (bottom). () Immunoblots showing the steady-state amounts of Smad1 and Smad4 in MSCs treated without (−) or with (+) 1 μM CD1530 for 12 h. () Id1-luc reporter activity in control MSCs treated with or without rBMP-2 in the presence or absence of CD1530. Error bars represent s.d. for four wells. () Control and CD1530-pretreated GFP-expressing MSCs were mixed ! with rBMP-2–Matrigel and implanted in nude mice subcutaneously, and ectopic tissue masses were analyzed by μCT, H&E staining, osteocalcin (OC) immunostaining and GFP fluorescence signal. Bottom, merged GFP and OC images. Scale bars, 5 mm (top row); 250 μm (second row); 150 μm (third row). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Orthopaedic Surgery, Thomas Jefferson University College of Medicine, Philadelphia, Pennsylvania, USA. * Kengo Shimono, * Wei-en Tung, * Christine Macolino, * Amber Hsu-Tsai Chi, * Johanna H Didizian, * Christina Mundy, * Motomi Enomoto-Iwamoto, * Maurizio Pacifici & * Masahiro Iwamoto * Io Therapeutics, Irvine, California, USA. * Roshantha A Chandraratna * School of Dentistry, University of Michigan, Ann Arbor, Michigan, USA. * Yuji Mishina * Present address: The Children's Hospital of Philadelphia, Division of Orthopaedic Surgery, Philadelphia, Pennsylvania, USA. * Kengo Shimono, * Wei-en Tung, * Johanna H Didizian, * Motomi Enomoto-Iwamoto, * Maurizio Pacifici & * Masahiro Iwamoto Contributions M.I. and M.P. directed the project. K.S., W.T., C.M., A.H.-T.C., J.H.D., C.M. and M.E.-I. performed experiments, analyzed data and participated in experimental design. R.A.C. provided expertise on retinoid biology. Y.M. provided expertise in mouse genetics. M.P., K.S. and M.I. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Masahiro Iwamoto or * Maurizio Pacifici Author Details * Kengo Shimono Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-en Tung Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Macolino Search for this author in: * NPG journals * PubMed * Google Scholar * Amber Hsu-Tsai Chi Search for this author in: * NPG journals * PubMed * Google Scholar * Johanna H Didizian Search for this author in: * NPG journals * PubMed * Google Scholar * Christina Mundy Search for this author in: * NPG journals * PubMed * Google Scholar * Roshantha A Chandraratna Search for this author in: * NPG journals * PubMed * Google Scholar * Yuji Mishina Search for this author in: * NPG journals * PubMed * Google Scholar * Motomi Enomoto-Iwamoto Search for this author in: * NPG journals * PubMed * Google Scholar * Maurizio Pacifici Contact Maurizio Pacifici Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Iwamoto Contact Masahiro Iwamoto Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (815K) Supplementary Figures 1–5 and Supplementary Methods Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - A new treasure in the breast cancer gene hunt
- Nat Med 17(4):422-423 (2011)
Nature Medicine | News and Views A new treasure in the breast cancer gene hunt * Paul Spellman1 * Joe Gray2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:422–423Year published:(2011)DOI:doi:10.1038/nm0411-422Published online07 April 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. A new potential breast cancer oncogene, ZNF703, has been identified in the chromosomal region 8p12 in humans, which is commonly amplified in an aggressive subtype of breast cancer. ZNF703 is a transcriptional repressor and regulates many genes that are involved in multiple aspects of the cancer phenotype, such as increased proliferation, invasion and an altered balance of progenitor stem cells. 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 * Paul Spellman is at the Lawrence Berkeley National Laboratory, Berkeley, California, USA, and the US National Cancer Institute, Bethesda, Maryland, USA. * Joe Gray is at Oregon Health and Science University, Portland, Oregon, USA. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joe Gray Author Details * Paul Spellman Search for this author in: * NPG journals * PubMed * Google Scholar * Joe Gray Contact Joe Gray Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Axonal injury in reverse
- Nat Med 17(4):423-426 (2011)
Nature Medicine | Letter A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis * Ivana Nikić1 * Doron Merkler2, 3 * Catherine Sorbara1 * Mary Brinkoetter4 * Mario Kreutzfeldt2, 3 * Florence M Bareyre1 * Wolfgang Brück2 * Derron Bishop4 * Thomas Misgeld5, 6, 7 * Martin Kerschensteiner1, 7 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:495–499Year published:(2011)DOI:doi:10.1038/nm.2324Received27 July 2010Accepted07 February 2011Published online27 March 2011 Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits1, 2. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view2, 3, 4, 5, 6, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacol! ogical experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy. View full text Figures at a glance * Figure 1: In vivo imaging of FAD. () Confocal projection showing axons (white), activated macrophages/microglia (magenta) and T cells (cyan) in an acute spinal EAE lesion. Some axons appear normal (stage 0), whereas others are swollen (1) or fragmented (2). () Pseudocolored axons isolated from those shown in : normal appearing (0, green), swollen (1, yellow), and fragmented (2, red). () Frequency (in % ± s.e.m.) of axon stages in normal spinal cord (N) and in EAE lesions (0–30 d after EAE onset; differences at all EAE time points compared to control are significant, P < 0.001 to 0.05, one-tailed t test). () Multiphoton time-lapse images of a stage 1 axon (white) in EAE and activated macrophages/microglia (magenta). Time is shown as h:min; meningeal second harmonics (a scattering process) signal is green). The axon first breaks (red arrowhead) near a small swelling (yellow arrowhead) at a putative node of Ranvier before fragmenting (gray arrowheads). () Fate of stage 1 axons imaged 1–3 d after the peak o! f EAE (significant progression from 1–3 d, P < 0.05, chi-square test for trend). () Time-lapse images of recovering stage 1 axon (time is as shown in ). Scale bar in ,, 10 μm; scale bar in , 25 μm; scale bar in , 10 μm. * Figure 2: Early FAD stages show mitochondrial alterations but no demyelination. (–) Electron micrograph of a stage 1 axon with a paranodal swelling and preserved myelin (pseudocolored orange-brown; paranodal loops magnified and marked by arrows in ). The axon contains intact looking (green, example magnified in ) and swollen (red, magnified in ) mitochondria. () Top, confocal image of a myelinated stage 1 axon (white; FluoroMyelin, orange, arrows; nuclei, magenta). Bottom, magnified view of myelinated stage 1 axon (nuclear staining omitted). () Light microscopic quantification of axon myelination (stage 0–2 axons) at the onset of weight loss (n = 74 axons) and 2 d after clinical EAE onset (first clinical sign of EAE, minimum score of 0.5) (n = 111 axons). () Shape factor histograms of mitochondria from electron microscopy images of control (top) and stage 1 EAE axons (bottom; n = 149 and 138 mitochondria, respectively). Green indicates proportion of mitochondria with normal ultrastructure; red represents disrupted mitochondria. () Mitochondrial shap! e versus mitochondrial membrane potential in EAE lesions (green, normal; red, low (< mean − 2 s.d. of control), n = 235 mitochondria in four mice). () Confocal quantification of mean mitochondrial shape factor in axons from EAE and normal control (N) mice (n = 23–138 mitochondria per axon in stage 0 (green; myelination status was scored in additional stage 0 axons) and 1 (yellow); in stage 2 (red) all mitochondria were scored). () Left, confocal image of axons (gray) and their mitochondria (cyan) in EAE (nuclei, magenta). Right, shape of mitochondria (color coded, see scale at right) in axons at different stages of FAD selected from left panel. Stage and mean mitochondrial shape factor (SF ± s.e.m.) of the axon segment are indicated above each axon. Scale bar in , 2 μm; scale bars in –, 0.5 μm; scale bar in (top), 10 μm; scale bar in (bottom), 5 μm; scale bar in (left and right images), 10 μm. * Figure 3: Activated macrophage/microglia-derived reactive species induce FAD. () Confocal reconstruction of an axon that courses through an EAE lesion (nuclei, magenta) in a mouse with sparse axon labeling (white) and dense labeling of axonal mitochondria (cyan). Enlarged areas below show color-coded (scale on right) shape of mitochondria in the labeled axon outside (left) and inside (right) the lesion. () Quantification of mitochondrial shape in different segments of longitudinally reconstructed axons (segments of each axon connected with dashed line, n = 4 axons) outside (green) and inside (red) of EAE lesions. Each triangle represents a mitochondrion, and circles indicate mean mitochondrial shape factor for the axon segment (± s.e.m.; mitochondria are significantly shorter inside versus outside; P < 0.05, paired t test). () Infiltration density (± s.e.m.) around control (N, gray) and EAE stage 0 (green), 1 (yellow) and 2 (red) axon segments (n = 13–83 segments; infiltration density is significantly increased in stage 0 vs. control axons, stage ! 1 vs. stage 0 axons and stage 2 vs. stage 1 axons; t test). () Cell density (± s.e.m.) around stage 1 axon segments that either recovered (green) or persisted in a swollen state (yellow) during in vivo imaging and were fixed afterward for analysis of cellular infiltration (n = 60 segments; density is significantly lower around recovered axons; t test). () In vivo measurement of H2O2 concentration (detected with Amplex) in the dorsal spinal cord of healthy (N, left) and EAE (2 d after onset) mice. () Quantification of H2O2 concentration (± s.e.m; n = 3–5 mice per time point; levels are significantly increased at 0 d and 2 d after EAE onset; t test). N, control; Pre, preclinical mice; d, days after EAE onset. () In vivo two-photon time-lapse of spinal axons (gray) and their mitochondria (cyan) after H2O2 application (330 mM) (time, h:min). Right, magnification of small cluster of mitochondria that swell over time (arrows). () Frequency of stage 0 (green), 1 (yellow) and 2! (red) axons before and up to 5 h after in vivo H2O2 applicati! on (100 mM; n = 53 axons). Superimposed is the change over time in mean mitochondrial shape (± s.e.m., n = 15 axons; t test). () Percentage (± s.e.m.) of EAE axons in different stages of FAD after ROS and RNS scavenger or vehicle treatment (starting at weight loss, analyzed by t test). () Fate of stage 1 axons after 2 d of ROS and RNS scavenger or vehicle treatment (started 2–3 d after EAE onset, n = 22–26 axons; chi-square test). Scale bar in , 50 μm; scale bar in , 250 μm (calibration bar); scale bar in , 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 4: Axonal changes consistent with FAD are present in acute human multiple sclerosis lesions. () Representative axons in stages 0, 1 and 2 of FAD in an acute human multiple sclerosis lesion (Bielschowsky silver impregnation). () Prevalence of FAD stages in normal-appearing white matter (NAWM) and in acute multiple sclerosis lesions (n = 3 biopsies, > 450 axons per group; all stages are significantly different in lesion versus NAWM; one-tailed t test). () Confocal projections of an axon (top) that was located in NAWM around an active multiple sclerosis lesion, and examples of stage 0, 1 and 2 axons located inside the same lesion (quadruple immunostaining: axons, stained for neurofilament, white; myelin, stained for myelin basic protein, orange; nuclei, stained for NeuroTrace, magenta; mitochondria, stained for porin, cyan; mitochondria magnified in insets). () Myelination status of axons in NAWM and FAD stage 0 and 1 axons in multiple sclerosis lesions (n = 17–22 axons per group, from two to six biopsies). () Comparison of mean mitochondrial shape factors of axons i! n NAWM (N), and stage 0 and 1 multiple sclerosis axons (analyzed by t test). Scale bars in ,, 10 μm; scale bars in (insets), 1 μm. *P < 0.05; ***P < 0.001. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Thomas Misgeld & * Martin Kerschensteiner Affiliations * Research Unit Therapy Development, Institute of Clinical Neuroimmunology, Ludwig-Maximilians-Universität München, Munich, Germany. * Ivana Nikić, * Catherine Sorbara, * Florence M Bareyre & * Martin Kerschensteiner * Institute of Neuropathology, Georg-August University, Göttingen, Germany. * Doron Merkler, * Mario Kreutzfeldt & * Wolfgang Brück * Division of Clinical Pathology, Geneva University Hospital and Department of Pathology and Immunology, University of Geneva, Switzerland. * Doron Merkler & * Mario Kreutzfeldt * Department of Physiology, Indiana University School of Medicine-Muncie, Muncie, Indiana, USA. * Mary Brinkoetter & * Derron Bishop * Chair for Biomolecular Sensors, Center for Integrated Protein Sciences (Munich) at the Institute of Neuroscience, Technische Universität München, Munich, Germany. * Thomas Misgeld * Institute for Advanced Study, Technische Universität München, Munich, Germany. * Thomas Misgeld Contributions M. Kerschensteiner, T.M., D.B., D.M. and I.N. conceived the experiments. I.N. and C.S. did the imaging experiments. I.N., C.S., T.M. and M. Kerschensteiner did image analysis. M.B. and D.B. did and evaluated serial electron microscopy. I.N. and F.M.B. did therapy experiments. D.M., M. Kreutzfeldt and W.B. did histopathological evaluations of EAE and multiple sclerosis tissue. I.N., M. Kerschensteiner and T.M. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Martin Kerschensteiner or * Thomas Misgeld Author Details * Ivana Nikić Search for this author in: * NPG journals * PubMed * Google Scholar * Doron Merkler Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Sorbara Search for this author in: * NPG journals * PubMed * Google Scholar * Mary Brinkoetter Search for this author in: * NPG journals * PubMed * Google Scholar * Mario Kreutzfeldt Search for this author in: * NPG journals * PubMed * Google Scholar * Florence M Bareyre Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Brück Search for this author in: * NPG journals * PubMed * Google Scholar * Derron Bishop Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Misgeld Contact Thomas Misgeld Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Kerschensteiner Contact Martin Kerschensteiner Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (422K) In vivo multi-photon time-lapse that illustrates the degeneration of a transgenically labeled stage 1 axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cx3cr1GFP/+ mouse. Axonal degeneration is initiated near a putative node of Ranvier in close proximity to activated macrophages/microglia (magenta). * Supplementary Video 2 (2M) In vivo overview multi-photon time-lapse of the healthy lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse in which axons are labeled with YFP (gray). The video illustrates that no obvious morphological changes are induced by our imaging approach. 300 min, 11 frames. * Supplementary Video 3 (2M) In vivo overview multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse, in which axons are labeled with YFP (gray; in some axons, CFP-labeled mitochondria are visible due to spectral cross-talk) 2 d after the EAE onset. The video illustrates stage 1 to stage 2 transitions in three axons during the observation period. 300 min, 11 frames. * Supplementary Video 4 (270K) In vivo multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse in which the axons are labeled with YFP (gray) 3 d after the EAE onset. This video illustrates the recovery of a stage 1 axon during the observation period. 330 min, 11 frames. * Supplementary Video 5 (3M) Video sequence of a stage 1 EAE axon (shown in ) that illustrates the correlation between in vivo multi-photon imaging and ssTEM. * Supplementary Video 6 (188K) In vivo multi-photon microscopy time-lapse of an activated macrophage/microglia (one cell was manually pseudo-colored in magenta based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Cx3cr1GFP/+ × Thy1-CFP-S mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Note transition of the apposed axon from stage 0 to stage 1 during the time-lapse. Asterisk in first frame marks additional macrophage/microglia. 192 min, 20 frames. * Supplementary Video 7 (258K) In vivo multi-photon time-lapse of a T cell (one cell was manually pseudo-colored in cyan based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cd2-GFP mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Green asterisk in first frame marks additional T cell, gray asterisk marks axon fragment. 40 min, 27 frames. * Supplementary Video 8 (1M) In vivo multi-photon time-lapse of axonal (white) and mitochondrial (cyan) changes induced after application of H2O2 (330 mM) to the spinal cord of a Thy1-YFP-16 × Thy1-MitoCFP-P mouse. Note that axonal mitochondria change before the transition of the axons from stage 0 to stage 1. PDF files * Supplementary Text and Figures (918K) Supplementary Figures 1–5 and Supplementary Methods Read the full article * Instant access to this article: US$18Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - A Src link in schizophrenia
- Nat Med 17(4):425-427 (2011)
Nature Medicine | Article Schizophrenia susceptibility pathway neuregulin 1–ErbB4 suppresses Src upregulation of NMDA receptors * Graham M Pitcher1, 2 * Lorraine V Kalia1, 3, 5 * David Ng1, 5 * Nathalie M Goodfellow2 * Kathleen T Yee4 * Evelyn K Lambe2 * Michael W Salter1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:470–478Year published:(2011)DOI:doi:10.1038/nm.2315Received03 December 2010Accepted31 January 2011Published online27 March 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Abstract * Abstract * 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 Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β–ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral–CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst–induced phosphorylation of GluN2B by inhibit! ing Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function. View full text Figures at a glance * Figure 1: NRG1β-ErbB4 signaling prevents endogenous Src activation–induced potentiation of NMDAR-mediated synaptic responses in the hippocampal CA1. () Scatter plot of NMDAR EPSC peak amplitude over time (min) from three rat CA1 neurons recorded with control intracellular solution (ICS), ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA and NRG1β (2 nM) bath-applied beginning 20 min before whole-cell recording (black bar). Top, average NMDAR EPSC traces from the three neurons (scale bars, 45 ms; 50 pA) at the times indicated (1 and 2). () Summary scatter plot of NMDAR EPSC peak amplitude with control ICS (n = 20), EPQ(pY)EEIPIA (n = 13) or EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7; *P < 0.05 or **P < 0.01 versus EPQ(pY)EEIPIA). () Current-voltage (I-V) relationship for pharmacologically isolated NMDARs with control solution, EPQ(pY)EEIPIA or EPQ(pY)EEIPIA with NRG1β in ACSF. Right, superimposed NMDAR EPSC traces at membrane potentials from −80 to +60 mV. Scale bars, 150 ms; 250 pA. () Summary scatter plot of normalized peak NMDAR EPSC amplitude from CA1 neurons from WT mice during intracellular administrati! on of ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in ACSF (n = 18), from neurons from Erbb4−/−HER4heart mice during intracellular administration of EPQ(pY)EEIPIA with NRG1β (2 nM) (n = 7) or from neurons from WT mice during intracellular administration of EPQ(pY)EEIPIA with bath-applied NRG1β (2 nM) and AG1478 (n = 16). Top, representative average NMDAR EPSCs at the indicated times (1 and 2) and after D-APV (scale bars, 50 ms; 50 pA). () Summary histogram of EPQ(pY)EEIPIA-induced increase in NMDAR EPSC amplitude at 30 min of recording from WT neurons with ICS containing EPQ(pY)EEIPIA (n = 13), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording (n = 18), from CA1 pyramidal neurons from Erbb4−/−HER4heart mice with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7) or from WT CA1 pyramidal neurons with ICS containing EPQ(pY)EEIPIA with NRG1β and AG1478 in ACSF (n = 16). ***P < 0.001 versus wild-type. In al! l figures, group data are mean ± s.e.m. In voltage-clamp expe! riments the holding potential was −60 mV, except where otherwise indicated. * Figure 2: NRG1β has no effect on basal NMDAR-mediated synaptic responses in hippocampal CA1 or in prefrontal cortex but prevents endogenous Src activation-induced potentiation of NMDAR EPSCs at prefrontal cortex synapses. () Summary scatter plot of peak NMDAR EPSC amplitude over time from mouse CA1 neurons recorded with control ICS (n = 10) and NRG1β (2 nM) present in ACSF beginning at 10 min (black bar). Top, average NMDAR EPSCs from a representative neuron (scale bars, 50 ms; 50 pA) at the times indicated (1 and 2) and after D-APV. () Summary histogram of NMDAR EPSC decay from the CA1 neurons recorded in at the 10-min time point immediately before NRG1β administration and at the 40-min time point during NRG1β perfusion. Results are percentage of NMDAR EPSC decay with mean decay during first 2 min of recording before NRG1β administration normalized to 100% (dotted line). () Summary histogram of pharmacologically isolated NMDAR fEPSP responses (n = 14) before or during PD158780 application. Results are percentage of NMDAR fEPSP slope at the beginning of the recording normalized to 100% (dotted line). () Scatter plot of NMDAR EPSC peak amplitude from four prefrontal cortex neurons: with co! ntrol ICS, with control ICS with NRG1β (6 nM) in ACSF, with ICS containing EPQ(pY)EEIPIA or with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (black bar). Top, average NMDAR EPSC traces from the four neurons (scale bars, 100 ms; 40 pA) at the times indicated (1 and 2). () Summary histogram of normalized NMDAR EPSC amplitude at 30 min of recording from prefrontal cortex neurons with control ICS (n = 11), ICS containing EPQ(pY)EEIPIA (n = 9), control ICS with NRG1β present in ACSF (n = 7) or ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 5). ***P < 0.001. * Figure 3: NRG1β prevents but does not reverse endogenous Src-induced synaptic potentiation. () Scatter plot of EPSP slope over time from three rat CA1 neurons recorded with control ICS, ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in the ACSF beginning 20 min before whole-cell recording (black bar). Right, average EPSP traces from the three neurons (scale bars, 50 ms; 10 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1 stratum radiatum. Right, average fEPSPs (scale bars, 30 ms; 0.5 mV) at the times indicated (1 and 2). () Summary scatter plot of EPSP slope with control ICS (n = 16), ICS containing EPQ(pY)EEIPIA (n = 9) or ICS containing EPQ(pY)EEIPIA with NRG1β in the ACSF (n = 6; *P < 0.05, **P < 0.01 or ***P < 0.001 versus EPQ(pY)EEIPIA). Bottom, averaged slope of fEPSPs recorded simultaneously during experiments when whole-cell recordings were carried out. () Scatter plot of EPSP slope over time from two WT mouse CA1 neurons recorded with ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) or vehicle administe! red to the ACSF during EPQ(pY)EEIPIA-induced potentiation of EPSP responses (black bar). Right, average EPSPs from the two neurons (scale bars, 100 ms; 15 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1. Right, average fEPSPs (scale bars, 10 ms; 0.5 mV) at the times indicated (1, 2 and 3). () Summary histogram of the EPQ(pY)EEIPIA-induced increase in EPSP slope at 30 min of recording from neurons with ICS containing EPQ(pY)EEIPIA (n = 19), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording at 0.2 nM (n = 16; **P < 0.01 versus vehicle) or 2 nM (n = 6; ***P < 0.001 versus vehicle) or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) treatment during EPQ(pY)EEIPIA-induced potentiation of EPSPs (n = 12). * Figure 4: NRG1β prevents but does not reverse TBS-induced LTP in CA1 hippocampus and has no effect in Src−/− mice. () Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) or another slice treated with vehicle (black bar). Top, average fEPSP traces (scale bars, 10 ms; 0.5 mV) at the times indicated (1 and 2) from the two individual experiments. Bottom, summary scatter plot of grouped normalized fEPSP slope from control slices (n = 23) or NRG1β-treated slices (n = 14). () Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) present in ACSF beginning 30 min after TBS (black bar). Top, average fEPSP traces (scale bars, 15 ms; 0.5 mV) at the times indicated (1, 2, 3 and 4) from the experiment plotted. Bottom, summary scatter plot of grouped normalized fEPSP slope (n = 19). () Summary scatter plot of grouped normalized fEPSP slope in slices from Src+/+ mice treated with vehicle (VEH, n = 13) or NRG1β (NRG, 2 nM; n = 14; P < 0.001 versus VEH) (black bar). Inset, average fEPSP traces at the ti! mes indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. Bottom, summary scatter plot of normalized fEPSP slope in slices from Src−/− mice treated with NRG1β (2 nM, n = 11) or vehicle (n = 14) (black bar). Inset, average fEPSP traces recorded at the times indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. () Summary histogram of TBS-induced increase in fEPSP slope 60 min after TBS in slices from Src+/+ and Src−/− mice with NRG1β (+) or vehicle (−) treatment. Data are percentage of tbLTP with vehicle normalized to 100%. ***P < 0.001 versus Src+/+ with vehicle. () Immunoblot analysis of ErbB4, GluN1 and GluN2, in CA1 from a Src+/+ mouse or a Src−/− littermate. GAPDH was loading control. * Figure 5: NRG1β reduces depolarization of CA1 neurons during the period of TBS. () The first four pulse-induced burst EPSP of TBS for control mouse (Src+/+) slices (n = 28), slices (from Src+/+ mice) pretreated with D-APV (n = 17), slices from Src−/− mice (n = 12) or slices from Src+/+ mice pretreated with NRG1β (2 nM; n = 19). Traces are the mean membrane potential from all recordings in response to the first four stimuli. Gray region, ± s.e.m. Vertical arrows, start of stimulation for each of four pulses delivered at 100 Hz. x axis, time (200 ms). Bottom, average pre-TBS baseline single stimulus–evoked EPSPs (scale bars, 100 ms; 10 mV). () Burst EPSPs for the entire duration of TBS from control slices (n = 28) or slices pretreated with NRG1β (n = 19). () Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in control slices (n = 28) or slices pretreated with NRG1β (n = 19; *P < 0.05 or **P < 0.01 versus control). () Summary scatter plot of grouped normalized EPSP slope over time. () Fi! rst four pulse-induced burst EPSP of TBS for neurons from control slices (from Src+/+ mice), slices (from Src+/+ mice) pretreated with NRG1β (2 nM) or slices (from Src+/+ mice) pretreated with NRG1β (2 nM) and AG1478. x axis, time (200 ms). Inset, average representative pre-TBS baseline single stimulus–evoked EPSPs from a control slice and a slice pretreated with NRG1β and AG1478 (scale bars, 100 ms; 8 mV). () Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in slices pretreated with NRG1β (n = 19) or with NRG1β and AG1478 (n = 25; *P < 0.05, **P < 0.01 or ‡P < 0.001 versus NRG1β). * Figure 6: NRG1β does not alter Src association with the NMDAR but reduces Src tyrosine kinase activity and prevents TBS-induced GluN2B phosphorylation in hippocampal CA1. () Immunoprecipitation (IP) of GluN2 subunits carried out from hippocampal proteins prepared from control (−) or NRG1β (2 nM)-treated (+) slices. Proteins that immunoprecipitated with antibody to GluN2B were probed with antibodies to GluN2B, PSD-95 or Src (left). In 'input' lanes, 60 μg of proteins without immunoprecipitation were loaded. () Histogram of Src kinase activity in CA1 lysates without (n = 4) or with (n = 4) NRG1β (2 nM) pretreatment. Src kinase activity normalized to activity of Src in control lysates (**P < 0.01, t‐test versus control). () Summary histogram of TBS-induced pYGluN2B from CA1 region collected from Src+/+ (n = 6) or Src−/− (n = 6; **P < 0.01, t-test versus Src+/+) mice. () Top image, representative immunoblots of lysates from rat CA1 region with TBS or without (baseline, BL) probed with antibody to phosphorylated tyrosine (pY). The same membrane was stripped and reprobed with GluN2B antibody. Top graph, four-point standard curve derived ! from serial dilutions of rat brain proteins. Bottom image, representative immunoblot of lysates from rat CA1 region with TBS or without (BL) and treated with NRG1β (2 nM) probed sequentially with antibodies to pY and GluN2B. Bottom graph, four-point standard curve derived from serial dilutions of rat brain lysate. Right, summary histogram of immunoblot analysis. Densiometric quantification was carried out for pY GluN2B from six immunoblot experiments for each condition. Band intensity was quantified as mean gray value and the ratio of pY GluN2B to total GluN2B was calculated. Bars correspond to mean ratios normalized to ratios obtained for baseline (BL; *P < 0.05, t‐test versus BL). () Top, four-point standard curve derived from serial dilutions of Src+/+ mouse brain lysate. Bottom, summary histogram of TBS-induced pY GluN2B from hippocampal CA1 region without (n = 4) or with (n = 4) NRG1β (2 nM) treatment or with NRG1β (2 nM) and AG1478 treatment (n = 4). **P < 0.01, ! t‐test versus control or versus NRG1β and AG1478. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Lorraine V Kalia & * David Ng Affiliations * Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada. * Graham M Pitcher, * Lorraine V Kalia, * David Ng & * Michael W Salter * Department of Physiology, University of Toronto, Ontario, Canada. * Graham M Pitcher, * Nathalie M Goodfellow, * Evelyn K Lambe & * Michael W Salter * Institute of Medical Sciences, University of Toronto, Ontario, Canada. * Lorraine V Kalia & * Michael W Salter * Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, USA. * Kathleen T Yee Contributions G.M.P. designed the project, conducted the hippocampal experiments, analyzed the data and wrote the manuscript. L.V.K. and D.N. carried out and analyzed biochemical experiments. E.K.L. oversaw and analyzed the prefrontal cortex experiments. N.M.G. carried out and analyzed the prefrontal cortex experiments. K.T.Y. maintained, housed and provided ErbB4 knockout mice. All authors participated in revising the manuscript and agreed to the final version. M.W.S. conceived the study, analyzed data, supervised the overall project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael W Salter Author Details * Graham M Pitcher Search for this author in: * NPG journals * PubMed * Google Scholar * Lorraine V Kalia Search for this author in: * NPG journals * PubMed * Google Scholar * David Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Nathalie M Goodfellow Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen T Yee Search for this author in: * NPG journals * PubMed * Google Scholar * Evelyn K Lambe Search for this author in: * NPG journals * PubMed * Google Scholar * Michael W Salter Contact Michael W Salter Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–5 and Supplementary Methods Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - The bone and beyond: 'Dem bones' are made for more than walking
- Nat Med 17(4):428-430 (2011)
Nature Medicine | Between Bedside and Bench The bone and beyond: 'Dem bones' are made for more than walking * L Darryl Quarles1Journal name:Nature MedicineVolume: 17,Pages:428–430Year published:(2011)DOI:doi:10.1038/nm0411-428Published online07 April 2011 Bone is an endocrine organ that reaches out to other tissues, orchestrating responses that may have a role in pathology and physiology at distant sites. In 'Bench to Bedside', L. Darryl Quarles discusses recent studies showing how a feedback loop between the bone hormone FGF-23 and renal phosphate excretion and vitamin D metabolism is integrated with the classical PTH–vitamin D axis in chronic kidney disease (CKD). This new paradigm may change the diagnosis and treatment of disordered mineral homeostasis of people with CKD. A recent epidemiological study argues a mechanistic link between bone loss and atherosclerosis, maladies usually linked to aging. In 'Bedside to Bench', Sundeep Khosla discusses how this clinical study underpins research showing that mediators of inflammation and oxidative stress share common mechanisms that may lead to this calcium shift during aging. 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 * L. Darryl Quarles is at The University of Tennessee Health Science Center, Memphis, Tennessee, USA. Competing financial interests L.D.C. is a consultant to Amgen, KAI Pharmaceuticals and Shire. He has also been a paid speaker for Amgen. Corresponding author Correspondence to: * L Darryl Quarles Author Details * L Darryl Quarles Contact L Darryl Quarles Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The bone and beyond: A shift in calcium
- Nat Med 17(4):430-431 (2011)
Nature Medicine | Between Bedside and Bench The bone and beyond: A shift in calcium * Sundeep Khosla1Journal name:Nature MedicineVolume: 17,Pages:430–431Year published:(2011)DOI:doi:10.1038/nm0411-430Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Aging is associated with calcium loss from bone and increased calcium deposition in blood vessels, but whether this shift in calcium is due to common pathogenetic mechanisms shared by osteoporosis and atherosclerosis or whether these diseases are merely comorbidities of aging has been unclear. For example, women with osteoporosis have a fourfold increased risk of cardiovascular events as compared to women without osteoporosis1. But whether this is due to a true association between these diseases independent of age has been unclear, as both osteoporosis and atherosclerosis become clinically evident starting in the sixth decade, and their incidence increases further with aging. Both diseases also affect multiple ethnic groups and both sexes, although osteoporosis is more common in women than in men. A recent epidemiological study from the Multi-Ethnic Study of Atherosclerosis (MESA) by Hyder et al.2 is of particular interest, as their results indicate that osteoporosis and atherosclerosis are closely related, independent of age and other confounders. The authors used quantitative computed tomography in 946 women and 963 men, each with a mean age of approximately 65 years, to assess lumbar spine volumetric bone mineral density (LS-vBMD), an index of skeletal mass, as well as coronary artery calcium (CAC) and abdominal aortic calcium (AAC) scores, which are indices of atherosclerosis. After adjusting the data for age, ethnicity, body mass index, hypertension, dyslipidemia, diabetes mellitus, smoking, alcohol consumption, physical activity, inflammatory biomarkers (interleukin-6 (IL-6), C-reactive protein and homocysteine) and sex hormones, decreased LS-vBMD was significantly associated with increased CAC score among women and increased AAC score among both women and men. ! Adjusting statistically for these multiple variables was crucial to show a potential direct link between LS-vBMD and CAC and AAC scores independent of other variables that may be affecting both bone loss and atherosclerosis. These findings are clinically relevant, as bone density is a well-established predictor of fracture risk3, and arterial calcium content increases the risk of cardiovascular events such as myocardial infarction and stroke, as well as of death from cardiovascular disease4. Notably, the results of this study show that the relationship between vBMD and vascular calcification scores was still present after adjustment for multiple confounding variables; however, whereas epidemiological studies such as this can generate plausible hypotheses, they cannot establish causality. What, then, are the possible mechanistic links between bone loss and vascular disease? This question is best addressed after perusing the hitherto identified mechanisms underlying both bone and vascular remodeling. Bone is constantly modified through resorption by osteoclasts and formation by osteoblasts (Fig. 1), a remodeling process that is triggered by osteocytes—osteoblast-lineage cells embedded in bone—that respond to local injury, such as a microcrack occurring during normal loading, and serve an important repair function. Increasing evidence indicates that bone remodeling occurs in the bone remodeling compartment, a closed cavity covered by a canopy of bone-lining cells (probably quiescent osteoblasts) that is penetrated by a capillary that provides perivascular progenitor cells that differentiate into functional osteoblasts5. Figure 1: Possible mechanistic interplay between bone remodeling and the atherosclerotic plaque. Osteocytes embedded in bone sense microcracks and trigger a repair response involving resorption of damaged bone by osteoclasts and refilling of the excavated bone by osteoblasts, which are probably derived from perivascular stem cells. Bone-lining cells provide a 'canopy' over the site of bone remodeling. Similarly, the atherosclerotic plaque is covered by a fibrous cap that includes vascular smooth muscle cells (closely related to osteoblasts) and macrophages (closely related to osteoclasts), with the plaque itself containing lymphocytes, macrophages and vascular smooth muscle cells, as well as cholesterol crystals and cell remnants. The plaque is penetrated by the vasa vasorum, small blood vessels that supply larger blood vessels and provide perivascular adventitial cells that can differentiate into vascular smooth muscle cells. Potential mechanisms, including age-related increases in inflammation and in oxidative stress, may shed light on the association shown in human s! tudies between osteoporosis and the calcifying repair response in the atherosclerotic plaque. * Full size image (155 KB) The atherosclerotic plaque shows striking similarities with the bone remodeling compartment (Fig. 1). A crucial point is that one of the outcomes of the remodeling processes within the plaque is calcium deposition, which not only is a marker for atherosclerosis but also may contribute to plaque instability and rupture, resulting in myocardial infarction6. Why, then, is vascular calcification associated with bone loss, as demonstrated by Hyder et al.2? 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 * Sundeep Khosla is in the Endocrine Research Unit, College of Medicine, Mayo Clinic, Rochester, Minnesota, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Sundeep Khosla Author Details * Sundeep Khosla Contact Sundeep Khosla Search for this author in: * NPG journals * PubMed * Google Scholar Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 - Research Highlights
- Nat Med 17(4):432-433 (2011)
Nature Medicine | Research Highlights Research Highlights * Victoria Aranda * Michael Basson * Kevin Da Silva * Juan Carlos López * Carolina Pola * Meera SwamiJournal name:Nature MedicineVolume: 17,Pages:432–433Year published:(2011)DOI:doi:10.1038/nm0411-432Published online07 April 2011 Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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. Immunology: New VISTAs on immune regulation The existence of a new inhibitory ligand that regulates the function of T cells is reported in The Journal of Experimental Medicine (, 577). This molecule, termed V-domain immunoglobulin suppressor of T cell activation (VISTA), may have functional relevance in autoimmune diseases and cancer. A series of stimulatory and inhibitory molecules tightly controls immune function, in particular T cell activation. Li Wang et al. identified VISTA, a new member of the well characterized B7 family of regulatory ligands. Antigen-presenting cells that express VISTA inhibit the proliferation of T cells and cytokine production, cellular responses that can be blocked by a VISTA-specific antibody. In vivo, interfering with the function of VISTA in mice promotes the development of experimental autoimmune encephalomyelitis, a model of multiple sclerosis. In addition, VISTA overexpression on cancer cells suppresses antitumor immune responses. Immunoregulatory molecules of the B7 family, such as PD-L1, have attracted interest as potential therapeutic targets for cancer and autoimmune conditions. VISTA is poised to continue this trend and catch the attention of the pharmaceutical industry. —JCL A tale of TNF and progranulin Progranulin, a growth factor involved in development, tissue repair and inflammation, binds the tumor necrosis factor (TNF) receptor, according to a new study (Science doi:10.1126/science.1199214). Chuan-ju Liu and his colleagues used a yeast two-hybrid screen to identify binding partners for progranulin. Progranulin competes with TNF for binding to the TNF receptor, inhibiting downstream signaling events. By mapping the minimal motifs required for binding of progranulin to this receptor, the authors generated a peptide that mimics this binding event. TNF is important in promoting disease progression in arthritis, and TNF inhibitors are widely used in the treatment of rheumatoid arthritis. In mouse models of arthritis, loss of progranulin exacerbated inflammatory damage and bone destruction. Administration of progranulin itself or the peptide mimic blocked disease progression and protected mice from joint destruction. Future research into the structural basis of this interaction may lead to new small-molecule inhibitors of TNF signaling for the treatment of autoimmune disease. —KDS Metabolism: A human-specific hit Two hallmarks of type 2 diabetes are insulin resistance and pancreatic beta cell dysfunction, but the relative contribution of these mechanisms to human diabetes is unknown. A recent study suggests that altered sialic acid composition in humans could compromise beta cell function (FASEB doi:10.1096/fj.10-175281). Most mammals have two types of cell surface sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), but humans carry an inactivating mutation in the CMP-Neu5Ac hydroxylase (CMAH) responsible for Neu5Ac production, so they only express Neu5Gc. To investigate this enzyme in diabetes, Sarah Kavaler et al. studied Cmah-knockout mice fed a high-fat diet and found that they had fasting hyperglycemia and glucose intolerance. This phenotype was not due to insulin resistance, but the Cmah-knockout mice had decreased pancreatic islet number and size, suggesting that they had impaired beta cell function. Although it is as yet unclear how changes in cell surface sialic acids affect pancreatic beta cells, this study highlights the importance of progressive beta cell dysfunction in the pathogenesis of type 2 diabetes. —MS Cancer: Splicing up CD44 The cancer stem cell (CSC) theory posits that biologically distinct cell populations exist within a tumor, spurring quests for 'stemness' markers as well as attempts to decipher how these markers functionally contribute to the unique properties of CSCs. Two recent studies provide a detailed but complicated picture of the CSC-specific roles of the surface protein CD44, a beacon for aggressive stem-like cells in many tumors. These findings suggest that CD44 isoforms generated by alternative splicing might regulate distinct aspects of the CSC phenotype. Rhonda L. Brown and her colleagues report that inducers of the epithelial-to-mesenchymal transition (EMT) trigger a CD44 isoform switch in breast cancer cells by downregulating the splicing factor that normally produces the variable isoform, CD44v (J. Clin. Invest. doi:10.1172/JCI44540). This leads to increased abundance of the CD44s variant, which then activates pathways that drive the acquisition of mesenchymal properties, considered a feature of CSCs. CD44's isoform-specific control of EMT seems to be important for the survival of tumor cells in vivo, and this connection is also observed in human breast tumors. By contrast, Takatsugu Ishimoto et al. suggest that CD44v may also confer stem-associated properties (Cancer Cell, 387–400). Using cellular and mouse models of gastrointestinal cancer, the authors show that CD44v stabilizes a cell membrane transporter crucial for the generation of antioxidant compounds. The transporter's activity helps tumor cells grow under oxidative stress, a property that has been also ascribed to CSCs. This function of CD44v also contributes to tumor progression in vivo, so it will be interesting to determine its relevancy in human malignancies. These two reports highlight the importance of dissecting the functional contributions of CSC markers to tumorigenesis. —VA Infectious disease: Interferons exploit lipids Type I interferons are an initial line of defense against viral infection. Mathieu Blanc et al. reveal a new facet to the antiviral activity of these cytokines—suppression of the sterol biosynthetic pathway (PLoS Biol., e1000598). This suppression is mediated by signaling of the type I interferon receptor IFNAR-1 through the tyrosine kinase Tyk2, and leads to decreased amounts of the transcription factor SREBP2 that controls the expression of many enzymes in the sterol biosynthetic pathway. The authors showed that suppression of this pathway is important for interferon-b's antiviral effect in cytomegalovirus-infected cells; for example, the suppressive effect was negated by treatment with mevalonate, an upstream metabolite in this pathway. The researchers also provided evidence that the relevant branch of the pathway in affecting viral growth does not involve sterols, but rather the isoprenoid compounds used for protein prenylation. Type I interferons can in this respect be considered to mimic statins, which target the sterol pathway and are known to have antiviral effects. —MB A two-faced variant? Individuals infected with hepatitis A virus (HAV) show high variability in their susceptibility to liver disease. A new study shows that a human genetic variant previously associated with protection against asthma and allergy also increases susceptibility to HAV-induced hepatitis, providing an unexpected link between these conditions (J. Clin. Invest., 1111–1118). James Cavallini/Photo Researchers, Inc. Hye Young Kim et al. found that severe, HAV-induced liver disease was associated with a polymorphism in the gene encoding the HAV receptor TIM1, leading to a six-amino-acid insertion that increases the efficiency of TIM1 binding to HAV. View full text Read the full article * Instant access to this article: US$32Buy now * Subscribe to Nature Medicine for full access: SubscribeLogin for existing subscribers Additional access options: * Use a document delivery service * Rent this article from DeepDyve * Login via Athens * Purchase a site license * Institutional access * 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 * Victoria Aranda Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Basson Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Da Silva Search for this author in: * NPG journals * PubMed * Google Scholar * Juan Carlos López Search for this author in: * NPG journals * PubMed * Google Scholar * Carolina Pola Search for this author in: * NPG journals * PubMed * Google Scholar * Meera Swami Search for this author in: * NPG journals * PubMed * Google Scholar - Enhancing ties between academia and industry to improve health
- Nat Med 17(4):434-436 (2011)
Nature Medicine | Commentary Enhancing ties between academia and industry to improve health * S Claiborne Johnston1, 2 * Stephen L Hauser1 * Susan Desmond-Hellmann3 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:434–436Year published:(2011)DOI:doi:10.1038/nm0411-434Published online07 April 2011 Abstract * Abstract * 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 Concerns about conflicts of interest have driven a wedge between academia and the pharmaceutical and devices industries. Although elevated concern for bias is justified, particularly when academics may affect drug sales, partnerships between industry and academia are essential to achieve the full promise of health improvement from the public investment in biomedical research. New models for such partnerships are developing and should be encouraged. View full text Author information * Abstract * Author information Affiliations * Department of Neurology, University of California, San Francisco. * S Claiborne Johnston & * Stephen L Hauser * Clinical and Translational Science Institute, University of California, San Francisco. * S Claiborne Johnston * Office of the Chancellor, University of California, San Francisco, San Francisco, California, USA. * Susan Desmond-Hellmann Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * S Claiborne Johnston Author Details * S Claiborne Johnston Contact S Claiborne Johnston Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen L Hauser Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Desmond-Hellmann Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Time to 'walk the walk' about industry ties to enhance health
- Nat Med 17(4):437-438 (2011)
Nature Medicine | Commentary Time to 'walk the walk' about industry ties to enhance health * Thomas P Stossel1 * Lance K Stell2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:437–438Year published:(2011)DOI:doi:10.1038/nm0411-437Published online07 April 2011 Abstract * Abstract * 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 Overwhelming evidence that relationships between universities, physicians and the medical products industry benefit patients explains the ubiquitous calls to encourage such relationships. Yet accumulating 'conflict of interest' regulations in academic health centers, government and industry have had the opposite effect. Justifications underlying the regulations lack quantitative rigor, and the rules they enforce impose costly bureaucratic requirements of dubious benefit. Evidence shows that they have diminished the collaborations deemed beneficial to health enhancement. View full text Author information * Abstract * Author information Affiliations * Thomas P. Stossel is in the Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. * Lance K. Stell is in the Department of Philosophy, Davidson College, Davidson, North Carolina, USA. Competing financial interests T.P.S. is a founding scientist, director and consultant to Bioaegis Therapeutics, a startup biotechnology company developing intellectual property licensed by his employer, Brigham and Women's Hospital, based on his research. He is also a director of Velico Medical Corporation, to which Brigham and Women's Hospital has also licensed intellectual property based on his research. He serves on a Merck, Sharp and Dohme advisory board. Corresponding author Correspondence to: * Thomas P Stossel Author Details * Thomas P Stossel Contact Thomas P Stossel Search for this author in: * NPG journals * PubMed * Google Scholar * Lance K Stell Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease
- Nat Med 17(4):439-447 (2011)
Nature Medicine | Review The role of spreading depression, spreading depolarization and spreading ischemia in neurological disease * Jens P Dreier1Journal name:Nature MedicineVolume: 17,Pages:439–447Year published:(2011)DOI:doi:10.1038/nm.2333Published online07 April 2011 Abstract * Abstract * 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 The term spreading depolarization describes a wave in the gray matter of the central nervous system characterized by swelling of neurons, distortion of dendritic spines, a large change of the slow electrical potential and silencing of brain electrical activity (spreading depression). In the clinic, unequivocal electrophysiological evidence now exists that spreading depolarizations occur abundantly in individuals with aneurismal subarachnoid hemorrhage, delayed ischemic stroke after subarachnoid hemorrhage, malignant hemispheric stroke, spontaneous intracerebral hemorrhage or traumatic brain injury. Spreading depolarization is induced experimentally by various noxious conditions including chemicals such as potassium, glutamate, inhibitors of the sodium pump, status epilepticus, hypoxia, hypoglycemia and ischemia, but it can can also invade healthy, naive tissue. Resistance vessels respond to it with tone alterations, causing either transient hyperperfusion (physiological hemo! dynamic response) in healthy tissue or severe hypoperfusion (inverse hemodynamic response, or spreading ischemia) in tissue at risk for progressive damage, which contributes to lesion progression. Therapies that target spreading depolarization or the inverse hemodynamic response may potentially treat these neurological conditions. View full text Figures at a glance * Figure 1: Mechanisms of spreading depolarization in the neuron. Top, in the healthy brain impermeant negatively charged proteins inside the neuron cause small cations such as sodium and calcium to enter from the extracellular space3, 10, producing a small dendritic inward current (Iin, pink arrow). Compensation for this constant inward current by dendritic outward current (Iout, green arrow) of the sodium pump establishes the so-called double Gibbs-Donnan equilibrium of the physiological ion distribution, characterized by iso-osmolality across the membrane and steep physiological ion gradients9, 18. Bottom, the core process of spreading depolarization is failure of sodium and calcium pumps to provide sufficient dendritic outward currents to balance the persistent inward currents through pink and purple channels3, 7. If net dendritic current turns inward (persistent influx of sodium and calcium is more than the outflux of potassium), the double is shifted toward an almost simple Gibbs-Donnan equilibrium, characterized by near-complete los! s of electrochemical energy, almost passive ion distribution across the membrane, intracellular hyperosmolality with cellular swelling and distortion of dendritic spines6 and extracellular hypo-osmolality with extracellular volume (ECV) shrinkage (blue arrow: water follows sodium and calcium influx). The hallmark of this process is near-complete sustained depolarization from ~−70 to ~−10 mV. Pink membrane channels: classic candidates for neuronal influx of calcium and sodium during spreading depolarization in healthy, naive tissue such as NMDA receptor–controlled channels, slowly inactivating sodium and calcium-sensitive nonspecific cation channels7. Purple channels: additional candidate channels for inward flux in presence of noxious stimuli; classic candidates are AMPA/kainate receptor–controlled channels14; other more recent candidates are acid-sensing81 and transient receptor potential channels as well as pannexin hemichannels78. Green channel: delayed rectifier! and SK potassium channels that provide outward fluxes antagon! izing the depolarization. * Figure 2: Normal (spreading hyperemia) and inverse (spreading ischemia) function of the neurovascular unit in response to spreading depolarization in the human brain. The neurovascular unit consists of neurons, astrocytes, endothelium, pericytes and vascular smooth muscle. During spreading depolarization, neurons swell. In brain tissue at risk, spreading depolarization can cause vasoconstriction and tissue ischemia (left). The slow potential change (SPC) is a direct extracellular (EC) electrocorticographic index of the spreading depolarization4. Spreading depression of brain electrical activity is only a secondary electrocorticographic index of spreading depolarization and is observed as a suppression of fast potential changes. Under physiological conditions, there is a normal neurovascular response defined by vasodilatation and increased rCBF in response to spreading depolarization that causes spreading hyperemia, which is measured with subdural optode/laser-Doppler flowmetry (right). This provides tissue clearance from metabolites and increased oxidative substrate supply to fuel energy-dependent membrane pumps, which re‐establish the ! normal polarized state of the neurons and ion homeostasis. Thus, a short-lasting SPC and short-lasting spreading depression of activity are typical during the normal hyperemic response. Under pathological conditions, however, an inverse neurovascular response occurs with a corrupted interplay of neurovascular unit elements that results in severe vasoconstriction in response to spreading depolarization (right). The subsequent spreading ischemia describes the consequent spreading depolarization–induced perfusion deficit when it leads to a prolonged SPC39. The prolonged SPC, indicating prolonged neuronal depolarization, results from mismatch between energy demand and supply, which causes insufficiency of membrane pumps to repolarize the neurons. Similar to the SPC, spreading depression of activity is prolonged when spreading ischemia is coupled to spreading depolarization. Herein, spreading ischemia is relatively short lived, but it can last for more than 2 hours in animals ! and humans46, 52. * Figure 3: Spreading ischemia in the rat brain. These stills from a movie show the virtual disappearance of pial arteries (*) and the propagating paleness of the rat brain surface during a typical spreading ischemia, seen through the operation microscope40. Of note, the SPC starts somewhat earlier than the fall of rCBF. This indicates that the spreading depolarization precedes the perfusion deficit, in contrast to the sequence of events during a nonspreading ischemia in which the spreading depolarization follows the perfusion deficit after some minutes8, 40. * Figure 4: Vicious cycle underlying spreading ischemia. After aneurismal subarachnoid hemorrhage, a blood clot covers the brain surface and alters the cortical microenvironment and blood vessel reactivity while the blood constituents disintegrate. Basal potassium rise and basal NO decline synergistically augment the effects of vasoconstrictors while they inhibit the effects of vasodilators released in response to spreading depolarization. Consequent vasoconstriction in response to spreading depolarization causes a severe decrease of rCBF, termed spreading ischemia, which propagates in the tissue together with spreading depolarization. Once ischemia is established, it will be maintained by the vicious cycle of the inverse hemodynamic response. This cycle consists of decline in oxidative substrate supply while energy demand is increased, consequent fall of tissue ATP with reduced sodium pump activity while glutamate transporters show reverse transport and reduced extracellular clearance. There is also failure of neuronal repolariza! tion and, therefore, continued release of vasoconstrictors, which maintains the perfusion deficit while the perfusion deficit maintains the depolarization. When spreading depolarization is prolonged by spreading ischemia, intraneuronal calcium surge and other death signals are prolonged. When the presence of those signals outlasts the so-called commitment point, neurons will die. The inverse hemodynamic response to spreading depolarization is a promising target for therapeutic intervention. Na++, K+ ATPase, sodium pump. Author information * Abstract * Author information Affiliations * Center for Stroke Research, Departments of Experimental Neurology and Neurology, Charité University Medicine Berlin, Berlin, Germany. * Jens P Dreier Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Jens P Dreier Author Details * Jens P Dreier Contact Jens P Dreier Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Metabolite profiles and the risk of developing diabetes
- Nat Med 17(4):448-453 (2011)
Nature Medicine | Article Metabolite profiles and the risk of developing diabetes * Thomas J Wang1, 2, 3 * Martin G Larson3, 4 * Ramachandran S Vasan3, 5 * Susan Cheng2, 3, 6 * Eugene P Rhee1, 7, 8 * Elizabeth McCabe2, 3 * Gregory D Lewis1, 2, 8 * Caroline S Fox3, 9, 10 * Paul F Jacques11 * Céline Fernandez12 * Christopher J O'Donnell2, 3, 8 * Stephen A Carr8 * Vamsi K Mootha8, 13, 14 * Jose C Florez8, 13 * Amanda Souza8 * Olle Melander15 * Clary B Clish8 * Robert E Gerszten1, 2, 8 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:448–453Year published:(2011)DOI:doi:10.1038/nm.2307Received07 April 2010Accepted19 January 2011Published online20 March 2011 Abstract * Abstract * 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 Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines and other polar metabolites were profiled in baseline specimens by liquid chromatography–tandem mass spectrometry (LC-MS). Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly significant associations with future diabetes: isoleucine, leucine, valine, tyrosine and phenylalanine. A combination of three amino acids predicted future diabetes (with a more than fivefold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential key role of amino acid metabolism early in the pathogenesis of diabetes and s! uggest that amino acid profiles could aid in diabetes risk assessment. View full text Author information * Abstract * Author information * Supplementary information Affiliations * Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Thomas J Wang, * Eugene P Rhee, * Gregory D Lewis & * Robert E Gerszten * Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Thomas J Wang, * Susan Cheng, * Elizabeth McCabe, * Gregory D Lewis, * Christopher J O'Donnell & * Robert E Gerszten * Framingham Heart Study of the National Heart, Lung, and Blood Institute and Boston University School of Medicine, Framingham, Massachusetts, USA. * Thomas J Wang, * Martin G Larson, * Ramachandran S Vasan, * Susan Cheng, * Elizabeth McCabe, * Caroline S Fox & * Christopher J O'Donnell * Department of Mathematics and Statistics, Boston University, Boston, Massachusetts, USA. * Martin G Larson * Cardiology Section, Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts, USA. * Ramachandran S Vasan * Division of Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Susan Cheng * Renal Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Eugene P Rhee * Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. * Eugene P Rhee, * Gregory D Lewis, * Christopher J O'Donnell, * Stephen A Carr, * Vamsi K Mootha, * Jose C Florez, * Amanda Souza, * Clary B Clish & * Robert E Gerszten * Division of Endocrinology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Caroline S Fox * National Heart, Lung, and Blood Institute, Division of Intramural Research, Bethesda, Maryland, USA. * Caroline S Fox * Jean Mayer US Department of Agriculture Human Nutrition Research Center, Tufts University, Boston, Massachusetts, USA. * Paul F Jacques * Department of Experimental Medical Science, Lund University, Malmö, Sweden. * Céline Fernandez * Diabetes Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Vamsi K Mootha & * Jose C Florez * Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Vamsi K Mootha * Department of Clinical Sciences, Lund University, Malmö, Sweden. * Olle Melander Contributions T.J.W. conceived of the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. A.S. and E.P.R., under the direction of C.B.C., developed the metabolic profiling platform, performed mass spectrometry experiments and analyzed the data. S.A.C. and V.K.M. helped in the establishment of the metabolite profiling platform and manuscript revision. G.D.L. contributed to data analysis and manuscript generation. M.G.L., R.S.V., S.C. and E.M. helped in experimental design, performed statistical analyses and assisted in manuscript generation. C.J.O. and C.S.F. helped in experimental design and manuscript revision. P.F.J. directed the dietary analyses in the Framingham Heart Study and contributed to manuscript revision. J.C.F. assisted in the interpretation of the data and contributed to manuscript revision. O.M. and C.F. performed the replication analyses in the Malmö Diet and Cancer cohort and contributed to manuscript revision. R.E.G. conceived of! the study, designed the experiments, analyzed and interpreted the data and wrote the manuscript. Competing financial interests T.J.W., R.S.V., M.G.L., V.K.M. and R.E.G. are named as co-inventors on a patent application to the US Patent Office pertaining to metabolite predictors of diabetes. J.C.F. has received consulting honoraria from Publicis Healthcare, Merck, bioStrategies, XOMA and Daiichi-Sankyo and has been a paid invited speaker at internal scientific seminars hosted by Pfizer and Alnylam Pharmaceuticals. Corresponding authors Correspondence to: * Thomas J Wang or * Robert E Gerszten Author Details * Thomas J Wang Contact Thomas J Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Martin G Larson Search for this author in: * NPG journals * PubMed * Google Scholar * Ramachandran S Vasan Search for this author in: * NPG journals * PubMed * Google Scholar * Susan Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Eugene P Rhee Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth McCabe Search for this author in: * NPG journals * PubMed * Google Scholar * Gregory D Lewis Search for this author in: * NPG journals * PubMed * Google Scholar * Caroline S Fox Search for this author in: * NPG journals * PubMed * Google Scholar * Paul F Jacques Search for this author in: * NPG journals * PubMed * Google Scholar * Céline Fernandez Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher J O'Donnell Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen A Carr Search for this author in: * NPG journals * PubMed * Google Scholar * Vamsi K Mootha Search for this author in: * NPG journals * PubMed * Google Scholar * Jose C Florez Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Souza Search for this author in: * NPG journals * PubMed * Google Scholar * Olle Melander Search for this author in: * NPG journals * PubMed * Google Scholar * Clary B Clish Search for this author in: * NPG journals * PubMed * Google Scholar * Robert E Gerszten Contact Robert E Gerszten Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (127K) Supplementary Figure 1, Supplementary Tables 1–4 and Supplementary Methods Additional data - Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists
- Nat Med 17(4):454-460 (2011)
Nature Medicine | Article Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonists * Kengo Shimono1, 4 * Wei-en Tung1, 4 * Christine Macolino1 * Amber Hsu-Tsai Chi1 * Johanna H Didizian1, 4 * Christina Mundy1 * Roshantha A Chandraratna2 * Yuji Mishina3 * Motomi Enomoto-Iwamoto1, 4 * Maurizio Pacifici1, 4 * Masahiro Iwamoto1, 4 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:454–460Year published:(2011)DOI:doi:10.1038/nm.2334Received15 September 2010Accepted18 February 2011Published online03 April 2011 Abstract * Abstract * 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 Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-li! ke kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans. View full text Figures at a glance * Figure 1: RAR agonists block chondrogenesis and intramuscular rBMP-2-driven heterotopic ossification. () Micromass cultures of mesenchymal cells from E11.5 mouse limb treated with increasing doses of all-trans-retinoic acid (RA) or the RAR-γ agonist NRX204647 and stained with alcian blue on day 8. () Similar micromass cultures prepared from E11.5 RAR-γ–null or wild-type (WT) limb mesenchymal cells and treated with all-trans-retinoic acid (100 nM) for 8 d. () Micromass cultures prepared from dual RAR-α– and RAR-β–deficient (αβ-def) or wild-type limb mesenchymal cells and treated with all-trans-retinoic acid as above. () Heterotopic ossification masses induced by implantation of rBMP-2-filled collagen sponge into a microsurgically created pocket in the calf muscles. The mice were treated with vehicle, all-trans-retinoic acid (12 mg per kg body weight per day) or NRX204647 (1.2 mg per kg body weight per day) by gavage. Large round mineralized heterotopic ossification masses were visible by μCT in mice that received vehicle, markedly decreased in mice treated with r! etinoic acid and absent in NRX204647-treated mice. Ectopic tissues were sectioned and examined by Masson trichrome (MT) and alcian blue (AB) staining, immunofluorescence for myosin heavy chain (MHC: red) and osteocalcin (OC: green) and TRAP staining. Scale bar for μCT, 5 mm; for histology, 200 μm. () Heterotopic ossification tissue induced by subcutaneous implantation of rBMP-2/Matrigel mixture. Treatment and analysis of ectopic tissue were as above. Scale bar for μCT, 5 mm; for alcian blue, 2.5 mm. * Figure 2: Effectiveness of different retinoids against heterotopic ossification. () Chemical structures of synthetic RAR-γ agonists and natural 13-cis-retinoic acid. () Suppression of heterotopic ossification by retinoids evaluated by measuring BV/TV in ectopic masses in control versus retinoid-treated mice by μCT on day 12. Values from control groups were set at 100% and used to calculate relative values in experimental groups. Each group had ≥ 8 samples; data shown as means ± s.e.m. (*P < 0.01 versus control). () Macro views (top), soft X-ray radiograms (middle) and histological sections stained with H&E (bottom) of ectopic masses collected from mice treated with vehicle or CD1530. Scale bar in top and middle images, 1 cm; bottom images, 100 μm. () Selectivity of RAR-γ agonist action. Subcutaneous heterotopic ossification was triggered in wild-type, heterozygous RAR-γ (HT) and RAR-γ–null (KO) mice, and mice were treated with RAR-γ agonist CD1530 (4 mg per kg body weight per day) or vehicle for 12 d. *P < 0.01 versus control. () Evaluation o! f rebound effects. Mice implanted with rBMP-2–Matrigel mixture subcutaneously were treated with vehicle, CD1530 or RAR-α agonist NRX195183 for 10 days. Treatment was stopped, and heterotopic ossification was evaluated by μCT at later time points. () Window of opportunity tests. Mice implanted as above were left untreated up to day 6 and were then treated with CD1530 or NRX195183 until day 12. * Figure 3: RAR-γ agonists block FOP-like heterotopic ossification. () ADTC5 cells expressing the strong constitutively active ALK2 Q207D or control empty vector were transfected with the BMP signaling reporter Id1-luc and then treated with 0, 10, 30 or 100 nM CD1530. Reporter activity was normalized to phRG-TK encoding Renilla luciferase. Error bars represent s.d. for triplicate samples. () ALK2 Q207D–transgenic mouse model of FOP-like heterotopic ossification. Consecutive soft X-ray images of the same mice, injected with Ad-Cre and cardiotoxin, taken at P7, P21 and P35 showing massive heterotopic ossification in vehicle-treated mice (double arrowheads), but markedly less in companion mice treated with CD1530 (4.0 mg per kg body weight per day). Scale bar, 1 cm. () μCT images showing massive heterotopic ossification in vehicle-treated ALK2 Q207D–transgenic mice injected with Ad-Cre and cardiotoxin (double arrowheads) but not in companion CD1530-treated mice. Scale bar, 1 cm. () Bright-field (BF) and fluorescent images showing ectopic s! keletal tissue (positive for alizarin complexon, AR) in vehicle-treated ALK2 Q207D–transgenic mice injected with Ad-Cre and cardiotoxin but not in those treated with CD1530. Positive GFP fluorescence confirmed that Ad-Cre had activated transgene expression in all mice. There was minimal GFP and AR fluorescence in mice that had not been injected with Ad-Cre and cardiotoxin. Scale bar, 5 mm. * Figure 4: Mechanisms of RAR-γ agonist action. () Id1-luc reporter activity in ATDC5 cells. Error bars represent s.d. for triplicate samples. () Immunoblots showing phosphorylated Smad1, Smad5 and Smad8 (p-Smad1,5,8) protein amounts in control (DMSO) ATDC5 cells treated with rBMP-2 and in cells treated with CD1530 (top) and overall Smad1 amounts in control cells and in CD1530-treated cells (bottom). Membranes were reblotted with α-tubulin–specific antibodies for normalization. () Dual Smad1 and β-actin immunofluorescence staining and nuclear DAPI staining showing that Smad1 was largely cytoplasmic in control cells and relocated to the nucleus during rBMP-2 treatment, but there was minimal detectable Smad1 signal in cytoplasm or nucleus of cells concurrently treated with CD1530. Red, Smad1; green, β-actin; blue, nuclei. () Immunoblots similar to those in showing that the overall amounts of Smad1, Smad4 and Smad5 with or without CD1530 and rBMP-2 treatment. () Immunoblots showing that the decreases in Smad1 levels eli! cited by CD1530 treatment were counteracted by co-treatment with the proteasome inhibitors AW9155 or PI-108. * Figure 5: RAR-γ agonists reprogram the differentiation potentials of skeletal progenitor cells. (,) ATDC5 cells were grown in the presence of vehicle (control) or 1 μM CD1530 for 2 d, rinsed, replated and then grown for an additional 7 d with or without 100 ng ml−1 rBMP-2. Control cells responded well to rBMP-2 and underwent chondrogenic differentiation revealed by strong alcian blue () and alkaline phosphatase () staining, but the cells pretreated with CD1530 did not and failed to stain. () Similar experiments with GFP-expressing MSCs derived from mouse bone marrow show that the cells underwent differentiation upon rBMP-2 treatment (top) but CD1530 pretreated MSCs did not and failed to stain with alizarin red (bottom). () Immunoblots showing the steady-state amounts of Smad1 and Smad4 in MSCs treated without (−) or with (+) 1 μM CD1530 for 12 h. () Id1-luc reporter activity in control MSCs treated with or without rBMP-2 in the presence or absence of CD1530. Error bars represent s.d. for four wells. () Control and CD1530-pretreated GFP-expressing MSCs were mixed ! with rBMP-2–Matrigel and implanted in nude mice subcutaneously, and ectopic tissue masses were analyzed by μCT, H&E staining, osteocalcin (OC) immunostaining and GFP fluorescence signal. Bottom, merged GFP and OC images. Scale bars, 5 mm (top row); 250 μm (second row); 150 μm (third row). Author information * Abstract * Author information * Supplementary information Affiliations * Department of Orthopaedic Surgery, Thomas Jefferson University College of Medicine, Philadelphia, Pennsylvania, USA. * Kengo Shimono, * Wei-en Tung, * Christine Macolino, * Amber Hsu-Tsai Chi, * Johanna H Didizian, * Christina Mundy, * Motomi Enomoto-Iwamoto, * Maurizio Pacifici & * Masahiro Iwamoto * Io Therapeutics, Irvine, California, USA. * Roshantha A Chandraratna * School of Dentistry, University of Michigan, Ann Arbor, Michigan, USA. * Yuji Mishina * Present address: The Children's Hospital of Philadelphia, Division of Orthopaedic Surgery, Philadelphia, Pennsylvania, USA. * Kengo Shimono, * Wei-en Tung, * Johanna H Didizian, * Motomi Enomoto-Iwamoto, * Maurizio Pacifici & * Masahiro Iwamoto Contributions M.I. and M.P. directed the project. K.S., W.T., C.M., A.H.-T.C., J.H.D., C.M. and M.E.-I. performed experiments, analyzed data and participated in experimental design. R.A.C. provided expertise on retinoid biology. Y.M. provided expertise in mouse genetics. M.P., K.S. and M.I. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Masahiro Iwamoto or * Maurizio Pacifici Author Details * Kengo Shimono Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-en Tung Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Macolino Search for this author in: * NPG journals * PubMed * Google Scholar * Amber Hsu-Tsai Chi Search for this author in: * NPG journals * PubMed * Google Scholar * Johanna H Didizian Search for this author in: * NPG journals * PubMed * Google Scholar * Christina Mundy Search for this author in: * NPG journals * PubMed * Google Scholar * Roshantha A Chandraratna Search for this author in: * NPG journals * PubMed * Google Scholar * Yuji Mishina Search for this author in: * NPG journals * PubMed * Google Scholar * Motomi Enomoto-Iwamoto Search for this author in: * NPG journals * PubMed * Google Scholar * Maurizio Pacifici Contact Maurizio Pacifici Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Iwamoto Contact Masahiro Iwamoto Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (815K) Supplementary Figures 1–5 and Supplementary Methods Additional data - Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways
- Nat Med 17(4):461-469 (2011)
Nature Medicine | Article Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways * Siyuan Zhang1 * Wen-Chien Huang1 * Ping Li1 * Hua Guo1 * Say-Bee Poh1 * Samuel W Brady1, 2 * Yan Xiong1 * Ling-Ming Tseng1 * Shau-Hsuan Li1 * Zhaoxi Ding1 * Aysegul A Sahin3 * Francisco J Esteva1, 2, 4 * Gabriel N Hortobagyi4 * Dihua Yu1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:461–469Year published:(2011)DOI:doi:10.1038/nm.2309Received03 June 2010Accepted21 January 2011Published online13 March 2011 Abstract * Abstract * 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 Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzuma! b-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance. View full text Figures at a glance * Figure 1: SRC hyperactivation is a key signaling alteration in acquired trastuzumab-resistant cells. () MTS assay comparing cell proliferation of indicated parental breast cancer cell lines and their corresponding acquired TtzmR sublines upon treatment with freshly added trastuzumab (Ttzm, 2 μg ml−1) for 4 d. () Tumor volume of mammary fat pad xenografts derived from either BT474 parental (P) or TtzmR subline upon treatment of IgG control or Ttzm (10 mg per kg body weight, intraperitoneally) weekly. Tumor volume at various times of treatment is presented as percentage of original tumor size at day 0 of treatment. () Representative histograms from flow cytometric analysis of EGFR and ERBB2 abundance in BT474 parental and TtzmR cells. () Relative amounts of EGFR, ERBB2, HER3 and IGF-1R in the indicated parental and TtzmR cells analyzed by flow cytometry. () Immunoblots comparing major cell signaling changes between the indicated parental and TtzmR sublines. P indicates phosphorylation; for example, P-EGFR-Y1068 is EFGR phosphorylated at Tyr1068 () Immunoblots assessing the! impact of overexpression of EGFR and IGF-1R in BT474 parental (BT474.P) cells on signaling. () MTS assay evaluating trastuzumab resistance of BT474 cells overexpressing EGFR or IGF-1R, treated as in . () Immunoblots of EGFR and phosphorylated SRC after shRNA-mediated EGFR knockdown in BT474.TtzmR cells. () MTS assay evaluating sensitivity of TtzmR to trastuzumab after EGFR knockdown. Cells were treated as in . () Left, immunoblots of knock-down of SRC in BT474.TtzmR cells by SRC shRNA. Right, MTS assay assessing trastuzumab sensitivity of TtzmR cells after SRC knockdown. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 2: SRC is activated in PTEN-deficient de novo trastuzumab-resistant cells. () MTS assay comparing cell proliferation of the indicated parental cell lines and their corresponding PTEN knockdown sublines upon trastuzumab treatment (Ttzm, 2 μg ml−1, 4 d). PTEN knockdown by either PTEN antisense (PTEN.as) oligonucleotides or PTEN shRNA is described in Online Methods. PTEN nonsense oligonucleotides, PTEN.ns. () Immunoblots assessing the effects of PTEN knockdown on phosphorylation of SRC Tyr416 (P-SRC-Y416) and other signals. () Immunoblots examining effects of inhibition of PI3K-AKT pathway on P-SRC-Y416 in PTEN knockdown cells. () Immunoblots assessing P-SRC-Y416 after reconstitution of wild-type (WT) PTEN, PTEN C124S or PTEN G129E mutants in PTEN-deficient MDA-MB-468 cells. () Coimmunoprecipitation assay to test for interactions between PTEN and SRC in BT474.P cells. () In vitro PTEN phosphatase assay detecting capability of purified GST-PTEN proteins (WT, C124S or G129E) to directly dephosphorylate the P-SRC-Y416 and P-SRC-Y527 phosphopeptides. (! ) Immunoblots evaluating the efficiency of SRC knockdown by SRC shRNA in BT474 PTEN knockdown cells. () MTS assay assessing trastuzumab sensitivity of PTEN knockdown cells with or without SRC knockdown. Cells were treated as in . All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 3: SRC is a key modulator of trastuzumab response. () Immunoblots comparing SRC phosphorylation status in the indicated cells expressing a constitutively active SRC mutant (Y527F) or a kinase-dead SRC mutant (K295R). () MTS assay assessing trastuzumab sensitivity of cells transfected with SRC Y527F or SRC K295R mutant. Cells were treated as in Figure 1a. Error bars, s.e.m. () 3D tumor spheroid assay comparing response to trastuzumab treatment of BT474.GFP and BT474.SRC Y527F. 3D tumor spheroid assay was carried out as described in Online Methods. Scale bar, 100 μm. () Top, representative BT474 orthotopic xenograft tumors. Scale bar, 1 cm. Bottom, volume of mammary fat pad xenograft tumors derived from either GFP-labeled BT474 parental (GFP) or SRC Y527F–expressing cells upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is percentage of original tumor size at day zero of treatment. Error bars, s.e.m. Ttzm-treated GFP group versus SRC Y527F group (ANOVA, P < 0.001, two-sided). () Correlation betwee! n clinical response rate and amount of tumor phospho-SRC-Y416 (pSRC) in patients who received first-line trastuzumab-based therapy. Complete response (CR), partial response (PR) and stable disease (SD) were grouped together and compared with SD. Patient response was compared by Fisher's exact test (P = 0.011, two-sided). () Low versus high tumor phospho-SRC-Y416 abundance and overall survival of patients who received first-line trastuzumab-based therapy. Difference of overall survival was analyzed by Kaplan-Meier survival model with log-rank test (P = 0.044, two-sided). * Figure 4: SRC inhibition induced signaling alterations in multiple trastuzumab-resistant models. () Immunoblots detecting EGF-induced EGFR dimerization in BT474.TtzmR cells stably infected with control shRNA or SRC shRNA. Cross-linking of cell membrane EGFR is described in Online Methods. MW, molecular weight. () Immunoblots comparing EGFR phosphorylation upon EGF treatment in BT474.TtzmR cells with or without SRC knockdown. () Immunoblots assessing EGFR signaling in BT474.TtzmR cells upon treatment with the SRC inhibitor saracatinib with or without EGF treatment. Cells were pretreated with saracatinib or vehicle 1 h before EGF stimulation. () Immunoblots assessing abundance of HER3 phosphorylation and other signaling events upon treatment with saracatinib alone, trastuzumab alone or combination treatment. () Immunoblots evaluating HER3 activation in response to trastuzumab treatment after stable knockdown of SRC in TtzmR cells by shRNA. (–) Immunoblots showing the impact of trastuzumab plus saracatinib on AKT signaling in multiple trastuzumab-resistant models: BT474.! TtzmR (), PTEN knockdown (), or constitutively active SRC mutant (SRC Y527F) expressing (). * Figure 5: Trastuzumab treatment plus SRC inhibition overcomes multiple resistance mechanisms in vitro. () MTS assay examining the effect of SRC inhibition in combination with trastuzumab treatment in the indicated four trastuzumab-resistant models. BT474.TtzmR cells, cells overexpressing IGF-1R and EGFR, and PTEN.shRNA cells were treated as described in Online Methods. () MTS assay evaluating the effects of trastuzumab, saracatinib or combination treatment in control BT474-GFP and trastuzumab-resistant cells overexpressing SRC Y527F. () 3D tumor spheroid assay comparing the cell proliferation of BT474.TtzmR cells upon treatment with trastuzumab alone, saracatinib alone or combination treatment. Tumor spheroid assay was carried out as described in Online Methods. Scale bar, 100 μm. () MTS assay comparing SRC inhibition by saracatinib and AKT inhibition by triciribine on overcoming trastuzumab resistance. () TUNEL assay examining induction of apoptosis by saracatinib and trastuzumab combined treatment in control (Con.shRNA) and PTEN knockdown (PTEN.shRNA) cells. TUNEL-positive! cells were detected by flow cytometry. () The induction of DNA fragmentation (indicated by sub-G1 population detected by flow cytometry) by combined treatment with saracatinib and trastuzumab in BT474.PTEN knockdown cells (PTEN.as) and BT474.SRC constitutively active (Y527F) cells. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates. * Figure 6: Trastuzumab plus saracatinib combinatorial treatment overcomes trastuzumab resistance in vivo. () Top, representative immunofluorescence images of SRC knockdown in PTEN.shRNA xenografts using intratumoral injection of SRC.shRNA-containing virus. Scale bar, 100 μm. Bottom, volume of trastuzumab-resistant PTEN-deficient tumors with or without SRC knockdown upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is presented as percentage of original tumor size at day zero of treatment. () Top, representative immunohistochemistry (IHC) images of in vivo inhibition of SRC-Y416 phosphorylation by saracatinib or AKT-S473 phosphorylation by triciribine in BT474.TtzmR xenograft tumors. Scale bar, 100 μm. Bottom, TtzmR xenograft tumor volume in response to different treatments. () Left, representative in vivo luciferase images of mice at day 0 and 21 days after indicated treatment. Left side of animal, BT474 control.shRNA tumors; right side, BT474 PTEN.shRNA tumors. Right, tumor volume in response to different treatments. () Left, representative IHC stain! ing of AKT-S473 phosphorylation after different treatments (vehicle or trastuzumab plus saracatinib) in BT474.PTEN.shRNA xenograft tumors. Scale bar, 50 μm. Right, overall AKT-S473 phosphorylation IHC staining intensity between trastuzumab-alone group and combination-treatment group. Phospho-AKT (pAKT) staining was compared between each group by Fisher's exact test (P = 0.049, two-sided). () Top, representative tumor sections with TUNEL staining. Scale bar, 50 μm. Bottom, in situ TUNEL staining of apoptotic cells in tumors treated as indicated. All error bars, s.e.m. All in vivo data were generated from a minimum of five replicates. () Model of SRC as a common node downstream of multiple resistance pathways and conquering trastuzumab resistance by targeting SRC. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA. * Siyuan Zhang, * Wen-Chien Huang, * Ping Li, * Hua Guo, * Say-Bee Poh, * Samuel W Brady, * Yan Xiong, * Ling-Ming Tseng, * Shau-Hsuan Li, * Zhaoxi Ding, * Francisco J Esteva & * Dihua Yu * Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, USA. * Samuel W Brady, * Francisco J Esteva & * Dihua Yu * Department of Pathology, MD Anderson Cancer Center, Houston, Texas, USA. * Aysegul A Sahin * Department of Breast Medical Oncology, MD Anderson Cancer Center, Houston, Texas, USA. * Francisco J Esteva & * Gabriel N Hortobagyi Contributions S.Z., W.-C.H. and D.Y. designed experiments and analyzed data; S.Z., W.-C.H., H.G., P.L., S.-B.P., S.W.B., Y.X., L.-M.T. and Z.D. carried out experiments; S.Z., H.G. and S.-H.L. did statistical analysis of clinical data; A.A.S. collected tumor samples and evaluated immunohistochemistry staining with H.G.; G.N.H. and F.J.E. collected clinical patient information and analyzed patient response data; S.Z., S.W.B. and D.Y. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dihua Yu Author Details * Siyuan Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Wen-Chien Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Ping Li Search for this author in: * NPG journals * PubMed * Google Scholar * Hua Guo Search for this author in: * NPG journals * PubMed * Google Scholar * Say-Bee Poh Search for this author in: * NPG journals * PubMed * Google Scholar * Samuel W Brady Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Ling-Ming Tseng Search for this author in: * NPG journals * PubMed * Google Scholar * Shau-Hsuan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaoxi Ding Search for this author in: * NPG journals * PubMed * Google Scholar * Aysegul A Sahin Search for this author in: * NPG journals * PubMed * Google Scholar * Francisco J Esteva Search for this author in: * NPG journals * PubMed * Google Scholar * Gabriel N Hortobagyi Search for this author in: * NPG journals * PubMed * Google Scholar * Dihua Yu Contact Dihua Yu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (524K) Supplementary Figures 1–22, Supplementary Table 1 and Supplementary Methods Additional data - Schizophrenia susceptibility pathway neuregulin 1–ErbB4 suppresses Src upregulation of NMDA receptors
- Nat Med 17(4):470-478 (2011)
Nature Medicine | Article Schizophrenia susceptibility pathway neuregulin 1–ErbB4 suppresses Src upregulation of NMDA receptors * Graham M Pitcher1, 2 * Lorraine V Kalia1, 3, 5 * David Ng1, 5 * Nathalie M Goodfellow2 * Kathleen T Yee4 * Evelyn K Lambe2 * Michael W Salter1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:470–478Year published:(2011)DOI:doi:10.1038/nm.2315Received03 December 2010Accepted31 January 2011Published online27 March 2011 Abstract * Abstract * 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 Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β–ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral–CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst–induced phosphorylation of GluN2B by inhibit! ing Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function. View full text Figures at a glance * Figure 1: NRG1β-ErbB4 signaling prevents endogenous Src activation–induced potentiation of NMDAR-mediated synaptic responses in the hippocampal CA1. () Scatter plot of NMDAR EPSC peak amplitude over time (min) from three rat CA1 neurons recorded with control intracellular solution (ICS), ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA and NRG1β (2 nM) bath-applied beginning 20 min before whole-cell recording (black bar). Top, average NMDAR EPSC traces from the three neurons (scale bars, 45 ms; 50 pA) at the times indicated (1 and 2). () Summary scatter plot of NMDAR EPSC peak amplitude with control ICS (n = 20), EPQ(pY)EEIPIA (n = 13) or EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7; *P < 0.05 or **P < 0.01 versus EPQ(pY)EEIPIA). () Current-voltage (I-V) relationship for pharmacologically isolated NMDARs with control solution, EPQ(pY)EEIPIA or EPQ(pY)EEIPIA with NRG1β in ACSF. Right, superimposed NMDAR EPSC traces at membrane potentials from −80 to +60 mV. Scale bars, 150 ms; 250 pA. () Summary scatter plot of normalized peak NMDAR EPSC amplitude from CA1 neurons from WT mice during intracellular administrati! on of ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in ACSF (n = 18), from neurons from Erbb4−/−HER4heart mice during intracellular administration of EPQ(pY)EEIPIA with NRG1β (2 nM) (n = 7) or from neurons from WT mice during intracellular administration of EPQ(pY)EEIPIA with bath-applied NRG1β (2 nM) and AG1478 (n = 16). Top, representative average NMDAR EPSCs at the indicated times (1 and 2) and after D-APV (scale bars, 50 ms; 50 pA). () Summary histogram of EPQ(pY)EEIPIA-induced increase in NMDAR EPSC amplitude at 30 min of recording from WT neurons with ICS containing EPQ(pY)EEIPIA (n = 13), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording (n = 18), from CA1 pyramidal neurons from Erbb4−/−HER4heart mice with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 7) or from WT CA1 pyramidal neurons with ICS containing EPQ(pY)EEIPIA with NRG1β and AG1478 in ACSF (n = 16). ***P < 0.001 versus wild-type. In al! l figures, group data are mean ± s.e.m. In voltage-clamp expe! riments the holding potential was −60 mV, except where otherwise indicated. * Figure 2: NRG1β has no effect on basal NMDAR-mediated synaptic responses in hippocampal CA1 or in prefrontal cortex but prevents endogenous Src activation-induced potentiation of NMDAR EPSCs at prefrontal cortex synapses. () Summary scatter plot of peak NMDAR EPSC amplitude over time from mouse CA1 neurons recorded with control ICS (n = 10) and NRG1β (2 nM) present in ACSF beginning at 10 min (black bar). Top, average NMDAR EPSCs from a representative neuron (scale bars, 50 ms; 50 pA) at the times indicated (1 and 2) and after D-APV. () Summary histogram of NMDAR EPSC decay from the CA1 neurons recorded in at the 10-min time point immediately before NRG1β administration and at the 40-min time point during NRG1β perfusion. Results are percentage of NMDAR EPSC decay with mean decay during first 2 min of recording before NRG1β administration normalized to 100% (dotted line). () Summary histogram of pharmacologically isolated NMDAR fEPSP responses (n = 14) before or during PD158780 application. Results are percentage of NMDAR fEPSP slope at the beginning of the recording normalized to 100% (dotted line). () Scatter plot of NMDAR EPSC peak amplitude from four prefrontal cortex neurons: with co! ntrol ICS, with control ICS with NRG1β (6 nM) in ACSF, with ICS containing EPQ(pY)EEIPIA or with ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (black bar). Top, average NMDAR EPSC traces from the four neurons (scale bars, 100 ms; 40 pA) at the times indicated (1 and 2). () Summary histogram of normalized NMDAR EPSC amplitude at 30 min of recording from prefrontal cortex neurons with control ICS (n = 11), ICS containing EPQ(pY)EEIPIA (n = 9), control ICS with NRG1β present in ACSF (n = 7) or ICS containing EPQ(pY)EEIPIA with NRG1β in ACSF (n = 5). ***P < 0.001. * Figure 3: NRG1β prevents but does not reverse endogenous Src-induced synaptic potentiation. () Scatter plot of EPSP slope over time from three rat CA1 neurons recorded with control ICS, ICS containing EPQ(pY)EEIPIA or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) in the ACSF beginning 20 min before whole-cell recording (black bar). Right, average EPSP traces from the three neurons (scale bars, 50 ms; 10 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1 stratum radiatum. Right, average fEPSPs (scale bars, 30 ms; 0.5 mV) at the times indicated (1 and 2). () Summary scatter plot of EPSP slope with control ICS (n = 16), ICS containing EPQ(pY)EEIPIA (n = 9) or ICS containing EPQ(pY)EEIPIA with NRG1β in the ACSF (n = 6; *P < 0.05, **P < 0.01 or ***P < 0.001 versus EPQ(pY)EEIPIA). Bottom, averaged slope of fEPSPs recorded simultaneously during experiments when whole-cell recordings were carried out. () Scatter plot of EPSP slope over time from two WT mouse CA1 neurons recorded with ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) or vehicle administe! red to the ACSF during EPQ(pY)EEIPIA-induced potentiation of EPSP responses (black bar). Right, average EPSPs from the two neurons (scale bars, 100 ms; 15 mV). Bottom, scatter plots of simultaneously recorded fEPSPs from CA1. Right, average fEPSPs (scale bars, 10 ms; 0.5 mV) at the times indicated (1, 2 and 3). () Summary histogram of the EPQ(pY)EEIPIA-induced increase in EPSP slope at 30 min of recording from neurons with ICS containing EPQ(pY)EEIPIA (n = 19), ICS containing EPQ(pY)EEIPIA with NRG1β present in ACSF beginning 20 min before whole-cell recording at 0.2 nM (n = 16; **P < 0.01 versus vehicle) or 2 nM (n = 6; ***P < 0.001 versus vehicle) or ICS containing EPQ(pY)EEIPIA with NRG1β (2 nM) treatment during EPQ(pY)EEIPIA-induced potentiation of EPSPs (n = 12). * Figure 4: NRG1β prevents but does not reverse TBS-induced LTP in CA1 hippocampus and has no effect in Src−/− mice. () Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) or another slice treated with vehicle (black bar). Top, average fEPSP traces (scale bars, 10 ms; 0.5 mV) at the times indicated (1 and 2) from the two individual experiments. Bottom, summary scatter plot of grouped normalized fEPSP slope from control slices (n = 23) or NRG1β-treated slices (n = 14). () Scatter plot of normalized fEPSP slope over time for a rat hippocampal slice treated with NRG1β (2 nM) present in ACSF beginning 30 min after TBS (black bar). Top, average fEPSP traces (scale bars, 15 ms; 0.5 mV) at the times indicated (1, 2, 3 and 4) from the experiment plotted. Bottom, summary scatter plot of grouped normalized fEPSP slope (n = 19). () Summary scatter plot of grouped normalized fEPSP slope in slices from Src+/+ mice treated with vehicle (VEH, n = 13) or NRG1β (NRG, 2 nM; n = 14; P < 0.001 versus VEH) (black bar). Inset, average fEPSP traces at the ti! mes indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. Bottom, summary scatter plot of normalized fEPSP slope in slices from Src−/− mice treated with NRG1β (2 nM, n = 11) or vehicle (n = 14) (black bar). Inset, average fEPSP traces recorded at the times indicated (1 and 2) (scale bars, 10 ms; 0.5 mV) from two representative experiments. () Summary histogram of TBS-induced increase in fEPSP slope 60 min after TBS in slices from Src+/+ and Src−/− mice with NRG1β (+) or vehicle (−) treatment. Data are percentage of tbLTP with vehicle normalized to 100%. ***P < 0.001 versus Src+/+ with vehicle. () Immunoblot analysis of ErbB4, GluN1 and GluN2, in CA1 from a Src+/+ mouse or a Src−/− littermate. GAPDH was loading control. * Figure 5: NRG1β reduces depolarization of CA1 neurons during the period of TBS. () The first four pulse-induced burst EPSP of TBS for control mouse (Src+/+) slices (n = 28), slices (from Src+/+ mice) pretreated with D-APV (n = 17), slices from Src−/− mice (n = 12) or slices from Src+/+ mice pretreated with NRG1β (2 nM; n = 19). Traces are the mean membrane potential from all recordings in response to the first four stimuli. Gray region, ± s.e.m. Vertical arrows, start of stimulation for each of four pulses delivered at 100 Hz. x axis, time (200 ms). Bottom, average pre-TBS baseline single stimulus–evoked EPSPs (scale bars, 100 ms; 10 mV). () Burst EPSPs for the entire duration of TBS from control slices (n = 28) or slices pretreated with NRG1β (n = 19). () Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in control slices (n = 28) or slices pretreated with NRG1β (n = 19; *P < 0.05 or **P < 0.01 versus control). () Summary scatter plot of grouped normalized EPSP slope over time. () Fi! rst four pulse-induced burst EPSP of TBS for neurons from control slices (from Src+/+ mice), slices (from Src+/+ mice) pretreated with NRG1β (2 nM) or slices (from Src+/+ mice) pretreated with NRG1β (2 nM) and AG1478. x axis, time (200 ms). Inset, average representative pre-TBS baseline single stimulus–evoked EPSPs from a control slice and a slice pretreated with NRG1β and AG1478 (scale bars, 100 ms; 8 mV). () Summary scatter plot of peak amplitude of burst EPSPs in response to each of the four stimuli-elicited bursts in slices pretreated with NRG1β (n = 19) or with NRG1β and AG1478 (n = 25; *P < 0.05, **P < 0.01 or ‡P < 0.001 versus NRG1β). * Figure 6: NRG1β does not alter Src association with the NMDAR but reduces Src tyrosine kinase activity and prevents TBS-induced GluN2B phosphorylation in hippocampal CA1. () Immunoprecipitation (IP) of GluN2 subunits carried out from hippocampal proteins prepared from control (−) or NRG1β (2 nM)-treated (+) slices. Proteins that immunoprecipitated with antibody to GluN2B were probed with antibodies to GluN2B, PSD-95 or Src (left). In 'input' lanes, 60 μg of proteins without immunoprecipitation were loaded. () Histogram of Src kinase activity in CA1 lysates without (n = 4) or with (n = 4) NRG1β (2 nM) pretreatment. Src kinase activity normalized to activity of Src in control lysates (**P < 0.01, t‐test versus control). () Summary histogram of TBS-induced pYGluN2B from CA1 region collected from Src+/+ (n = 6) or Src−/− (n = 6; **P < 0.01, t-test versus Src+/+) mice. () Top image, representative immunoblots of lysates from rat CA1 region with TBS or without (baseline, BL) probed with antibody to phosphorylated tyrosine (pY). The same membrane was stripped and reprobed with GluN2B antibody. Top graph, four-point standard curve derived ! from serial dilutions of rat brain proteins. Bottom image, representative immunoblot of lysates from rat CA1 region with TBS or without (BL) and treated with NRG1β (2 nM) probed sequentially with antibodies to pY and GluN2B. Bottom graph, four-point standard curve derived from serial dilutions of rat brain lysate. Right, summary histogram of immunoblot analysis. Densiometric quantification was carried out for pY GluN2B from six immunoblot experiments for each condition. Band intensity was quantified as mean gray value and the ratio of pY GluN2B to total GluN2B was calculated. Bars correspond to mean ratios normalized to ratios obtained for baseline (BL; *P < 0.05, t‐test versus BL). () Top, four-point standard curve derived from serial dilutions of Src+/+ mouse brain lysate. Bottom, summary histogram of TBS-induced pY GluN2B from hippocampal CA1 region without (n = 4) or with (n = 4) NRG1β (2 nM) treatment or with NRG1β (2 nM) and AG1478 treatment (n = 4). **P < 0.01, ! t‐test versus control or versus NRG1β and AG1478. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Lorraine V Kalia & * David Ng Affiliations * Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario, Canada. * Graham M Pitcher, * Lorraine V Kalia, * David Ng & * Michael W Salter * Department of Physiology, University of Toronto, Ontario, Canada. * Graham M Pitcher, * Nathalie M Goodfellow, * Evelyn K Lambe & * Michael W Salter * Institute of Medical Sciences, University of Toronto, Ontario, Canada. * Lorraine V Kalia & * Michael W Salter * Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, USA. * Kathleen T Yee Contributions G.M.P. designed the project, conducted the hippocampal experiments, analyzed the data and wrote the manuscript. L.V.K. and D.N. carried out and analyzed biochemical experiments. E.K.L. oversaw and analyzed the prefrontal cortex experiments. N.M.G. carried out and analyzed the prefrontal cortex experiments. K.T.Y. maintained, housed and provided ErbB4 knockout mice. All authors participated in revising the manuscript and agreed to the final version. M.W.S. conceived the study, analyzed data, supervised the overall project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael W Salter Author Details * Graham M Pitcher Search for this author in: * NPG journals * PubMed * Google Scholar * Lorraine V Kalia Search for this author in: * NPG journals * PubMed * Google Scholar * David Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Nathalie M Goodfellow Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen T Yee Search for this author in: * NPG journals * PubMed * Google Scholar * Evelyn K Lambe Search for this author in: * NPG journals * PubMed * Google Scholar * Michael W Salter Contact Michael W Salter Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (7M) Supplementary Figures 1–5 and Supplementary Methods Additional data - Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity
- Nat Med 17(4):479-487 (2011)
Nature Medicine | Article Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity * Tracy L Flach1, 7 * Gilbert Ng1, 7 * Aswin Hari1 * Melanie D Desrosiers1 * Ping Zhang2 * Sandra M Ward2 * Mark E Seamone3 * Akosua Vilaysane3 * Ashley D Mucsi1 * Yin Fong1 * Elmar Prenner4 * Chang Chun Ling2 * Jurg Tschopp5 * Daniel A Muruve3 * Matthias W Amrein6 * Yan Shi1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:479–487Year published:(2011)DOI:doi:10.1038/nm.2306Received26 July 2010Accepted18 January 2011Published online13 March 2011 Abstract * Abstract * 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 As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4+ T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function–associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behav! e as a general sensor for solid structures. View full text Figures at a glance * Figure 1: Alum shows affinity for DC surface without inducing phagocytosis. () Left, serum IgG1 OVA-specific antibody titers 2 weeks after immunization with the indicated type of alum as adjuvant. Right, similar to the left except that clinical alum AlOH and AlPO4 were tested. () An SEM image of CsAl mounted on an AFM tip with epoxy. () Typical binding forces between a DC2.4 cell and four forms of alum. Blank refers to a tip with no alum attached. The number in parentheses indicates number of independent repeats of that operation (a representative curve for each is shown). At top right is a schematic depiction of the assay. The bottom right graph is a comparison of force curves between an MSU tip and an alum tip in their interaction with a DC2.4 cell. DC2.4 cell interaction with an MSU (or latex, not shown) tip is characterized by a gradual increase in binding strength and by a point of loss of retraction force curve indicated by the arrow (point of irreversible binding: phagocytosis). () SEM of DC2.4 cells cultured for 2 h with either CsAl (top) or! MSU (bottom). * Figure 2: Alum targets only DCs. () The binding forces of the indicated cells with a CsAl tip as compared to a blank tip. B cells (A20, Bjab, Raj and Ramos), macrophages (RAW 264.7, Ana Ry 1 and J744) and DCs (DC2.4 and PMA-transformed THP-1) were cultured on glass disks by direct adhesion or via a poly-D-lysine substrate coating. () Similar to except that GM-CSF plus IL-4 and DAP supernatant were used to differentiate BMDCs and macrophages, respectively, and magnetic cell sorting beads were used to purify splenic B cells. All examined with an Imject tip. () Environmental SEM image and EDS analysis of alum (CsAl precipitate) content in DC2.4 cells treated with alum for 2 h. Cells were washed three times without further treatment or gold sputter coating before data collection. In areas of high carbon reading, the aluminum (natural contaminant) in the glass is shielded from the electron beams due to the cell mass. () Similar to except that BMDCs from C57BL/6 or TLR4-knockout mice were contacted by AlOH. () Si! milar to except that DC2.4 cells were pretreated with 10 μg ml−1 of LPS for 30 min before the assay. * Figure 3: Alum's direct affinity for membrane lipids. () CsAl precipitate crystals were stained with equal molar concentrations of either phosphatidylcholine (PC), nitro-2-1,3-benzoxadiazol-4-yl (NBD), phosphatidylethanolamine (PE, NBD), sphingomyelin (Sph, BODIPY), or cholesterol (Chol, BODIPY). The bound fluorescence intensity was analyzed by FACS. () A synthetic cholesterol, SW.I.30, was attached to a gold-coated AFM tip via the thiol group at the end of the aliphatic chain extension. Top, a schematic depiction of the experiments. Left graph, the reading of the maximal attraction forces for SW.I.30. n represents the number of independent repeats. Control is a similar synthesis with the aliphatic extension from the hydroxyl group on the head of cholesterol. Right graph, a direct comparison between both cholesterol (SW.I.30) and synthetic sphingomyelin (PZ-3019) in their biological orientation in the membrane with reference to alum crystal contact. Blank, an unmodified tip. PZ-3019 is depicted at the bottom. () Synthetic bilay! er membranes from defined membrane lipids were laid on a glass surface for contact by an Imject tip. The red indicates the upper leaflet for contact. Average binding forces were recorded; the number on the top indicates the number of repeats. Error bars are s.e.m. * Figure 4: Alum triggers membrane lipid sorting and the activation of the Src-ITAM-Syk-PI3K pathway for DC activation. () Left, similar to Figure 1c except the CsAl binding with DC2.4 cell was recorded in the presence of the indicated inhibitors. Blank is a tip without alum. All other readings were recorded with an alum functionalized tip. Right, similar to the left except that an AlOH tip was used. () Similar to except that BMDCs cultured from wild-type (C57BL/6) or the indicated knockouts; Syk, Src (Hck, Fgr and Lyn triple-deficient) or ITAM (FcRγ and DAP12 double-deficient (Fcer1g−/− Tyrobp−/−)) were used in place of wild-type DCs without inhibitors. () Western blot analysis of total and phosphorylated Erk1/2 in DC2.4 and RAW 264.7 cells treated with alum. () Left, binding forces between DC2.4 or RAW 264.7 cells and AlOH-coated tips in the presence of the ndicated Erk inhibitor or activator. Right, similar to except DC2.4 and RAW 264.7 cells were pretreated with ferrocene and PD098059 before alum stimulation. () The draining LN CD11c+ cells were analyzed for Alexa levels 36 h aft! er subcutaneous injection of Alexa-OVA and alum mixture as described in the Online Methods. The numbers indicate the CD11c+Alexa+ cells as a percentage of the total cells. * Figure 5: Alum-mediated DC binding and activation requires the ITAM-Syk pathway but not Nlrp3-ASC. () Left, similar to Figure 4a except BMDCs from wild-type C57BL/6, Pycard−/− or Nlrp3−/− mice were used. Right, similar to graph on left except that an AlOH tip was used. () FACS analysis of CD40, CD80 and CD86 expression on BMDCs from wild-type, Nlrp3−/− and Pycard−/− mice left untreated or activated with MSU, bacteria (BAC, killed DH5αEscherichia coli) or CsAl precipitate for 24 h. () ELISA results of TNF-α (left) and IL-1β (right) production from DCs left untreated or activated with CpG, E. coli or CsAl precipitate for 6h. The loss of IL-1β production in Pycard−/− and Nlrp3−/− DCs confirms their deficiencies. () Similar to except BMDCs from wild-type, Syk-, Src- or ITAM-deficient Fcer1g−/− Tyrobp−/− DCs were used. MSU, known to trigger very little or no TNF-α production from BMDCs (our own observation), was used as a baseline control. * Figure 6: DCs following alum contact gain strong adhesion to CD4 T cells. () Alexa levels in untreated or BMDCs incubated with CsAl precipitate, Alexa-OVA and conjugated CsAl precipitate–Alexa-OVA as measured by FACS after 2 h. Piceatannol or cytochalasin B was also incubated with CsAl–Alexa-OVA conjugates. () Top left, splenic B or CD4+ cell binding by DC2.4 cells treated with CsAl precipitate for 4 h. Excess alum was washed away before the binding assay. The top right graph is similar to the left except that BMDCs from TLR4-knockout mice were treated with AlOH. Bottom right, identical data to those at top left depicted in a bar graph for better statistical representation; one-way analysis of variance (ANOVA) for all four is 0.014. () Similar to bottom right panel except that DCs were pretreated with soluble OVA for 2 h before the reading and that the approaching cell was a magnetic cell sorting–purified splenic OT-II T cell. One-way ANOVA for all four is 0.022. () Left, BMDC surface expression of ICAM-1 after treatment with AlOH, CsAl or C! pG for 24 h was analyzed by FACS with YN1/1.7 antibody. Right, similar to except that untreated, alum-treated wild-type or alum-treated Icam1−/− BMDCs were used to make contact with either wild-type CD4+ T cells or LFA-1–knockout CD4+ T cells. () SEM image of a magnetic cell sorting–purified untreated splenic DC (left) and a DC treated with alum with subsequent Histopaque gradient purification (top right). Bottom right, a DC recovered from the pellet. () Serum OVA-specific IgG1 titers measured by ELISA 2 weeks after C57BL/6 mice were intravenously immunized with OVA and DC treated with CsAl precipitate and purified with a Histopaque gradient. Standard immunization control is CsAl-based standard OVA immunization. Control is an untreated mouse serum. () Similar to , except that Nlrp3−/− DCs were used. () A proposed mechanism of how alum invokes the humoral immune response in vaccination. In response to the alum-antigen mixture, DC membrane lipids serve as a surrog! ate receptor to the crystal surface. The ensuing lipid sorting! triggers the aggregation of ITAM containing molecules, Syk recruitment and PI3K activation. However, alum does not enter DCs; it instead delivers the antigen into the DCs via endocytic uptake. DCs process the antigen in their MHC class II compartment and at the same time become activated as a consequence of the inflammatory phagocytosis. The activated DCs strongly engage CD4+ cells via ICAM-1 and LFA-1 binding, leading to the subsequent cognate B cell activation. Here cross-presentation of MHC class I antigens is absent, resulting in no CTL induction. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Tracy L Flach & * Gilbert Ng Affiliations * Immunology Research Group, Department of Microbiology & Infectious Diseases, and Snyder Institute, University of Calgary, Calgary, Alberta, Canada. * Tracy L Flach, * Gilbert Ng, * Aswin Hari, * Melanie D Desrosiers, * Ashley D Mucsi, * Yin Fong & * Yan Shi * Department of Chemistry and Alberta Ingenuity Center for Carbohydrate Science, University of Calgary, Calgary, Alberta, Canada. * Ping Zhang, * Sandra M Ward & * Chang Chun Ling * Department of Medicine, University of Calgary, Calgary, Alberta, Canada. * Mark E Seamone, * Akosua Vilaysane & * Daniel A Muruve * Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. * Elmar Prenner * Department of Biochemistry, University of Lausanne, Epalinges, Switzerland. * Jurg Tschopp * Department of Biology and Anatomy, University of Calgary, Calgary, Alberta, Canada. * Matthias W Amrein Contributions Y.S. designed experiments with input from M.W.A. and wrote the manuscript with assistance from T.L.F. and D.A.M. T.L.F. performed the experiments unless indicated otherwise below. G.N. performed EDS and SEM assays and developed methods for lipid-crystal binding analysis. A.H. and A.D.M. performed CD4-DC binding analysis and EDS and SEM work and contributed to bilayer lipid synthesis. M.D.D. performed antibody induction and cytokine studies with assistance from Y.F. S.M.W., P.Z. and C.C.L. performed aliphatic chain extension on cholesterol and sphingomyelin. M.E.S. and A.V. performed western blotting. E.P. provided Langmuir trough and technical assistance. J.T. and D.A.M. provided inflammasome-deficient mice and technical assistance and consultation. M.W.A. supervised all aspects of work involving AFM. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Yan Shi Author Details * Tracy L Flach Search for this author in: * NPG journals * PubMed * Google Scholar * Gilbert Ng Search for this author in: * NPG journals * PubMed * Google Scholar * Aswin Hari Search for this author in: * NPG journals * PubMed * Google Scholar * Melanie D Desrosiers Search for this author in: * NPG journals * PubMed * Google Scholar * Ping Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Sandra M Ward Search for this author in: * NPG journals * PubMed * Google Scholar * Mark E Seamone Search for this author in: * NPG journals * PubMed * Google Scholar * Akosua Vilaysane Search for this author in: * NPG journals * PubMed * Google Scholar * Ashley D Mucsi Search for this author in: * NPG journals * PubMed * Google Scholar * Yin Fong Search for this author in: * NPG journals * PubMed * Google Scholar * Elmar Prenner Search for this author in: * NPG journals * PubMed * Google Scholar * Chang Chun Ling Search for this author in: * NPG journals * PubMed * Google Scholar * Jurg Tschopp Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel A Muruve Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias W Amrein Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Shi Contact Yan Shi Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7, Supplementary Methods and Supplementary Data Additional data - RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis
- Nat Med 17(4):488-494 (2011)
Nature Medicine | Article RGMa modulates T cell responses and is involved in autoimmune encephalomyelitis * Rieko Muramatsu1, 2, 8 * Takekazu Kubo3, 8 * Masahiro Mori4 * Yuka Nakamura1, 2 * Yuki Fujita1, 2 * Tsugio Akutsu5 * Tatsusada Okuno6 * Junko Taniguchi4 * Atsushi Kumanogoh6 * Mari Yoshida7 * Hideki Mochizuki2, 5 * Satoshi Kuwabara4 * Toshihide Yamashita1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:488–494Year published:(2011)DOI:doi:10.1038/nm.2321Received11 August 2010Accepted02 February 2011Published online20 March 2011 Abstract * Abstract * 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 In multiple sclerosis, activated CD4+ T cells initiate an immune response in the brain and spinal cord, resulting in demyelination, degeneration and progressive paralysis. Repulsive guidance molecule-a (RGMa) is an axon guidance molecule that has a role in the visual system and in neural tube closure. Our study shows that RGMa is expressed in bone marrow–derived dendritic cells (BMDCs) and that CD4+ T cells express neogenin, a receptor for RGMa. Binding of RGMa to CD4+ T cells led to activation of the small GTPase Rap1 and increased adhesion of T cells to intracellular adhesion molecule-1 (ICAM-1). Neutralizing antibodies to RGMa attenuated clinical symptoms of mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and reduced invasion of inflammatory cells into the CNS. Silencing of RGMa in MOG-pulsed BMDCs reduced their capacity to induce EAE following adoptive transfer to naive C57BL/6 mice. CD4+ T cells isolated from mic! e treated with an RGMa-specific antibody showed diminished proliferative responses and reduced interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4 and IL-17 secretion. Incubation of PBMCs from patients with multiple sclerosis with an RGMa-specific antibody reduced proliferative responses and pro-inflammatory cytokine expression. These results demonstrate that an RGMa-specific antibody suppresses T cell responses, and suggest that RGMa could be a promising molecular target for the treatment of multiple sclerosis. View full text Figures at a glance * Figure 1: RGMa activates Rap1 and regulates CD4+ T cell adhesion. () Quantitative RT-PCR showing relative expression level of mRNA encoding RGMa in LPS-stimulated BMDCs at the indicated concentrations for 24 h. () Western blot analysis of RGMa (50-kDa and 35-kDa bands; top rows) and α-tubulin (bottom row). Relative expression of RGMa in the BMDCs. () Binding of human RGMa-Fc to splenic CD4+ T cells. () Top, representative western blot images obtained with a Rap1 pull-down assay. The bottom graph shows the relative Rap1 activity, as determined by the band intensity of RalGDS-bound Rap1 normalized to that of total Rap1 in the lysates. () CD4+ T cell adhesion to ICAM-1 in the presence and absence of GGTI-298, a selective Rap1 inhibitor. Error bars are the mean ± s.e.m. of three or four independent experiments. *P < 0.05 and **P < 0.01 by one-way analysis of variance followed by Tukey's test for , and and by Student's t test for . * Figure 2: Expression of RGMa and neogenin in MOG-induced EAE and multiple sclerosis tissue. () Frozen sections of the spleen immunostained for RGMa (labeled with Alexa Fluor 568) and CD11c (labeled with Alexa Fluor 488) in EAE and control mice. The graph shows the relative expression of RGMa in CD11c+ cells in the lymph node, spleen and spinal cord before (control) and 1, 2 and 3 weeks after immunization with MOG. n = 37–51 cells for each mouse. *P < 0.05 and **P < 0.01 by one-way analysis of variance followed by Tukey's test. () The sections (same sections as shown in ) of the spleen immunostained for RGMa (labeled with Alexa Fluor 568) and plasmacytoid dendritic cells (mPDCA-1) (labeled with Alexa Fluor 488). The graph shows the relative expression of RGMa in mPDCA-1+ cells. n = 40–48 cells for each mouse. () Expression of neogenin (labeled with Alexa Fluor 568) in CD4+ T cells (labeled with Alexa Fluor 488) in the spleen. The graph shows the relative expression of neogenin in CD4+ T cells. () In situ Rap1 pull-down assay (labeled with Alexa Fluor 568) in CD4! + T cells (labeled with Alexa Fluor 488) in cervical spinal cord tissue sections of EAE and control mice. () Multiple sclerosis brain and spinal cord tissues sections double-labeled for RGMa (with Alexa Fluor 488) in combination with CD83 or CD209 (DC-SIGN) (labeled with Alexa Fluor 568) n = 8. () Expression of neogenin (labeled with Alexa Fluor 488) in human CD3+ cells (labeled with Alexa Fluor 568) in relapsing-remitting multiple sclerosis and healthy control PBMCs. () Relative fluorescence intensity of neogenin in the immunohistochemical analysis. Error bars represent the mean ± s.e.m. of 3 or 4 independent experiments. Scale bars in –, 50 μm for low (overlay images in , , and ) and 10 μm for high (all other images) magnification images; scale bar in , 5 μm. * Figure 3: RGMa-specific antibody treatment reduces the severity of MOG-induced EAE. () Clinical EAE disease scores (EAE score) in mice treated with control IgGs (n = 16) and RGMa-specific antibodies (anti-RGMa; n = 9). Data represent the mean ± s.e.m. *P < 0.05, and **P < 0.01 by Mann-Whitney's U test. The arrows represent the time points of antibody administration. () Incidence of EAE clinical signs in MOG-induced EAE mice treated with control IgGs or RGMa-specific antibodies. () The average day of disease onset between the two treatment groups. () The mean ± s.e.m. of the maximum EAE score of each mouse with EAE. () The mean ± s.e.m. of the cumulative EAE scores. *P < 0.05 and **P < 0.01 by Student's t test. NS, not significant. () H&E staining of the cervical spinal cord in RGMa-specific antibody– and control IgG–treated mice. () Histological scores (inflammatory index; see Supplementary Methods) for the inflammatory lesions. Error bars represent the mean ± s.e.m. (control IgG, n = 6; RGMa-specific antibody, n = 5). *P < 0.01 by Student's t test.! () Representative images of CD4+, CD11b+, F4/80+, B220+, CD11c+ and mPDCA-1+ cells in the spinal cord of control IgG- and RGMa-specific antibody–treated EAE mice. (,) FluoroMyelin () and APP () staining in the spinal cord of IgG- and RGMa-specific antibody–treated mice. Scale bars in ,,, 200 μm; scale bar in , 100 μm. * Figure 4: RGMa-specific antibody treatment suppresses T cell responses in EAE. () EAE scores in mice after adoptive transfer of MOG-stimulated BMDCs with or without RGMa knockdown. () EAE scores in mice after adoptive transfer of re-stimulated CD4+ T cells from donor mice immunized with MOG with or without the RGMa-specific antibody treatment (anti-RGMa). () Number of GFP-labeled CD4+ T cells in the brain sections of control recipient mice and mice treated with RGMa-specific antibody at day 10 after adoptive transfer of the re-stimulated EGFP-labeled CD4+ T cells. () Percentage of CD4+ T cells obtained from MOG-EAE mice that adhered to ICAM-1 in the presence of RGMa-specific or control antibodies in vitro. () Transmigration of CD4+ T cells across b-End3 cells. Left, CD4+ T cells from EAE mice treated in vivo with control IgG or RGMa-specific antibody; right, CD4+ T cells from EAE mice treated in vitro with control IgG or RGMa-specific antibody. () Top, representative western blots obtained by Rap1 pull-down assay. Bottom, relative Rap1 activity. () EAE! scores after intraperitoneal injection of RGMa-specific antibody or control IgG in SJL/J mice immunized with PLP. Number of mice in each group: control siRNA: 5, RGMa siRNA: 5 (); control IgG: 7, RGMa-specific antibody: 7 (); control IgG: 5, RGMa-specific antibody: 5 (); control IgG: 4, RGMa-specific antibody: 4 (), each group: 4 (); each group: 3 (); and control IgG: 11, RGMa-specific antibody: 9 (). Error bars in all panels represent the means ± s.e.m; *P < 0.05 and **P < 0.01 as compared with control group by Mann-Whitney's U test for , and and Student's t test for –. * Figure 5: T cell proliferation and cytokine production from MOG-EAE mice and PBMCs from humans with multiple sclerosis. () Top graph, MOG-specific proliferative responses in splenic cells obtained from mice treated with RGMa-specific antibody (anti-RGMa) or control IgG at d 21 after MOG immunization. Each bar represents the mean ± s.e.m. (control IgG, n = 6; RGMa-specific antibody, n = 5). Bottom graphs, production of IL-2, IFN-γ, IL-17, IL-4, IL-10 and TGF-β by spleen cells obtained from mice treated with RGMa-specific antibody or control IgGs. Each bar represents the mean ± s.e.m. (control IgG, n = 6; RGMa-specific antibody, n = 5). *P < 0.05, and **P < 0.01 by Student's t-test. () Relative levels of cell proliferation (top two graphs) and relative levels of cytokine mRNA (bottom graphs) from PBMCs stimulated with PMA plus ionomycin in the presence or absence of the RGMa-specific antibody. PBMCs were obtained from individuals with relapsing-remitting multiple sclerosis (8 in clinical relapse; 9 in clinical remission). Each error bar represents the mean ± s.e.m; *P < 0.05 and **P < 0.01! by Student's t test. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Rieko Muramatsu & * Takekazu Kubo Affiliations * Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan. * Rieko Muramatsu, * Yuka Nakamura, * Yuki Fujita & * Toshihide Yamashita * Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo, Japan. * Rieko Muramatsu, * Yuka Nakamura, * Yuki Fujita, * Hideki Mochizuki & * Toshihide Yamashita * Department of Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan. * Takekazu Kubo & * Toshihide Yamashita * Department of Neurology, Graduate School of Medicine, Chiba University, Chiba, Japan. * Masahiro Mori, * Junko Taniguchi & * Satoshi Kuwabara * Department of Neurology, Kitasato University School of Medicine, Kanagawa, Japan. * Tsugio Akutsu & * Hideki Mochizuki * Department of Immunopathology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan. * Tatsusada Okuno & * Atsushi Kumanogoh * Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan. * Mari Yoshida Contributions T.K. performed preliminary experiments for expression analysis, behavioral and histological analysis of EAE, and cytokine production, and contributed to conceiving the study. Later, R.M. took over the work and performed all experiments, with the exception of the portions indicated below. Y.N. performed EAE induction, adoptive transfer experiments, immunohistochemical analyses and spinal cord injury experiments. Y.F. performed the lymphocyte binding assay, Rap1 activity assay and cytokine analysis. M.M., J.T. and S.K. performed experiments with PBMCs. T.O. helped with irradiation experiments. M.M., T.A., J.T., M.Y., H.M. and S.K. performed experiments with autopsy samples. T.K and T.Y. conceived the project and developed the hypothesis. T.K, R.M., A.K. and T.Y. designed the experiments. A.K. and T.O. discussed the hypothesis and helped with data interpretation. T.Y. coordinated and directed the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Toshihide Yamashita Author Details * Rieko Muramatsu Search for this author in: * NPG journals * PubMed * Google Scholar * Takekazu Kubo Search for this author in: * NPG journals * PubMed * Google Scholar * Masahiro Mori Search for this author in: * NPG journals * PubMed * Google Scholar * Yuka Nakamura Search for this author in: * NPG journals * PubMed * Google Scholar * Yuki Fujita Search for this author in: * NPG journals * PubMed * Google Scholar * Tsugio Akutsu Search for this author in: * NPG journals * PubMed * Google Scholar * Tatsusada Okuno Search for this author in: * NPG journals * PubMed * Google Scholar * Junko Taniguchi Search for this author in: * NPG journals * PubMed * Google Scholar * Atsushi Kumanogoh Search for this author in: * NPG journals * PubMed * Google Scholar * Mari Yoshida Search for this author in: * NPG journals * PubMed * Google Scholar * Hideki Mochizuki Search for this author in: * NPG journals * PubMed * Google Scholar * Satoshi Kuwabara Search for this author in: * NPG journals * PubMed * Google Scholar * Toshihide Yamashita Contact Toshihide Yamashita 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 - A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis
- Nat Med 17(4):495-499 (2011)
Nature Medicine | Letter A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis * Ivana Nikić1 * Doron Merkler2, 3 * Catherine Sorbara1 * Mary Brinkoetter4 * Mario Kreutzfeldt2, 3 * Florence M Bareyre1 * Wolfgang Brück2 * Derron Bishop4 * Thomas Misgeld5, 6, 7 * Martin Kerschensteiner1, 7 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:495–499Year published:(2011)DOI:doi:10.1038/nm.2324Received27 July 2010Accepted07 February 2011Published online27 March 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits1, 2. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view2, 3, 4, 5, 6, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacol! ogical experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy. View full text Figures at a glance * Figure 1: In vivo imaging of FAD. () Confocal projection showing axons (white), activated macrophages/microglia (magenta) and T cells (cyan) in an acute spinal EAE lesion. Some axons appear normal (stage 0), whereas others are swollen (1) or fragmented (2). () Pseudocolored axons isolated from those shown in : normal appearing (0, green), swollen (1, yellow), and fragmented (2, red). () Frequency (in % ± s.e.m.) of axon stages in normal spinal cord (N) and in EAE lesions (0–30 d after EAE onset; differences at all EAE time points compared to control are significant, P < 0.001 to 0.05, one-tailed t test). () Multiphoton time-lapse images of a stage 1 axon (white) in EAE and activated macrophages/microglia (magenta). Time is shown as h:min; meningeal second harmonics (a scattering process) signal is green). The axon first breaks (red arrowhead) near a small swelling (yellow arrowhead) at a putative node of Ranvier before fragmenting (gray arrowheads). () Fate of stage 1 axons imaged 1–3 d after the peak o! f EAE (significant progression from 1–3 d, P < 0.05, chi-square test for trend). () Time-lapse images of recovering stage 1 axon (time is as shown in ). Scale bar in ,, 10 μm; scale bar in , 25 μm; scale bar in , 10 μm. * Figure 2: Early FAD stages show mitochondrial alterations but no demyelination. (–) Electron micrograph of a stage 1 axon with a paranodal swelling and preserved myelin (pseudocolored orange-brown; paranodal loops magnified and marked by arrows in ). The axon contains intact looking (green, example magnified in ) and swollen (red, magnified in ) mitochondria. () Top, confocal image of a myelinated stage 1 axon (white; FluoroMyelin, orange, arrows; nuclei, magenta). Bottom, magnified view of myelinated stage 1 axon (nuclear staining omitted). () Light microscopic quantification of axon myelination (stage 0–2 axons) at the onset of weight loss (n = 74 axons) and 2 d after clinical EAE onset (first clinical sign of EAE, minimum score of 0.5) (n = 111 axons). () Shape factor histograms of mitochondria from electron microscopy images of control (top) and stage 1 EAE axons (bottom; n = 149 and 138 mitochondria, respectively). Green indicates proportion of mitochondria with normal ultrastructure; red represents disrupted mitochondria. () Mitochondrial shap! e versus mitochondrial membrane potential in EAE lesions (green, normal; red, low (< mean − 2 s.d. of control), n = 235 mitochondria in four mice). () Confocal quantification of mean mitochondrial shape factor in axons from EAE and normal control (N) mice (n = 23–138 mitochondria per axon in stage 0 (green; myelination status was scored in additional stage 0 axons) and 1 (yellow); in stage 2 (red) all mitochondria were scored). () Left, confocal image of axons (gray) and their mitochondria (cyan) in EAE (nuclei, magenta). Right, shape of mitochondria (color coded, see scale at right) in axons at different stages of FAD selected from left panel. Stage and mean mitochondrial shape factor (SF ± s.e.m.) of the axon segment are indicated above each axon. Scale bar in , 2 μm; scale bars in –, 0.5 μm; scale bar in (top), 10 μm; scale bar in (bottom), 5 μm; scale bar in (left and right images), 10 μm. * Figure 3: Activated macrophage/microglia-derived reactive species induce FAD. () Confocal reconstruction of an axon that courses through an EAE lesion (nuclei, magenta) in a mouse with sparse axon labeling (white) and dense labeling of axonal mitochondria (cyan). Enlarged areas below show color-coded (scale on right) shape of mitochondria in the labeled axon outside (left) and inside (right) the lesion. () Quantification of mitochondrial shape in different segments of longitudinally reconstructed axons (segments of each axon connected with dashed line, n = 4 axons) outside (green) and inside (red) of EAE lesions. Each triangle represents a mitochondrion, and circles indicate mean mitochondrial shape factor for the axon segment (± s.e.m.; mitochondria are significantly shorter inside versus outside; P < 0.05, paired t test). () Infiltration density (± s.e.m.) around control (N, gray) and EAE stage 0 (green), 1 (yellow) and 2 (red) axon segments (n = 13–83 segments; infiltration density is significantly increased in stage 0 vs. control axons, stage ! 1 vs. stage 0 axons and stage 2 vs. stage 1 axons; t test). () Cell density (± s.e.m.) around stage 1 axon segments that either recovered (green) or persisted in a swollen state (yellow) during in vivo imaging and were fixed afterward for analysis of cellular infiltration (n = 60 segments; density is significantly lower around recovered axons; t test). () In vivo measurement of H2O2 concentration (detected with Amplex) in the dorsal spinal cord of healthy (N, left) and EAE (2 d after onset) mice. () Quantification of H2O2 concentration (± s.e.m; n = 3–5 mice per time point; levels are significantly increased at 0 d and 2 d after EAE onset; t test). N, control; Pre, preclinical mice; d, days after EAE onset. () In vivo two-photon time-lapse of spinal axons (gray) and their mitochondria (cyan) after H2O2 application (330 mM) (time, h:min). Right, magnification of small cluster of mitochondria that swell over time (arrows). () Frequency of stage 0 (green), 1 (yellow) and 2! (red) axons before and up to 5 h after in vivo H2O2 applicati! on (100 mM; n = 53 axons). Superimposed is the change over time in mean mitochondrial shape (± s.e.m., n = 15 axons; t test). () Percentage (± s.e.m.) of EAE axons in different stages of FAD after ROS and RNS scavenger or vehicle treatment (starting at weight loss, analyzed by t test). () Fate of stage 1 axons after 2 d of ROS and RNS scavenger or vehicle treatment (started 2–3 d after EAE onset, n = 22–26 axons; chi-square test). Scale bar in , 50 μm; scale bar in , 250 μm (calibration bar); scale bar in , 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 4: Axonal changes consistent with FAD are present in acute human multiple sclerosis lesions. () Representative axons in stages 0, 1 and 2 of FAD in an acute human multiple sclerosis lesion (Bielschowsky silver impregnation). () Prevalence of FAD stages in normal-appearing white matter (NAWM) and in acute multiple sclerosis lesions (n = 3 biopsies, > 450 axons per group; all stages are significantly different in lesion versus NAWM; one-tailed t test). () Confocal projections of an axon (top) that was located in NAWM around an active multiple sclerosis lesion, and examples of stage 0, 1 and 2 axons located inside the same lesion (quadruple immunostaining: axons, stained for neurofilament, white; myelin, stained for myelin basic protein, orange; nuclei, stained for NeuroTrace, magenta; mitochondria, stained for porin, cyan; mitochondria magnified in insets). () Myelination status of axons in NAWM and FAD stage 0 and 1 axons in multiple sclerosis lesions (n = 17–22 axons per group, from two to six biopsies). () Comparison of mean mitochondrial shape factors of axons i! n NAWM (N), and stage 0 and 1 multiple sclerosis axons (analyzed by t test). Scale bars in ,, 10 μm; scale bars in (insets), 1 μm. *P < 0.05; ***P < 0.001. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Thomas Misgeld & * Martin Kerschensteiner Affiliations * Research Unit Therapy Development, Institute of Clinical Neuroimmunology, Ludwig-Maximilians-Universität München, Munich, Germany. * Ivana Nikić, * Catherine Sorbara, * Florence M Bareyre & * Martin Kerschensteiner * Institute of Neuropathology, Georg-August University, Göttingen, Germany. * Doron Merkler, * Mario Kreutzfeldt & * Wolfgang Brück * Division of Clinical Pathology, Geneva University Hospital and Department of Pathology and Immunology, University of Geneva, Switzerland. * Doron Merkler & * Mario Kreutzfeldt * Department of Physiology, Indiana University School of Medicine-Muncie, Muncie, Indiana, USA. * Mary Brinkoetter & * Derron Bishop * Chair for Biomolecular Sensors, Center for Integrated Protein Sciences (Munich) at the Institute of Neuroscience, Technische Universität München, Munich, Germany. * Thomas Misgeld * Institute for Advanced Study, Technische Universität München, Munich, Germany. * Thomas Misgeld Contributions M. Kerschensteiner, T.M., D.B., D.M. and I.N. conceived the experiments. I.N. and C.S. did the imaging experiments. I.N., C.S., T.M. and M. Kerschensteiner did image analysis. M.B. and D.B. did and evaluated serial electron microscopy. I.N. and F.M.B. did therapy experiments. D.M., M. Kreutzfeldt and W.B. did histopathological evaluations of EAE and multiple sclerosis tissue. I.N., M. Kerschensteiner and T.M. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Martin Kerschensteiner or * Thomas Misgeld Author Details * Ivana Nikić Search for this author in: * NPG journals * PubMed * Google Scholar * Doron Merkler Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Sorbara Search for this author in: * NPG journals * PubMed * Google Scholar * Mary Brinkoetter Search for this author in: * NPG journals * PubMed * Google Scholar * Mario Kreutzfeldt Search for this author in: * NPG journals * PubMed * Google Scholar * Florence M Bareyre Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Brück Search for this author in: * NPG journals * PubMed * Google Scholar * Derron Bishop Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Misgeld Contact Thomas Misgeld Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Kerschensteiner Contact Martin Kerschensteiner Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (422K) In vivo multi-photon time-lapse that illustrates the degeneration of a transgenically labeled stage 1 axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cx3cr1GFP/+ mouse. Axonal degeneration is initiated near a putative node of Ranvier in close proximity to activated macrophages/microglia (magenta). * Supplementary Video 2 (2M) In vivo overview multi-photon time-lapse of the healthy lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse in which axons are labeled with YFP (gray). The video illustrates that no obvious morphological changes are induced by our imaging approach. 300 min, 11 frames. * Supplementary Video 3 (2M) In vivo overview multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse, in which axons are labeled with YFP (gray; in some axons, CFP-labeled mitochondria are visible due to spectral cross-talk) 2 d after the EAE onset. The video illustrates stage 1 to stage 2 transitions in three axons during the observation period. 300 min, 11 frames. * Supplementary Video 4 (270K) In vivo multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16 (× Thy1-MitoCFP-P) mouse in which the axons are labeled with YFP (gray) 3 d after the EAE onset. This video illustrates the recovery of a stage 1 axon during the observation period. 330 min, 11 frames. * Supplementary Video 5 (3M) Video sequence of a stage 1 EAE axon (shown in ) that illustrates the correlation between in vivo multi-photon imaging and ssTEM. * Supplementary Video 6 (188K) In vivo multi-photon microscopy time-lapse of an activated macrophage/microglia (one cell was manually pseudo-colored in magenta based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Cx3cr1GFP/+ × Thy1-CFP-S mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Note transition of the apposed axon from stage 0 to stage 1 during the time-lapse. Asterisk in first frame marks additional macrophage/microglia. 192 min, 20 frames. * Supplementary Video 7 (258K) In vivo multi-photon time-lapse of a T cell (one cell was manually pseudo-colored in cyan based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cd2-GFP mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Green asterisk in first frame marks additional T cell, gray asterisk marks axon fragment. 40 min, 27 frames. * Supplementary Video 8 (1M) In vivo multi-photon time-lapse of axonal (white) and mitochondrial (cyan) changes induced after application of H2O2 (330 mM) to the spinal cord of a Thy1-YFP-16 × Thy1-MitoCFP-P mouse. Note that axonal mitochondria change before the transition of the axons from stage 0 to stage 1. PDF files * Supplementary Text and Figures (918K) Supplementary Figures 1–5 and Supplementary Methods Additional data - Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy
- Nat Med 17(4):500-503 (2011)
Nature Medicine | Letter Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy * Eric A Collisson1, 2, 10 * Anguraj Sadanandam1, 3, 10 * Peter Olson4, 9 * William J Gibb1, 9 * Morgan Truitt4 * Shenda Gu1 * Janine Cooc5 * Jennifer Weinkle1 * Grace E Kim6 * Lakshmi Jakkula1 * Heidi S Feiler1 * Andrew H Ko2 * Adam B Olshen7 * Kathleen L Danenberg5 * Margaret A Tempero2 * Paul T Spellman1 * Douglas Hanahan3, 4 * Joe W Gray1, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:500–503Year published:(2011)DOI:doi:10.1038/nm.2344Received23 February 2010Accepted04 March 2011Published online03 April 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis1. Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast2 and lung3 cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical ! outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies. View full text Figures at a glance * Figure 1: Subtypes of PDA in tumors and cell lines and their prognostic significance. () Heat map showing three subtypes of PDA in DWD-merged UCSF and GSE15471 (ref. 5) PDA microarray data sets using the PDAssigner gene set. () Kaplan-Meier survival curve comparing survival of individuals with classical (red), QM-PDA (blue) and exocrine-like (green) subtypes. P value is by log-rank test. () Heat map showing three subtypes of PDA in a DWD-merged core clinical and human PDA cell line microarray data sets using the PDAssigner gene set. () Heat map showing three subtypes of PDA in a DWD-merged core clinical and mouse PDA cell line microarray data sets using PDAssigner gene set. The bars on the side denote PDAssigner genes upregulated in classical (violet), QM-PDA (gray) and exocrine-like (green). See Supplementary Table 3 for gene descriptions. * Figure 2: Classical PDA subtype and the GATA6 transcription factor. () Relative log expression of GATA6 in PDA cell lines, transduced with shRNA against GATA6 (shGATA6) or control (shLuc), as determined by quantitative RT-PCR. () Colonies per low-powered field (LPF) in PDA cell lines transduced with shRNA against GATA6 or control. Error bars indicate s.d. * Figure 3: Classical subtype cells depend on KRas. () PDA lines (all with GTPase-inactivating KRAS mutations) were transduced with lentiviruses encoding either control (shLUC) or KRAS-targeting (shKRAS) RNAi. Relative proliferation is plotted. Error bars indicate s.d. () Box plot of relative proliferation of classical and QM-PDA human PDA cell lines. P values by the Kruskal-Wallis test. * Figure 4: Drug responses differ by subtype. (,) Half-maximal inhibitory concentration (IC50) in negative log10 of drug concentration is plotted for each cell line tested with gemcitabine () and erlotinib (). (,) Box plots of IC50 of classical and QM-PDA human PDA cell lines for gemcitabine () and erlotinib (). P values represent statistics using Kruskal-Wallis test. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE17891 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Eric A Collisson & * Anguraj Sadanandam Affiliations * Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA. * Eric A Collisson, * Anguraj Sadanandam, * William J Gibb, * Shenda Gu, * Jennifer Weinkle, * Lakshmi Jakkula, * Heidi S Feiler, * Paul T Spellman & * Joe W Gray * Division of Hematology and Oncology, University of California–San Francisco (UCSF), San Francisco, California, USA. * Eric A Collisson, * Andrew H Ko & * Margaret A Tempero * Swiss Institute for Experimental Cancer Research, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland. * Anguraj Sadanandam & * Douglas Hanahan * Diabetes Center and Department of Biochemistry and Biophysics, UCSF, San Francisco, California, USA. * Peter Olson, * Morgan Truitt & * Douglas Hanahan * Response Genetics, Los Angeles, California, USA. * Janine Cooc & * Kathleen L Danenberg * Department of Pathology, UCSF, San Francisco, California, USA. * Grace E Kim * Department of Epidemiology and Biostatistics and Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA. * Adam B Olshen * Department of Biomedical Engineering, Oregon Health and Science University, Portland, Oregon, USA. * Joe W Gray * Present addresses: Pfizer, La Jolla, California, USA (P.O.) and Genomic Health, Redwood City, California, USA (W.J.G.). * Peter Olson & * William J Gibb Contributions E.A.C. and A.S. designed, conducted and interpreted experiments and wrote the manuscript. P.O., W.J.G., M.T., S.G., J.C., J.W., L.J., and H.S.F. performed experiments. K.L.D. and P.T.S. provided support and interpreted experiments. G.E.K. and A.H.K. coordinated clinical sample acquisition. A.B.O. provided statistical expertise. M.A.T. provided support, interpreted experiments and coordinated clinical sample acquisition. D.H. and J.W.G. designed and interpreted experiments, wrote the manuscript and supervised the project. Competing financial interests J.C. and K.L.D. are employees of Response Genetics. P.O. is an employee of Pfizer. W.J.G. is an employee of Genomic Health. Corresponding author Correspondence to: * Joe W Gray Author Details * Eric A Collisson Search for this author in: * NPG journals * PubMed * Google Scholar * Anguraj Sadanandam Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Olson Search for this author in: * NPG journals * PubMed * Google Scholar * William J Gibb Search for this author in: * NPG journals * PubMed * Google Scholar * Morgan Truitt Search for this author in: * NPG journals * PubMed * Google Scholar * Shenda Gu Search for this author in: * NPG journals * PubMed * Google Scholar * Janine Cooc Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Weinkle Search for this author in: * NPG journals * PubMed * Google Scholar * Grace E Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Lakshmi Jakkula Search for this author in: * NPG journals * PubMed * Google Scholar * Heidi S Feiler Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew H Ko Search for this author in: * NPG journals * PubMed * Google Scholar * Adam B Olshen Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen L Danenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret A Tempero Search for this author in: * NPG journals * PubMed * Google Scholar * Paul T Spellman Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas Hanahan Search for this author in: * NPG journals * PubMed * Google Scholar * Joe W Gray Contact Joe W Gray Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information Excel files * Supplementary Table 1 (287K) Variable and NMF genes for core clinical PDA microarray data sets * Supplementary Table 2 (37K) Patient identifiers and subtypes for different PDA microarray data sets * Supplementary Table 3 (111K) DWD combined matrix containing samples from UCSF and Badea et al., gene expression microarray datasets and 62 PDA assigner genes * Supplementary Table 4 (33K) Patient characteristics and statistical analysis * Supplementary Table 5 (115K) DWD combined matrix containing score clinical samples and human cell lines gene expression microarray datasets and 62 PDA assigner genes * Supplementary Table 6 (90K) DWD combined matrix containing score clinical samples and mouse cell lines gene expression microarray datasets and 62 PDA assigner genes PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–7 and Supplementary Methods Additional data - RGB marking facilitates multicolor clonal cell tracking
- Nat Med 17(4):504-509 (2011)
Nature Medicine | Technical Report RGB marking facilitates multicolor clonal cell tracking * Kristoffer Weber1 * Michael Thomaschewski1 * Michael Warlich1, 2 * Tassilo Volz2 * Kerstin Cornils1 * Birte Niebuhr3 * Maike Täger3 * Marc Lütgehetmann2 * Jörg-Matthias Pollok4 * Carol Stocking3 * Maura Dandri2 * Daniel Benten2 * Boris Fehse1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:504–509Year published:(2011)DOI:doi:10.1038/nm.2338Received10 May 2010Accepted01 December 2010Published online27 March 2011 Abstract * Abstract * 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 We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector–mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a usef! ul tool for cell clonality studies in tissue regeneration and pathology. View full text Figures at a glance * Figure 1: The principle of vector-mediated RGB marking. () Based on color theories, mixing red, green and blue at one intensity should result in four mixed colors: yellow, violet, turquoise and white. () If the basic colors are mixed at all possible intensities, a full spectrum of rainbow colors is generated. () Concurrent vector-mediated introduction of three fluorescent proteins emitting red, green and blue, for example, mCherry (red), Venus (yellow-green) and Cerulean (blue) should in principle lead to seven types of transduced cells. According to the RGB principle, simultaneous expression of different basic colors in the cells could be expected to result in mixed colors (compare also Supplementary Fig. 2). () Lentiviral vector-mediated RGB marking of the indicated target cells using LeGO-C2, LeGO-V2 and LeGO-Cer2 (Supplementary Fig. 1). Shown are overlays of three color photographs taken consecutively with red, green and blue filters using a fluorescence microscope. All cells were cultured for approximately 72 h after plating! ; whereas 293T clones have stayed together during culture, FH-hTERT clones have spread over the tissue culture vessel. Primary hepatocytes have not replicated during culture. * Figure 2: LeGO-mediated RGB marking facilitates analysis of polyclonal liver regeneration. () In vitro analysis of primary hepatocytes confirming efficient RGB marking and generation of numerous different mixed colors. () Liver sections from uPA-SCID mice 1 month after transplantation with RGB-marked primary mouse hepatocytes indicate polyclonal engraftment. () A liver section regenerated by engrafted RGB-marked primary hepatocytes is depicted (same experiment as in ). The apparently one big blue clone (marked by an asterisk in the Cerulean image) in fact consists of three different clones that engrafted in the same area (overlay). These clones (indicated by arrows) are easily distinguishable on the basis of RGB marking, as they express one (blue middle clone), two (cyan top clone) or all three colors (white bottom clone) at the same time. All images represent overlays of three individual color photographs taken consecutively with red, green and blue filters using a fluorescence microscope. * Figure 3: Efficient RGB marking of colony-forming hematopoietic stem and progenitor cells. () RGB marking of HSCs and HPCs in vitro. () RGB-marked progenitors that generated different types of colonies in methyl cellulose (see also Supplementary Fig. 4). () Spleen of a C57BL/J6 mouse 10 d after transplantation of syngeneic RGB-marked HSCs and HPCs. CFU-S are easily distinguishable. () RGB marking of CFU-S in vivo. Images in , and represent overlays of three individual color photographs taken consecutively with red, green and blue filters using a fluorescence microscope. The image in was taken with a standard digital camera. * Figure 4: Stable RGB marking of carcinogenic cell clones in vitro and in vivo. () LeGO vector-mediated RGB marking of BON carcinoma cells in vitro. () Homogenous coloring of single-cell derived RGB-marked BON clones expanded in vitro. () Outgrowth of multiple liver tumors in NOD-SCID mice (shown are whole-liver sections) after transplantation of the two clones presented in . Images in and were taken with a standard fluorescence microscope, image with a confocal microscope. () Vector insertion site–specific PCR reactions confirming the identity of engrafted tumors with the ex vivo–expanded founder clones on the molecular level (for example, D3-specific PCR was positive on a tumor derived from clone D3, but negative on tumors derived from clones D5 and E3). M, marker ladder for DNA size. * Figure 5: Serial transplantation of RGB marked tumors. () Outgrowth of multiple RGB-marked liver tumors after transplantation of RGB-marked BON carcinoma cells (compare Fig. 4a). () Two examples of tumors (T1, T2) explanted from liver tissue. The insets show the same fields of view photographed with a phase-contrast microscope. () Cultured cells derived from explanted tumors displayed the same color as the initial tumors (T1 and T2). (,) Retransplantation of these tumor cells resulted in engraftment of uniformly colored tumors in secondary recipients resembling the color of the initial tumors T1 and T2. In , single tumors are shown, whereas shows whole-liver sections. () Infusion of mixed cells derived from tumors T1 and T2 resulted in three types of tumors: monochromatic violet (resembling T1) and yellow ones (resembling T2), as well as tumors containing cells of both violet and yellow colors. Images in , and were taken with a confocal microscope, whereas images in to were generated with a standard fluorescence microscope. () I! nsertion site–specific PCR reactions confirming clonal identity of serially transplanted tumors. Author information * Abstract * Author information * Supplementary information Affiliations * Research Department Cell and Gene Therapy, Clinic for Stem Cell Transplantation, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. * Kristoffer Weber, * Michael Thomaschewski, * Michael Warlich, * Kerstin Cornils & * Boris Fehse * Internal Medicine I, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. * Michael Warlich, * Tassilo Volz, * Marc Lütgehetmann, * Maura Dandri & * Daniel Benten * Heinrich-Pette-Institute, Leibniz-Institute for Experimental Virology, Hamburg, Germany. * Birte Niebuhr, * Maike Täger & * Carol Stocking * Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. * Jörg-Matthias Pollok Contributions K.W. designed the study, produced LeGO vectors and performed and analyzed gene transfer experiments in target cells in vitro and in vivo. M. Thomaschewski and M.W. isolated, transduced and transplanted primary hepatocytes and analyzed mice. M. Thomaschewski also performed mouse studies with BON tumor cells. T.V. and M.L. performed experiments in uPA mice. K.C. identified vector insertions by LM-PCR and performed specific PCRs. B.N., M. Täger and C.S. designed and performed experiments with mouse HSCs. J.-M.P. and M.L. provided and prepared primary human hepatocytes. M.D. provided the uPA-SCID model and supervised in vivo experiments in that model. D.B. designed and performed mouse studies. B.F. designed the study, analyzed and evaluated results and wrote the manuscript. All authors read and approved the final version of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Boris Fehse Author Details * Kristoffer Weber Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Thomaschewski Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Warlich Search for this author in: * NPG journals * PubMed * Google Scholar * Tassilo Volz Search for this author in: * NPG journals * PubMed * Google Scholar * Kerstin Cornils Search for this author in: * NPG journals * PubMed * Google Scholar * Birte Niebuhr Search for this author in: * NPG journals * PubMed * Google Scholar * Maike Täger Search for this author in: * NPG journals * PubMed * Google Scholar * Marc Lütgehetmann Search for this author in: * NPG journals * PubMed * Google Scholar * Jörg-Matthias Pollok Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Stocking Search for this author in: * NPG journals * PubMed * Google Scholar * Maura Dandri Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Benten Search for this author in: * NPG journals * PubMed * Google Scholar * Boris Fehse Contact Boris Fehse Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (680K) Supplementary Figures 1–4 and Supplementary Tables 1 and 2 Additional data - Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21
- Nat Med 17(4):510-513 (2011)
Nature Medicine | Technical Report Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21 * Elisavet A Papageorgiou1 * Alex Karagrigoriou2 * Evdokia Tsaliki1, 3, 4 * Voula Velissariou3 * Nigel P Carter5 * Philippos C Patsalis1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:510–513Year published:(2011)DOI:doi:10.1038/nm.2312Received05 May 2010Accepted16 November 2010Published online06 March 2011 Abstract * Abstract * 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 The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially methylated regions (DMRs). In this study, we present a strategy using the methylated DNA immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach ! provided correct diagnosis of 14 trisomy 21 and 26 normal cases. View full text Figures at a glance * Figure 1: Schematic illustration of the fetal-specific DNA methylation ratio approach. A fetus with trisomy 21 has an extra copy of the fetal-specific methylated region compared to a normal fetus. DNA methylation enrichment followed by real-time qPCR of a fetal-specific methylated region can allow relative quantification of the amount of fetal DNA in normal and trisomy 21 cases. * Figure 2: DNA methylation ratio values obtained from the 12 DMRs. The y axis represents the methylation ratio value. Each color dot represents the ratio value obtained from one of the 12 DMRs tested (EP1 to EP12). () DNA methylation ratio values obtained from the normal cases P6 and P13. () DNA methylation ratio values obtained from the trisomy 21 cases P29 and P35. * Figure 3: Box plot representation of the results obtained from four DMRs. EP1, EP4, EP7 and EP10 in 20 normal and 20 trisomy 21 cases. The box plots depict the five-number summaries, namely the minimum and maximum values, the upper (Q3) and lower (Q1) quartiles and the median. The median is identified by a line inside the box. The length of the box represents the interquartile range. Values more than three IQRs from either end of the box are labeled as extremes and are denoted by an asterisk (*). Values more than 1.5 IQRs but less than three IQRs from either end of the box are labeled as outliers (o). Author information * Abstract * Author information * Supplementary information Affiliations * Cytogenetics and Genomics Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus. * Elisavet A Papageorgiou, * Evdokia Tsaliki & * Philippos C Patsalis * Department of Mathematics and Statistics, University of Cyprus, Nicosia, Cyprus. * Alex Karagrigoriou * Department of Cytogenetics and Molecular Biology, Mitera Hospital, Athens, Greece. * Evdokia Tsaliki & * Voula Velissariou * Faculty of Biology, School of Sciences, National and Kapodistrian University of Athens, Athens, Greece. * Evdokia Tsaliki * Molecular Cytogenetics Department, The Wellcome Trust Sanger Institute, Cambridge, Hinxton, UK. * Nigel P Carter Contributions E.A.P. and E.T. carried out the experiments. E.A.P. wrote the manuscript. E.A.P. and A.K. performed the statistical analysis. E.T. and V.V. collected the majority of the samples in this study. N.P.C. provided input on the selection of the DMRs. P.C.P. was the principal investigator and has supervised the project. All authors reviewed, critiqued and offered comments on the text. Competing financial interests P.C.P. and E.A.P. have filed a US provisional patent for the fetal-specific DNA methylation ratio approach (application no. 61/405,421). Corresponding author Correspondence to: * Philippos C Patsalis Author Details * Elisavet A Papageorgiou Search for this author in: * NPG journals * PubMed * Google Scholar * Alex Karagrigoriou Search for this author in: * NPG journals * PubMed * Google Scholar * Evdokia Tsaliki Search for this author in: * NPG journals * PubMed * Google Scholar * Voula Velissariou Search for this author in: * NPG journals * PubMed * Google Scholar * Nigel P Carter Search for this author in: * NPG journals * PubMed * Google Scholar * Philippos C Patsalis Contact Philippos C Patsalis Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (180K) Supplementary Tables 1–6, Supplementary Data and Supplementary Methods Additional data - Cancer drugs: remedy required
- Nat Med 17(4):514 (2011)
Nature Medicine | Erratum Cancer drugs: remedy required Journal name:Nature MedicineVolume: 17,Page:514Year published:(2011)DOI:doi:10.1038/nm0411-514aPublished online07 April 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, 231 (2011); published online 7 March 2011; corrected after print 11 March 2011 In the version of this article initially published, the manufacturer of bevacizumab (Avastin) was reported incorrectly as Merck. The manufacturer of bevacizumab is Genentech/Roche. The error has been corrected in the HTML and PDF versions of the article. The article also states that the US Food and Drug Administration (FDA) approved bevacizumab on the basis of a phase 3 trial that showed a median increase in progression-free survival of 5.9 months, which is consistent with the published literature (N. Engl. J. Med.357, 2666–2676, 2007). An independent review of the data concluded that the progression-free survival difference was 5.5 months, which is listed on the FDA-approved label for bevacizumab. On December 15, the FDA's Office of New Drugs (OND) issued a decision memo on Avastin, which states that "it is the conclusion of OND that the breast cancer indication for Avastin be withdrawn." The FDA has granted Genentech/Roche a public hearing on the matter, to be held in June 2011. Additional data - Hugging tight in Huntington's
- Nat Med 17(4):514 (2011)
Nature Medicine | Erratum Hugging tight in Huntington's * Ashu Johri * Rajnish K Chaturvedi * M Flint BealJournal name:Nature MedicineVolume: 17,Page:514Year published:(2011)DOI:doi:10.1038/nm0411-514bPublished online07 April 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, 245–246 (2011); published online 7 March 2011; corrected after print 7 April 2011 In the version of this article initially published, the first author of the primary paper that the N&V referred to was incorrectly cited as Petrilli. The correct first author is Song. The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Ashu Johri Search for this author in: * NPG journals * PubMed * Google Scholar * Rajnish K Chaturvedi Search for this author in: * NPG journals * PubMed * Google Scholar * M Flint Beal Search for this author in: * NPG journals * PubMed * Google Scholar - Hugging tight in Huntington's
- Nat Med 17(4):514 (2011)
Nature Medicine | Corrigendum Hugging tight in Huntington's * Ashu Johri * Rajnish K Chaturvedi * M Flint BealJournal name:Nature MedicineVolume: 17,Page:514Year published:(2011)DOI:doi:10.1038/nm0411-514cPublished online07 April 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, 245–246 (2011); published online 7 March 2011; corrected after print 7 April 2011 In the version of this article initially published, the authors omitted to mention that L. Scorrano and colleagues had previously shown increased Drp1 activity and mitochondrial fission in cellular models of Huntington's disease and would now like to cite this work (EMBO Mol. Med.2, 490–503, 2010).The error has been corrected in the HTML and PDF versions of the article. Additional data Author Details * Ashu Johri Search for this author in: * NPG journals * PubMed * Google Scholar * Rajnish K Chaturvedi Search for this author in: * NPG journals * PubMed * Google Scholar * M Flint Beal Search for this author in: * NPG journals * PubMed * Google Scholar - Conversion of vascular endothelial cells into multipotent stem-like cells
- Nat Med 17(4):514 (2011)
Nature Medicine | Corrigendum Conversion of vascular endothelial cells into multipotent stem-like cells * Damian Medici * Eileen M Shore * Vitali Y Lounev * Frederick S Kaplan * Raghu Kalluri * Bjorn R OlsenJournal name:Nature MedicineVolume: 17,Page:514Year published:(2011)DOI:doi:10.1038/nm0411-514dPublished online07 April 2011 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.16, 1400–1406 (2010); published online 21 November 2010; corrected after print 7 April 2011 In the version of this article initially published, the flow cytometry plot in Figure 4b corresponding to the condition HCMEC and TGF-β2 was incorrect. This plot has been replaced with the correct one in the HTML and PDF versions of the article. Additional data Author Details * Damian Medici Search for this author in: * NPG journals * PubMed * Google Scholar * Eileen M Shore Search for this author in: * NPG journals * PubMed * Google Scholar * Vitali Y Lounev Search for this author in: * NPG journals * PubMed * Google Scholar * Frederick S Kaplan Search for this author in: * NPG journals * PubMed * Google Scholar * Raghu Kalluri Search for this author in: * NPG journals * PubMed * Google Scholar * Bjorn R Olsen Search for this author in: * NPG journals * PubMed * Google Scholar
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