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- Too soon to translate?
- Nat Med 17(7):751 (2011)
Nature Medicine | Editorial Too soon to translate? Journal name:Nature MedicineVolume: 17,Page:751Year published:(2011)DOI:doi:10.1038/nm0711-751Published online07 July 2011 An association between a retrovirus and chronic fatigue syndrome has courted controversy since it was first announced. In light of new data discounting this link, medical decisions made on its basis—some of which were encouraged by the patient advocate community—might have been premature. View full text Additional data - Overwhelmed drug regulators seek cure in cooperation
- Nat Med 17(7):753 (2011)
Article preview View full access options Nature Medicine | News Overwhelmed drug regulators seek cure in cooperation * Lucas LaursenJournal name:Nature MedicineVolume: 17,Page:753Year published:(2011)DOI:doi:10.1038/nm0711-753Published online07 July 2011 Regulatory authorities such as the US Food and Drug Administration and European Medicines Agency face continual criticism for their plodding pace of drug approval decisions. In 2009, the last year for which complete data are available, the median time for a standard review of a drug application in the US was 13 months—30% longer than the agency's target for such reviews. But even with this situation, it's undeniable that both agencies devote far more human and financial resources to the process than most other countries can afford to spend on their own. To keep up, many other medical regulatory authorities are banding together with one another to share the expertise and clinical results needed to make faster decisions. On 20 June, for example, New Zealand's Prime Minister John Key announced that the establishment of the Australia New Zealand Therapeutic Products Agency—stalled for several years—would proceed as planned. And, the month before, as the World Health Organization (WHO) held its annual assembly in Geneva, a concurrent meeting of health ministers from countries in the Gulf Cooperation Council approved pricing standards for drugs in its six member states from across the Arabian Peninsula. "There is increasing cooperation and work sharing between regulators," says Lembit Rägo, coordinator of Quality Assurance and Safety for Medicines at the WHO in Geneva and author of an April 2011 report on international harmonization of regulatory efforts (Clin. Pharmacol. Ther., 503–512, 2011). Delays are often worst in what the WHO calls "less-resourced countries," because in some cases those countries wait until a trusted authority such as the FDA or EMA has approved a drug before making a decision. The WHO has estimated that 90% of drug regulatory agencies in sub-Saharan Africa faced circumstances that hindered their function and could impede their ability to guarantee the safety of medicines within their borders. istockphoto Regulators heed advice. To make the situation even more daunting, many drugs that have potential use in developing countries—such as those for tropical disease—may not be priorities in North America and Europe. As such, they face longer delays between development and reaching their target markets. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Lucas Laursen Search for this author in: * NPG journals * PubMed * Google Scholar - New $10 million X Prize launched for tricorder-style medical device
- Nat Med 17(7):754 (2011)
Article preview View full access options Nature Medicine | News New $10 million X Prize launched for tricorder-style medical device * Hannah WatersJournal name:Nature MedicineVolume: 17,Page:754Year published:(2011)DOI:doi:10.1038/nm0711-754aPublished online07 July 2011 In the fictional universe of the Star Trek TV series, Starfleet doctors routinely diagnosed their patients on the spot using boxy, gray, handheld devices called tricorders. But gadgets like these could be available much sooner than the twenty-third century thanks to a newly announced 'X Prize' for mobile diagnostics, which boasts a $10 million award. Run by the telecom giant Qualcomm and the X Prize Foundation—a California nonprofit behind several big-money competitions in genomics, space exploration and clean-energy technologies—the aptly named 'Tricorder Prize' aims to reward the inventor of a single portable device that, without human input, can diagnose an array of diseases with the same level of accuracy as a panel of physicians. Although the specific medical conditions haven't yet been selected, they will be challenging, organizers say, ranging from metabolic syndromes to infectious diseases to neurological conditions. Yasuhide Fumoto The X Prize's inspiration. Last month, around 40 healthcare and medical technology experts met near Qualcomm's headquarters in San Diego to begin the six-month process of fleshing out the final requirements for winning the prize. According to Don Jones, vice president of business development for Qualcomm's health and life sciences division, everyone agrees on many of the basic parameters: the device has to be portable, minimally invasive, fast and scalable. But working out the finer points of the rules is proving time consuming. "Would drawing blood negate our premise of being noninvasive? What if the process for drawing blood is painless?" Jones asks. "It's a teeter-tottering that goes back and forth." Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Waters Search for this author in: * NPG journals * PubMed * Google Scholar - FDA approval signals more 'homework' on the horizon in trials
- Nat Med 17(7):754 (2011)
Article preview View full access options Nature Medicine | News FDA approval signals more 'homework' on the horizon in trials * Hannah WatersJournal name:Nature MedicineVolume: 17,Page:754Year published:(2011)DOI:doi:10.1038/nm0711-754bPublished online07 July 2011 A growing number of people use technology to share the minutiae of their lives with others, detailing everything from the calories they consume, to the distance they run, to their point in the menstrual cycle. And clinical researchers are increasingly trying to harness this compulsion to divulge biometric data online. Now, with a 7 June go-ahead from the US Food and Drug Administration (FDA) for the country's first completely at-home trial of a drug, the trend has come to the fore. In a proof-of-concept study, a group from the University of California–San Francisco and New York–based Pfizer will trod through a trial of the company's drug for overactive bladder, Detrol (tolterodine). The drug received approval for this indication in 1998; as such, the trial is simply to see whether a home-based trial can work. Participants will be recruited online and, with the exception of blood tests bookending the trial period performed by a visiting nurse, the participants are on their own. They will receive their pills in the mail and log their bladder activity through a mobile phone app. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Waters Search for this author in: * NPG journals * PubMed * Google Scholar - As E. coli continues to claim lives, new approaches offer hope
- Nat Med 17(7):755 (2011)
Article preview View full access options Nature Medicine | News As E. coli continues to claim lives, new approaches offer hope * Elie DolginJournal name:Nature MedicineVolume: 17,Page:755Year published:(2011)DOI:doi:10.1038/nm0711-755Published online07 July 2011 On 25 May—just as the deadliest outbreak of Escherichia coli on record was beginning to tear through Germany—a team of physicians happened to publish an experimental therapy that could save lives in future outbreaks of this kind. The article described how an antibody therapy called Soliris (eculizumab) had successfully reversed the kidney damage and neurological symptoms seen in three young E. coli–infected children suffering from hemolytic-uremic syndrome (HUS), a deadly complication also seen in many victims of the German outbreak (N. Engl. J. Med. doi:10.1056/NEJMc1100859, 2011). Soliris has been on the market since 2007 to treat a rare blood disorder called paroxysmal nocturnal hemoglobinuria, and it costs a staggering $400,000 per year. The antibody's manufacturer, Connecticut-based Alexion Pharmaceuticals, had been testing Soliris in people who develop HUS without any E. coli infection. It filed for regulatory approval for this indication in the US and Europe earlier this year and reported further positive clinical trial data at last month's Congress of the European Hematology Association in London. But spurred by requests from German physicians after the findings related to E. coli–induced HUS came out, starting on 30 May, Alexion worked with health authorities in Germany to dispense the drug on a compassionate-use basis at no charge. Since then, doctors across the country have administered Soliris to hundreds of those affected by the outbreak. And on 20 June, Alexion formally launched an open-label clinical trial in people sickened by E. coli who have received Soliris during the crisis. "A clinical trial is the best environment to ensure that the drug is administered in a controlled manner to support safety and potential efficacy," says Irving Adler, an Alexion spokesperson. But, according to Franz Schaefer, the lead author of the initial study and a nephrologist at the Center for Pediatrics and Adolescent Medicine in Heidelberg, Germany, the numbers coming in suggest that not everyone has responded as well as the kids in his team's study. "The response, as far as I've heard, is kind of mixed," he says. istockphoto New treatments target outbreak. Unfortunately, when it comes to the varieties of E. coli that produce shiga toxins, such as those seen in Germany, few treatment options exist. Antibiotics, for example, can further stimulate the release of these toxins, making HUS symptoms worse, but no drugs are licensed specifically to treat the disease. Instead, physicians commonly perform plasmapheresis, replacing people's own blood fluids with those taken from a donor to rid the body of the bacterial toxins. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Trial puts niacin—and cholesterol dogma—in the line of fire
- Nat Med 17(7):756 (2011)
Article preview View full access options Nature Medicine | News Trial puts niacin—and cholesterol dogma—in the line of fire * Elie DolginJournal name:Nature MedicineVolume: 17,Page:756Year published:(2011)DOI:doi:10.1038/nm0711-756Published online07 July 2011 The balance of 'good' and 'bad' cholesterol noted at routine checkups—and some of the drugs used to tip this balance—might not influence heart risk in the way widely thought. It's already known that statins, which lower levels of low-density lipoprotein (LDL), do not work for everybody. As such, doctors have long sought to complement these agents that reduce 'bad' cholesterol with medicines such as niacins and fibrates that raise levels of the 'good' stuff—namely, high-density lipoprotein (HDL) cholesterol. New evidence, however, suggests that simply elevating HDL cholesterol levels in the blood does not necessarily translate into clinical benefit for patients. "It's a beautiful hypothesis that HDL may be cardioprotective, and there are ample preclinical as well as observation data in support of that," says Sanjay Kaul, a cardiologist at the Cedars-Sinai Medical Center in Los Angeles. "But when we put it to real test, which is the gold-standard randomized clinical trial, none of the treatments have passed muster." The most recent failure came in May when the US National Heart, Lung and Blood Institute (NHLBI) prematurely halted the AIM-HIGH study. The 3,400-person trial, which examined high-dose extended-release niacin given together with statin therapy, was cut short after a preliminary data analysis found no additional benefits of the vitamin B–based drug in this patient population. "Maybe we've been too simplistic in thinking that raising HDL any way confers the same benefit as when it happens physiologically, and that's what we're grappling with," says the NHLBI's Patrice Desvigne-Nickens, a project officer for the trial. "AIM-HIGH poses the most substantial challenge yet to the HDL cholesterol hypothesis," says Dan Rader, a cardiologist at the University of Pennsylvania School of Medicine in Philadelphia who was not involved in the study. Michael Davidson, director of preventive cardiology at the University of Chicago and another trial onlooker, adds, "To those of us in the field, we thought it was the right kind of study—the right patient population to test the effect of niacin—and when the trial didn't work, it was both a disappointment and a surprise." Creative Commons Niacin questioned. Adding to the uncertainty, on 19 May, a week before the NHLBI pulled the plug on AIM-HIGH, an advisory committee to the US Food and Drug Administration (FDA) came to a damning conclusion on another HDL-raising drug. Off the back of the recent ACCORD trial, which showed that a fibrate drug from Abbott Laboratories called TriCor (fenofibrate) provided no added benefit to that of statins in people with diabetes, the panel unanimously recommended that the Chicago company launch a new trial of a similar agent called Tripilix, the only fibrate medicine currently approved for use in combination with statins. Importantly, the panel said the trial should be based on clinical outcomes and not just changes in triglycerides or HDL cholesterol levels. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Forty years on from Nixon's war, cancer research 'evolves'
- Nat Med 17(7):757 (2011)
Article preview View full access options Nature Medicine | News Forty years on from Nixon's war, cancer research 'evolves' * Nadia DrakeJournal name:Nature MedicineVolume: 17,Page:757Year published:(2011)DOI:doi:10.1038/nm0711-757Published online07 July 2011 Cancer researchers look to Darwin to improve tumor therapies. SAN FRANCISCO — Ever since US president Richard Nixon declared war on cancer in 1971, scientists and physicians have launched a full-on offensive against the disease, seeking to cure cancer by eradicating the multiplying enemy cells. But, with few exceptions, treatments haven't lived up to expectations. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Nadia Drake Search for this author in: * NPG journals * PubMed * Google Scholar - Federal court raises the bar for overturning patents
- Nat Med 17(7):758 (2011)
Article preview View full access options Nature Medicine | News Federal court raises the bar for overturning patents * Charlotte SchubertJournal name:Nature MedicineVolume: 17,Page:758Year published:(2011)DOI:doi:10.1038/nm0711-758aPublished online07 July 2011 A US federal appeals court ruling will make it harder for patent holders to lose their intellectual property protection because of charges—often based on small errors or omissions in patent applications—that they engaged in misconduct by misleading or deceiving the patent office. Now, except in egregious cases, such legal challenges can only succeed if the missing information would have affected whether the patent was issued in the first place. istockphoto New legal precedent. The ruling, issued by the US Court of Appeals for the Federal Circuit in Washington, DC in late May, pivoted around a patent for the design of disposable blood glucose test strips held by the Chicago drugmaker Abbott Laboratories. Several drug companies, including a subsidiary of Germany's Bayer, had argued that Abbott's patent was unenforceable because contradictory information had been filed with the US and European patent offices. In 2008, a lower court agreed and overturned the patent license, but the latest ruling reverses that decision and establishes a new legal precedent. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Charlotte Schubert Search for this author in: * NPG journals * PubMed * Google Scholar - Supreme Court decision on patent for HIV test unlikely to set major precedent
- Nat Med 17(7):758 (2011)
Article preview View full access options Nature Medicine | News Supreme Court decision on patent for HIV test unlikely to set major precedent * Hannah WatersJournal name:Nature MedicineVolume: 17,Page:758Year published:(2011)DOI:doi:10.1038/nm0711-758bPublished online07 July 2011 In the late 1980s, a postdoc researcher at Stanford University by the name of Mark Holodniy collaborated with Cetus Corporation, the primary patent holder of PCR, to develop a blood test for HIV that is now used in labs around the world. At the time, Holodniy signed a contract with both Cetus and Stanford; little did he know that by doing so he would set off a legal battle decades later. Roche Molecular Systems acquired marketing rights to the test from Cetus in 1991, and although Stanford applied for and received patents for the test its requests for a cut of the profits were ignored. When the California university sued for patent rights in 2005, the district court ruled in its favor, but, after Roche appealed, the Federal Circuit overturned the verdict. Finally, on 6 June, the US Supreme Court handed down a decision, leaving some specialists aghast about its implications for government-funded discovery. The 7-2 ruling in favor of Roche came down to contractual wording, with Holodniy's agreement with Cetus stating that he "will assign and do[es] hereby assign" his "right, title and interest" in discoveries made at Cetus to the company, whereas his contract with Stanford was less committal. Detractors argue that the decision undermines the Bayh-Dole Act, which permits universities such as Stanford that receive federal grants for research to apply for patents themselves instead of turning their discoveries over to the government. If this case sets a precedent and universities aren't guaranteed patents to their discoveries, some worry that the incentive to encourage faculty to innovate will be lost, collaborations outside academia will be discouraged and contracts will have to be stricter. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Hannah Waters Search for this author in: * NPG journals * PubMed * Google Scholar - Companies vie for a cut of the gene-editing market
- Nat Med 17(7):759 (2011)
Article preview View full access options Nature Medicine | News Companies vie for a cut of the gene-editing market * Daniel GrushkinJournal name:Nature MedicineVolume: 17,Page:759Year published:(2011)DOI:doi:10.1038/nm0711-759Published online07 July 2011 Researchers hoping to write the next chapter of biomedicine know that their progress will hinge largely on their ability to edit genes—cutting out unwanted DNA and manipulating the sequences they want to keep. Acolytes of gene-editing technology have recently been encouraged by early data about Sangamo BioSciences' new zinc finger nuclease–based therapies. In March, the California biotech reported preliminary phase 1 trial results showing that its HIV treatment, which uses editing to turn off the gene for the receptor on the cell membrane that the virus exploits, was safe and effective at improving people's T cell counts. Two months later, Sangamo scientists presented additional evidence at the American Society of Gene & Cell Therapy meeting in Seattle demonstrating in mouse and cell models the technology's promise in a number of other diseases, including hemophilia, so-called 'bubble boy disease' and a form of acquired blindness. Sangamo's success has fed a growing enthusiasm for genome engineering technologies. And now, a handful of biotechs with competing technologies and business models are vying to be the go-to company for gene-editing applications. But not all technologies are created equal, and there's debate within the research community about which technique is best for which purpose. "It's a bit like 'which one will win out—PCs or Macs?'," says Bert Vogelstein, a cancer researcher at the Johns Hopkins University School of Medicine in Baltimore. "The one people will choose is the one that's available, easiest, cheapest and the one they have experience with." Sangamo's zinc finger nucleases (ZFNs), which remain the best-known gene-editing technique for adult cells, can latch onto DNA at specific points and cleave unwanted parts out, in some cases repairing a disease-causing gene. In 2007, Sangamo licensed sales of the technology for research purposes to the St. Louis–based company Sigma-Aldrich, which now charges $25,000 for new constructs. Philip Gregory, Sangamo's chief scientific officer, admits that's a considerable price to pay. But he notes that even at that price point, demand is outpacing supply. "We've been asking Sigma to reduce the prices so that we could get the technology out there faster," he says. "But they claim to be overwhelmed with orders, anyway." Equinox Graphics / Photo Researchers, Inc. Companies hope for big profits from gene-editing technologies. Across the Atlantic, Horizon Discovery, a Cambridge, UK startup founded in 2007, is advancing its own gene-editing platform using another pitch. Although not as efficient as ZFNs, Horizon's approach is more flexible: it can add genes as easily as it can delete them, and the technology is easier to track. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Daniel Grushkin Search for this author in: * NPG journals * PubMed * Google Scholar - After GAVI pledges surpass goals, focus shifts to keeping promises
- Nat Med 17(7):760 (2011)
Article preview View full access options Nature Medicine | News After GAVI pledges surpass goals, focus shifts to keeping promises * Georgina KenyonJournal name:Nature MedicineVolume: 17,Page:760Year published:(2011)DOI:doi:10.1038/nm0711-760aPublished online07 July 2011 LONDON — In a move that surpassed expectations given the tough economic times, donors committed $4.3 billion to the GAVI Alliance at a 13 June pledging conference—exceeding the target of $3.7 billion. The funds secure the next five-year chapter of the Geneva-based public-private partnership aimed at stamping out preventable illness in the world's poorest countries—in particular, two of the biggest child killers, pneumonia and diarrhea. "Now poor kids will get the vaccines that rich kids get," Bill Gates, whose Bill & Melinda Gates Foundation is slated to give GAVI more than $1 billion, said at the meeting here. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Georgina Kenyon Search for this author in: * NPG journals * PubMed * Google Scholar - France adds weight to obesity research with new institute
- Nat Med 17(7):760 (2011)
Article preview View full access options Nature Medicine | News France adds weight to obesity research with new institute * Karen DenteJournal name:Nature MedicineVolume: 17,Page:760Year published:(2011)DOI:doi:10.1038/nm0711-760bPublished online07 July 2011 PARIS — Despite the idea of the so-called 'French paradox' that refers to the relatively slender waistlines in France vis-à-vis the country's rich foods, the nation is not immune to the worldwide obesity epidemic. According to French officials, 15% of the population is obese, and the numbers are rising. Faced with this reality, a new national obesity plan is in the works, making this area a top research priority for France in the upcoming years. istockphoto Obesity hurts 15% of France. On 25 May, France's Ministry of Higher Education and Research announced a commitment of €130 million ($185 million) as part of its future investment scheme dedicated solely toward obesity-related research. The sum, which will be dispensed over the next decade, represents a substantial increase over previous government plans to boost research in this field. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Karen Dente Search for this author in: * NPG journals * PubMed * Google Scholar - Pfizer breaks from merger mentality as others chase leads
- Nat Med 17(7):761 (2011)
Article preview View full access options Nature Medicine | News Pfizer breaks from merger mentality as others chase leads * Janelle WeaverJournal name:Nature MedicineVolume: 17,Page:761Year published:(2011)DOI:doi:10.1038/nm0711-761aPublished online07 July 2011 It was less than two years ago that the pharmaceutical giant Pfizer made headlines when it acquired the drugmaker Wyeth for a cool $68 billion. But these days Pfizer is generating a buzz for mulling over a different way to bump up its bottom line: shedding some of its nonpharmaceutical divisions. The potential plan has not won over all industry analysts, some of whom say that scooping up smaller companies with strong drug pipelines—particularly those in developing markets—still offers the best path to profits. The takeover of Genzyme by Sanofi this past spring was only the latest in a string of companies combining, going back to the Merck-Schering-Plough and Roche-Genentech fusions in 2009. These strategic rearrangements of industry titans have largely been a response to looming patent losses. Over the next two years, patents will expire on more than a dozen blockbuster drugs with combined annual sales of about $50 billion, according to the research organization EvaluatePharma in London. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Janelle Weaver Search for this author in: * NPG journals * PubMed * Google Scholar - A shortage link?
- Nat Med 17(7):761 (2011)
Article preview View full access options Nature Medicine | News A shortage link? * Janelle WeaverJournal name:Nature MedicineVolume: 17,Page:761Year published:(2011)DOI:doi:10.1038/nm0711-761bPublished online07 July 2011 On 19 May, US senator Herb Kohl, a Democrat representing Wisconsin, sent a letter to the chairman of the country's Federal Trade Commission (FTC) to urge the agency to examine the effect of mergers in the pharmaceutical industry on the nation's drug supply. In his letter, Kohl cites a recent Washington Post article reporting "an unprecedented surge in drug shortages in the United States that is endangering cancer patients, heart attack victims, accident survivors and a host of other ill people." A record 211 medications became scarce last year, triple the number in 2006, and at least 89 new shortages have emerged so far this year. "The megamergers of the past decade may be contributing to these critical drug deficiencies," Kohl states, adding that the disappearance of some companies and the restructuring of drug giants lead to diminished production of older and less profitable products. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Author Details * Janelle Weaver Search for this author in: * NPG journals * PubMed * Google Scholar - News in brief: Biomedical briefing
- Nat Med 17(7):762-763 (2011)
Article preview View full access options Nature Medicine | News News in brief: Biomedical briefing Journal name:Nature MedicineVolume: 17,Pages:762–763Year published:(2011)DOI:doi:10.1038/nm0711-762Published online07 July 2011 Worried about the dwindling drug arsenal in the face of ever-increasing antibiotic resistance, US lawmakers reintroduced legislation on 15 June to spur the development of new antimicrobial agents. The Generating Antibiotic Incentives Now (GAIN) bill, first proposed late last year, would provide pharmaceutical companies with extended market exclusivity and speedier regulatory reviews for new antibiotics. "It probably will make the difference for some folks to get involved," says Ramanan Laxminarayan, who directs the Extending the Cure project on antibiotic resistance at Resources for the Future, a Washington, DC–based policy organization. Not everyone agrees, though. For a dissenting opinion, see page 772. On 31 May, the World Health Organization's International Agency for Research on Cancer (IARC) stepped into the long-standing debate over whether cell phone use causes brain cancer, concluding that radio frequency electromagnetic fields—which include radio and television transmitters, as well as mobile phones—are "possibly carcinogenic" to people. "This classification reflects the fact that there's some evidence and a great deal of uncertainty," says Jonathan Samet, a University of Southern California epidemiologist who chaired the IARC's working group. Despite the billions of dollars spent fighting infectious diseases, only around 2% goes toward vaccine research. Aiming to boost that level, 15 scientists and vaccine advocates from around the world last month launched yet another group to increase vaccine funding. "There's a need for greater diversity in sources of funding," says Peter Hale, a vaccine advocate who started the Foundation for Vaccine Research. The Washington, DC–based organization plans to award grants to scientists using money donated from telethons and benefit concerts starting in the fall of next year. The US Food and Drug Administration (FDA) in late May approved the first new medicine in 25 years to treat diarrhea caused by the hospital-acquired superbug Clostridium difficile. In phase 3 trials, the antibacterial drug, called Dificid (fidaxomicin) and manufactured by San Diego–based Optimer Pharmaceuticals, significantly cut the rate of repeat infections compared with the older antibiotic vancomycin (see Nat. Med.17, 10, 2011). Alan Carr, a biotech analyst with Needham & Company in New York, predicts a "slow market uptake" at first. But after infectious disease society guidelines are updated, he believes worldwide sales may reach as high as $500 million per year. When Sanofi finalized its $20 billion takeover of Genzyme earlier this year, the drug giant announced that the Cambridge, Massachusetts biotech would retain its name and remain a standalone unit within the larger French parent company. But according to an employee bulletin dated 31 May, that unit will be a smaller one, made up only of Genzyme's core programs in multiple sclerosis and personalized genetic health, including rare diseases. Genzyme's current divisions in oncology, renal and biosurgery will be integrated into Sanofi's operations, the company said. At press time, no layoffs had been announced. Ronald Davis Recombinant DNA pioneer Ronald Davis, director of the Stanford Genome Technology Center in California, won this year's $500,000 genetics prize from the Peter and Patricia Gruber Foundation. Among his many accomplishments, Davis was the first to clone a eukaryotic gene, and he carried out the first targeted gene deletion experiments. "Ron has had so many contributions over the years," says Joseph Ecker, a molecular geneticist at the Salk Institute in La Jolla, California who worked with Davis as a post-doc in the mid-1980s. "Go look at his CV. It's just discovery after discovery, and we all just take it for granted that they came out of Ron's lab." See go.nature.com/JPBago for more. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - Straight talk with...Valery Danilenko
- Nat Med 17(7):764 (2011)
Nature Medicine | News Straight talk with...Valery Danilenko * Gary PeachJournal name:Nature MedicineVolume: 17,Page:764Year published:(2011)DOI:doi:10.1038/nm0711-764Published online07 July 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 Russian medicine is—at long last—undergoing a renaissance. The country's rocky economic ride following the collapse of the Soviet Union disrupted its research rubric and impoverished its healthcare system. Now, however, the nation's leadership is spearheading various initiatives to reverse the situation. One of them, the US-Russian Scientific Forum, established two years ago by a bilateral presidential commission, hopes to bring improvements by facilitating public-private research in biomedicine and innovative drugs. The Forum, which on the Russian side is represented by the country's Ministry of Health and Social Development and the Russian Academy of Sciences, among others, held its inaugural planning meeting in late April in Moscow. Valery Danilenko, who is helping to spearhead the effort and also leads the biotechnology division at the Vavilov Institute of General Genetics in Moscow, told Nature Medicine about the meeting and Russia's hopes for the Forum. The intervi! ew was conducted in Russian and translated by the interviewer, . View full text Additional data Author Details * Gary Peach Search for this author in: * NPG journals * PubMed * Google Scholar - The vagina catalogues
- Nat Med 17(7):765-767 (2011)
Nature Medicine | News | News Feature The vagina catalogues * Alison McCook1Journal name:Nature MedicineVolume: 17,Pages:765–767Year published:(2011)DOI:doi:10.1038/nm0711-765Published online07 July 2011 A group of scientists and dedicated women are participating in rigorous research to uncover secrets of a less-high-profile part of the microbiome. The goal: to learn more about a vaginal disease that most people have never heard of but affects millions of women each year with potentially life-threatening consequences to their unborn children. reports on the unique challenges of developing treatments for a condition with a feminine mystique. View full text Additional data Affiliations * Alison McCook is a science writer and editor in Philadelphia. Author Details * Alison McCook Search for this author in: * NPG journals * PubMed * Google Scholar - Good vibrations
- Nat Med 17(7):768-771 (2011)
Nature Medicine | News | News Feature Good vibrations * Elie Dolgin1Journal name:Nature MedicineVolume: 17,Pages:768–771Year published:(2011)DOI:doi:10.1038/nm0711-768Published online07 July 2011 Countless technologies aim to give scientists accurate readouts of the key components of biological samples from patients. But what if it's better to listen to a sample than to look at it? visits one company that's adapting a vibration detector developed for telecommunication satellites to make what could be the most sensitive commercial biosensor ever built. View full text Additional data Affiliations * Elie Dolgin is a news editor with Nature Medicine in New York. Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Biomarking the way
- Nat Med 17(7):771 (2011)
Article preview View full access options Nature Medicine | News | News Feature Biomarking the way * Elie Dolgin1Journal name:Nature MedicineVolume: 17,Page:771Year published:(2011)DOI:doi:10.1038/nm0711-771Published online07 July 2011 Less than a mile away from BioScale, another Lexington, Massachusetts–based company called T2 Biosystems is also developing a biomarker detector that similarly sidesteps the traditional use of light scan samples. Its platform, dubbed the T2Dx, uses both magnetic resonance and nanoparticles to rapidly test for pathogens in whole blood. "There's a big push to move to nonoptical detection," says Tom Lowery, vice president of diagnostics research and discovery with the company. "The question is, 'who has the data?'" Elie Dolgin T2's John McDonough and Tom Lowery. T2 thinks they do. At a recent meeting this spring, the company presented data showing that their technology detected fungal pathogens in spiked blood samples with high precision at levels as low as ten fungal cells per milliliter. And, a month later, at the general meeting of the American Society for Microbiology in New Orleans, scientists from T2 reported perfect concordance between the data obtained from the T2Dx platform and the results seen by classic microbiology culturing techniques in a small cohort of people with Candida albicans infections. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data Affiliations * Elie Dolgin is a news editor with Nature Medicine in New York. Author Details * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google Scholar - Antibiotic bill doesn't GAIN enough ground
- Nat Med 17(7):772 (2011)
Nature Medicine | News | Opinion Antibiotic bill doesn't GAIN enough ground * Paul G. Ambrose1Journal name:Nature MedicineVolume: 17,Page:772Year published:(2011)DOI:doi:10.1038/nm0711-772Published online07 July 2011 With an eye to tackling the growing problem of antimicrobial drug resistance, US lawmakers last month proposed new incentives to jump-start the ailing antibiotic industry. But the legislation as written is not likely to have the intended consequences, as it fails to adequately shield companies from competition with generic drugs. To truly entice investment and research from the drug industry, the bill needs to simplify the path to regulatory approval, provide greater protection from generic competition and aid drug companies with intellectual property extensions, tax relief and guaranteed market commitments. View full text Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Paul G. Ambrose is the president of the Institute for Clinical Pharmacodynamics in Latham, New York, and an honorary research fellow at the University of Oxford, UK. Author Details * Paul G. Ambrose Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Pain: a sufferer's view
- Nat Med 17(7):773 (2011)
Article preview View full access options Nature Medicine | Book Review Pain: a sufferer's view * Tony L. Yaksh1Journal name:Nature MedicineVolume: 17,Page:773Year published:(2011)DOI:doi:10.1038/nm0711-773Published online07 July 2011 The Pain Chronicles: Cures, Myths, Mysteries, Prayers, Diaries, Brain Scans, Healing, and the Science of Suffering Melanie Thernstrom Farrar, Straus and Giroux, 2010 384 pp., hardcover, $27.00 ISBN: 0865476810 Buy this book: USUKJapan Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In 1993, Melanie Thernstrom, author of The Pain Chronicles, suffered a mild stress injury that initiated chronic pain in her neck and shoulder. In this event, she joined the estimated 70-million-plus Americans who experience ongoing pain as a part of their daily existence, an observation coincident with the fact that analgesic drugs rank the highest, as measured by volume and incidence of purchase, in the over-the-counter drug market. By Thernstrom's description, it is evident that the experience had a dramatic and disabling impact on her quality of life. During this period, she visited dozens of physicians, psychiatrists, physical therapists and practitioners of alternative medicine with varying and often transient success. In 2001, she wrote an article in The New York Times magazine on chronic pain. Work on this article, along with her personal vested interest in the subject, led her to undertake a broad characterization of the concept of pain and its management. According! ly, she mixes her own perceptions, arising from her experiences, with those that she has acquired through interactions with pain patients and with physicians who treat pain. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Affiliations * Tony L. Yaksh is in the Department of Anesthesiology at the University of California–San Diego, La Jolla, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Tony L. Yaksh Author Details * Tony L. Yaksh Contact Tony L. Yaksh Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Effect of bitter tastants on human bronchi
- Nat Med 17(7):775 (2011)
Nature Medicine | Correspondence Effect of bitter tastants on human bronchi * Alyn H Morice1 * Robert T Bennett1 * Mubarak A Chaudhry2 * Michael E Cowen2 * Steven C Griffin2 * Mahmoud Loubani2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Page:775Year published:(2011)DOI:doi:10.1038/nm0711-775Published online07 July 2011 To the Editor: We read with interest the report of the effects of bitter tastants on airway smooth muscle by Deshpande et al.1 and the related News and Views article by Sanderson and Madison2. Deshpande et al.1 report a previously undescribed modulator of airway tone with a unique mode of relaxation in airway smooth muscle that may prove to be clinically significant. We attempted to reproduce the data presented using second-order human bronchi obtained from people with lung cancer after surgical resection. We set rings (n = 24 from nine subjects) at a passive tension of 2 g and contracted them with 1 mM methacholine or 1 mM acetylcholine. We did control relaxations using 10 μM isoprenaline (n = 9). In contrast to the report by Deshpande et al.1, isoprenaline induced rapid (7.9 ± 5.9 min) and potent relaxation of bronchi constricted with acetylcholine (145 ± 39% inhibition of maximum, n = 3) and methacholine (103 ± 49% inhibition of maximum, n = 6) (Fig. 1a). Their claim that bitter tas! te receptor agonists have three times the efficacy of b-agonists1 may result from the poor performance of isoprenaline in their report. Our own experience and that reported in the literature3, 4 is that isoprenaline is a potent and highly efficacious bronchodilator of human airway smooth muscle. We repeated the experiments reported by Deshpande et al.1 using three of the bitter tastants as bronchodilators in bronchi constricted with 1 mM methacholine. Saccharin (n = 5) produced no response up to concentrations of 3 mM (Fig. 1b). Quinine (1 mM; n = 5) and 1–3 mM chloroquine (n = 5) induced relaxation of bronchi (Fig. 1b). At these concentrations, we found that the average time for relaxation to baseline was 23 ± 6 min for quinine and 34 ± 16 min for chloroquine (Fig. 1b). After a mean washout time of 37 ± 10 min, the contractile response to methacholine was reduced (15 ± 19% and 27 ± 15% of the rings that were pre-exposed to quinine and chloroquine, respectively (Fig.! 1b)). Our inability to reverse the effects of bitter tastants! in the human bronchial preparation used in our study stands in contrast to the report by Deshpande et al.1, in which chloroquine mediated-relaxation was fully reversible in mouse tracheal rings. View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Cardiovascular and Respiratory Studies, University of Hull, Hull York Medical School, Hull, United Kingdom. * Alyn H Morice & * Robert T Bennett * Centre for Cardiology and Cardiothoracic Surgery, Castle Hill Hospital, Cottingham, United Kingdom. * Mubarak A Chaudhry, * Michael E Cowen, * Steven C Griffin & * Mahmoud Loubani Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alyn H Morice Author Details * Alyn H Morice Contact Alyn H Morice Search for this author in: * NPG journals * PubMed * Google Scholar * Robert T Bennett Search for this author in: * NPG journals * PubMed * Google Scholar * Mubarak A Chaudhry Search for this author in: * NPG journals * PubMed * Google Scholar * Michael E Cowen Search for this author in: * NPG journals * PubMed * Google Scholar * Steven C Griffin Search for this author in: * NPG journals * PubMed * Google Scholar * Mahmoud Loubani Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (61K) Supplementary Methods Additional data - Bronchodilator activity of bitter tastants in human tissue
- Nat Med 17(7):776 (2011)
Nature Medicine | Correspondence Bronchodilator activity of bitter tastants in human tissue * Maria G Belvisi1 * Nicole Dale1 * Mark A Birrell1 * Brendan J Canning2 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Page:776Year published:(2011)DOI:doi:10.1038/nm0711-776aPublished online07 July 2011 To the Editor: We read with interest the article by Deshpande et al.1 on bitter taste receptors (TAS2Rs) in the lungs and the expression of TAS2Rs on human airway smooth muscle (ASM). The authors describe experiments in which TAS2R agonists, such as chloroquine, evoked relaxation of mouse tracheal rings that was threefold greater in magnitude than that elicited by β-adrenergic receptor agonists1 (the current gold-standard bronchodilator therapy for asthma and chronic obstructive pulmonary disease2). We sought to investigate the uncharacteristically weak bronchodilator activity of isoproterenol in human tissue. View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Respiratory Pharmacology Group, Pharmacology and Toxicology Section, National Heart and Lung Institute Faculty of Medicine, Imperial College London, London, UK. * Maria G Belvisi, * Nicole Dale & * Mark A Birrell * Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA. * Brendan J Canning Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Maria G Belvisi Author Details * Maria G Belvisi Contact Maria G Belvisi Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Dale Search for this author in: * NPG journals * PubMed * Google Scholar * Mark A Birrell Search for this author in: * NPG journals * PubMed * Google Scholar * Brendan J Canning Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (119K) Supplementary Methods Additional data - Bronchodilator activity of bitter tastants in human tissue
- Nat Med 17(7):776-778 (2011)
Nature Medicine | Correspondence Bronchodilator activity of bitter tastants in human tissue * Deepak A Deshpande1 * Kathryn S Robinett1 * Wayne C H Wang1 * James S K Sham2 * Steven S An3 * Stephen B Liggett1, 4 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:776–778Year published:(2011)DOI:doi:10.1038/nm0711-776bPublished online07 July 2011 Deshpande et al. reply: Belvisi et al.1 show in human bronchi that the bitter taste receptor (TAS2R) agonist chloroquine evokes marked relaxation; this is in agreement with our findings in mouse airways2. They note, however, an equivalent efficacy (degree of maximal relaxation) with the b-agonist isoproterenol in human bronchi1. In our paper, we carried out the vast majority of intact airway physiology experiments in mice, in which we found that bitter tastants had a greater efficacy compared with isoproterenol2. We never stated that TAS2R agonists were more potent than b-agonists, and we note that the differences in the potency (the EC50) of isoproterenol and chloroquine are comparable in our study2 and that of Belvisi et al.1. In our paper, we specifically noted that the threefold greater efficacy of TAS2R agonists compared to the b-agonist isoproterenol referred to mouse airways2. With the limited number of human airways that we studied, we were not in a position to provide quantitative efficacy! data in human airways. We have now examined additional human bronchial rings after contraction with methacholine (Fig. 1a–c), and we can adequately compare the efficacy of bitter tastants and isoproterenol. We find 77 ± 4.3% relaxation by isoproterenol (n = 15 experiments), whereas chloroquine and quinine achieved essentially 100% relaxation (106 ± 4.7 and 99 ± 2.9%, respectively (Fig. 1d)). Accordingly, we still note a greater efficacy of bitter tastants compared with isoproterenol, but we recognize that it is certainly less than the threefold difference we observed in mice2; we attribute this difference to a species-specific effect. Thus, we agree that in the ex vivo human airway ring model, the efficacy of these TAS2R agonists is similar to that of isoproterenol. We would like to point out, however, that the full b-agonist, isoproterenol, is not used clinically. The most commonly used inhaled b-agonist is albuterol, a partial agonist, which evokes ~60% relaxation i! n ex vivo studies comparing human airways that have been preco! ntracted with muscarinic agonists and those precontracted with isoproterenol3, 4. Thus, from a clinical standpoint, it remains to be seen whether inhaled bitter tastants are more efficacious than this benchmark inhaled b-agonist. View full text Author information * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Deepak A Deshpande, * Kathryn S Robinett, * Wayne C H Wang & * Stephen B Liggett * Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. * James S K Sham * Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA. * Steven S An * Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Stephen B Liggett Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen B Liggett Author Details * Deepak A Deshpande Search for this author in: * NPG journals * PubMed * Google Scholar * Kathryn S Robinett Search for this author in: * NPG journals * PubMed * Google Scholar * Wayne C H Wang Search for this author in: * NPG journals * PubMed * Google Scholar * James S K Sham Search for this author in: * NPG journals * PubMed * Google Scholar * Steven S An Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen B Liggett Contact Stephen B Liggett Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (131K) Supplementary Methods Additional data - Downregulating DR6 to drive remyelination
- Nat Med 17(7):779-780 (2011)
Article preview View full access options Nature Medicine | Article Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination * Sha Mi1 * Xinhua Lee1 * Yinghui Hu1 * Benxiu Ji1 * Zhaohui Shao1 * Weixing Yang1 * Guanrong Huang1 * Lee Walus1 * Kenneth Rhodes1 * Bang Jian Gong1 * Robert H Miller2 * R Blake Pepinsky1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:816–821Year published:(2011)DOI:doi:10.1038/nm.2373Received27 December 2010Accepted06 April 2011Published online06 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal r! ole for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis. Figures at a glance * Figure 1: DR6 is expressed in oligodendrocytes. () RT-PCR quantification of relative DR6 mRNA expression in rat brain at different development stages (mRNA level in sample E18 = 1). () Western blot analysis of DR6 protein expression in rat brain at different development ages. () In situ hybridization analysis of DR6 mRNA expression in adult rat corpus callosum sections. Red, probed with DR6 antisense mRNA; green, stained with antibody to O4; yellow, merge of red and green. The arrowheads indicate O4+DR6+ cells, and blue is DAPI staining. Scale bars, 15 μm. () Immunocytochemical analysis of DR6 protein expression in oligodendrocytes. Red, stained with antibody to DR6; green, stained with antibodies to A2B5, PDGFRα and MBP; yellow, merge of red and green. Scale bars, 95 μm. () Western blot analysis of DR6 protein expression in A2B5+, PDGFRα+ and MBP+ oligodendrocyte cultures. β-actin expression was analyzed from the same samples as an internal control. () Quantification of DR6 protein expression from . Data are shown a! s means ± s.e.m. * Figure 2: DR6 antagonists promote A2B5+ OPC survival and differentiation. () Western blot analysis of MBP, MOG, cleaved casp3 and DR6 proteins in oligodendrocytes after treatment with DR6 or control siRNAs. β-actin expression was analyzed from the same samples as an internal control. () Western blot analysis of MBP, cleaved casp3 proteins in DR6 DN and DR6 FL lentivirus-infected A2B5+ OPC cultures. GFP expression was analyzed from the same samples as an internal control for lentivirus infection. () Quantification of percentage cleaved casp3+ oligodendrocytes after treatment with DR6 DN, DR6 FL and control virus. () Quantification of cleaved casp3+ cells in DR6 FL and control oligodendrocytes after treatment with casp3 and casp6 inhibitors. () Western blot analysis of cleaved casp3, MBP and MAG proteins in oligodendrocytes after treatment with N-APP or buffer control. () P2 oligodendrocytes from DR6 WT and DR6-null mice cultures stained with antibody to MBP. Scale bars, 25 μm. () Quantification of MBP+ mature oligodendrocytes in . () Quantificati! on of cleaved casp3+ oligodendrocytes from DR6 WT and null mice in P2 oligodendrocyte cultures. P values in and were determined by one-way analysis of variance (ANOVA followed by Tukey's test), and in and using the unpaired t test. β-actin expression was analyzed from the same samples as an internal control for all western blots. Data are shown as means ± s.e.m. * Figure 3: Inhibition of DR6 promotes oligodendrocyte survival, maturation and myelination. () Western blot analysis of MBP expression after P2 OPC treatment with DR6 antibodies (10 μg ml−1). () Immonocytochemical analysis to visualize MBP+ and cleaved casp3+ apoptotic oligodendrocytes after 5D10 or control isotype antibody treatment. Arrowheads indicate Casp3+ cells. Scale bars, 100 μm. () Quantification of MBP+ oligodendrocytes and cleaved casp3+ oligodendrocytes from . () Western blot analysis of cleaved casp3 and MBP proteins in 5D10- and control IgG–treated OPC cultures. () Immunocytochemical visualization of myelination in 5D10-, DR6 DN- and DR6 siRNA-treated DRG-OPC cocultures and the corresponding control-treated cocultures. Scale bars, 50 μm. Red, MBP staining. () Quantitative analysis of immunocytochemical staining of MBP+ myelinated axon clusters from . () Western blot analysis of MBP and MOG in cocultures treated with 5D10, DR6 FL and DR6 DN. () Western blot analysis of MBP, MAG and DR6 protein from WT and DR6-null mice cocultures. β-actin expre! ssion was analyzed from the same samples in and as an internal control. () Quantification of MBP+ axons from WT and DR6-null cocultures. P values in , and were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 4: Antibody to DR6 promotes functional recovery and remyelination in the rat EAE model. () EAE clinical score measurement after 5D10 and control antibody treatment. Arrows indicate treatment regimen. () Descending nerve conduction velocity measurements after 5D10 and control antibody treatment. () Electron microscopy analysis of remyelination in EAE rat spinal cords after 5D10 and control antibody treatments and in WT and DR6-null EAE mouse spinal cords. Remyelination is denoted by blue asterisks in lower-magnification images and red asterisks in higher-magnification images. () Quantification of myelinated axons from . P values were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 5: A DR6 antagonist promotes remyelination in brain slice cultures and in the LPC-induced spinal cord demyelination model. () Black gold staining (red) of myelinated axons in LPC-treated corpus callosum brain slice cultures. Brain slices were demyelinated by LPC followed by 5D10 and control IgG treatments for 3 d. Remyelination was visualized by light microscopy. Scale bar, 200 μm. () Quantification of myelin protein MAG by MSD analysis. P values were determined using one-way analysis of variance (nonparametric tests). () Toluidine blue staining and electron microscopy (EM) analysis of remyelination in LPC-demyelinated rat spinal cords after 5D10 and control antibody treatments and in LPC-demyelinated WT and DR6-null mouse spinal cords. Arrowheads, remyelinated axons; asterisks, axon pathology. () Quantification of myelinated axons from . P values in and were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 6: Oligodendrocyte maturation and myelination in DR6-null mice. () Quantification of immature PDGFRα+ oligodendrocytes from DR6 knockout and WT corpus callosum at age P15 and P30. () Quantification of CC1+ mature oligodendrocytes from DR6-null and WT corpus callosum at age P15 and P30. () Western blot analysis of MAG and MBP protein expression in DR6-null and WT mouse brain at P15 and P30. DR6 expression was used to confirm the DR6-null mice; β-actin expression was analyzed from the same samples as an internal control. () Quantification of MAG and MBP protein expression in P15 mice from . () EM showing visual () and quantitative () analysis of myelinated axon fibers of DR6 knockout and WT corpus callosum from P15 mice. Scale bars, 500 nM. () G ratio of myelinated axons from . () Colocalization of DR6 mRNA (in situ hybridization) and Olig2+ OPCs by immunohistochemical analysis in human normal and multiple sclerosis (MS) brains. Red, probed with DR6 antisense mRNA; green, stained with antibody to Olig2; yellow, merge of red and green. Sc! ale bars, 50 μm. () Quantification of DR6+Olig2+ cells in . P values in , , , , and were determined using the unpaired t test. Data are shown as means ± s.e.m. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Affiliations * Biogen Idec, Cambridge, Massachusetts, USA. * Sha Mi, * Xinhua Lee, * Yinghui Hu, * Benxiu Ji, * Zhaohui Shao, * Weixing Yang, * Guanrong Huang, * Lee Walus, * Kenneth Rhodes, * Bang Jian Gong & * R Blake Pepinsky * Center for Translational Neuroscience, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. * Robert H Miller Contributions S.M. supervised all experiments and wrote the paper. X.L., Y.H., B.J., Z.S., W.Y., G.H., L.W. and B.J.G. performed experiments. K.R. and R.B.P. provided helpful discussions, and R.H.M. and R.B.P. revised the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sha Mi Author Details * Sha Mi Contact Sha Mi Search for this author in: * NPG journals * PubMed * Google Scholar * Xinhua Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Yinghui Hu Search for this author in: * NPG journals * PubMed * Google Scholar * Benxiu Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui Shao Search for this author in: * NPG journals * PubMed * Google Scholar * Weixing Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Guanrong Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Lee Walus Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth Rhodes Search for this author in: * NPG journals * PubMed * Google Scholar * Bang Jian Gong Search for this author in: * NPG journals * PubMed * Google Scholar * Robert H Miller Search for this author in: * NPG journals * PubMed * Google Scholar * R Blake Pepinsky 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 1–6 Additional data - A downside to apoptosis in cancer therapy?
- Nat Med 17(7):780-782 (2011)
Article preview View full access options Nature Medicine | Article Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy * Qian Huang1, 2, 3, 12 * Fang Li2, 12 * Xinjian Liu2 * Wenrong Li2 * Wei Shi4 * Fei-Fei Liu4 * Brian O'Sullivan4 * Zhimin He2 * Yuanlin Peng5 * Aik-Choon Tan6 * Ling Zhou7 * Jingping Shen2 * Gangwen Han8 * Xiao-Jing Wang8, 9, 10 * Jackie Thorburn11 * Andrew Thorburn11 * Antonio Jimeno6, 9, 10 * David Raben2, 9 * Joel S Bedford5 * Chuan-Yuan Li2, 10, 11 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:860–866Year published:(2011)DOI:doi:10.1038/nm.2385Received17 November 2009Accepted27 April 2011Published online03 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2 (PGE2), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death–induced tumor repopulation pathway in which caspase 3 has a major role. Figures at a glance * Figure 1: In vitro and in vivo evidence for the generation of strong growth-stimulating signals in dying cells. () Stimulation of 4T1Fluc cellular growth in vitro by irradiated 4T1 cells. Top, growth of 4T1Fluc cells as observed by luciferase activities. The difference between each of the higher-dose irradiated groups (8, 10 and 12 Gy) and controls (0 Gy and no feeder) was statistically significant (P < 0.001, t test, n = 4). Bottom, representative images from bioluminescence imaging. () Top, relative growth of MEF-supported tumor cells versus tumor cells seeded alone. *P < 0.001, t test, n = 3. Bottom, representative bioluminescence images. (,) Effect of dying 4T1 () and MEF () cells on 4T1Fluc tumor cellular growth in vivo. Top, quantification of bioluminesent signals. Bottom, representative bioluminescent images of mice. In each of the two experiments, the difference between the two groups was highly significant (P < 0.001 from day 4 for , and from day 1 for , n = 5, one-way analysis of variance (ANOVA) test). In all cases, error bars indicate s.e.m. * Figure 2: The role of caspase 3 in cell death–induced tumor cell proliferation in vitro and in vivo. () Effects of dying wild-type (WT) and Casp3−/− MEF cells on different Fluc-labeled tumor cells. *P < 0.01, t test, n = 3. () The differences between the control groups and the Casp3-kn groups were statistically significant (P < 0.01 between each of the control and each of the caspase 3 knockdown clones, n = 3, t test). Bottom, western blot analysis of caspase 3 levels. () The effect of a dominant-negative caspase 3 (casp3DN) in dying, unlabeled 4T1 cells on 4T1Fluc tumor cell growth. Inset, western blot showing dominant-negative caspase 3 expression (casp3DN was hemagglutinin (HA) labeled). () Western blot analyses of key proteins involved in apoptosis. () The effect of dying wild-type and Casp3−/− MEF cells on growth of 4T1Fluc cells in mice. The difference between the two groups was highly significant statistically (P < 0.001 from day 3 on, n = 4, one-way ANOVA test). () The effect of caspase 3 knockdown in lethally irradiated 4T1 cells on growth of 4T1Fluc cells ! in vivo. The difference between the two groups was statistically significant from day 5 (P < 0.05, n = 5, one-way ANOVA test). In all panels, error bars represent s.e.m. * Figure 3: Relationship between caspase activation and growth of injected tumor cells in the irradiated tumor microenvironment. () Caspase 3 activation in 4T1 tumors as detected by a caspase 3 reporter. Left, schematic of the structure of a proteasome-based caspase 3 reporter (top left) and its mode of action (bottom left). Right, caspase 3 activities in 4T1 tumors transduced with the control as well as Casp3 reporter genes. The difference between the control and caspase 3 reporter groups are significant at days 3, 5 and 7 (P < 0.01, n = 5, t test). CMV, cytomegalovirus promoter; (Ubi)9, polyubiqutin domain consists of nine tandem copies of ubiquitin; pA, polyadenylation signal; DEVD, consensus caspase 3 cleavage site. () Top, growth of 4T1Fluc cells injected into irradiated and nonirradiated established tumors. The difference between the two groups was statistically significant (P < 0.05 from day 7, t test, n = 4). Bottom, representative images of tumor-bearing mice. () Immunofluorescence analysis of growth of intratumorally injected GFP-labeled cells and the indicated protein expression surrounding! the injected cells. SMA, smooth muscle actin, a marker for blood vessels. Scale bars, 100 μm. Error bars represent s.e.m. * Figure 4: A role for caspase 3–activated iPLA2 (Pla2g6) in facilitating cell death–stimulated tumor cell repopulation. () The effect of Pla2g6 levels in dying cells on the growth of 4T1Fluc cells in vitro. P < 0.01, n = 4, t test. () The effect of constitutively active (CA) iPLA2 ΔPla2g6 in Casp3−/− MEF cells in vivo. The differences between the luciferase signals were statistically significant (P < 0.01 on day 7, n = 5, t test). Inset, expression of truncated iPLA2. () The in vivo effect of iPLA2 knockdown in wild-type MEF cells. The difference between the two groups was statistically significant from day 3 (P < 0.02, from day 7, n = 5, one-way ANOVA). Inset, western blot analysis of shRNA knockdown of Pla2g6. Error bars represent s.e.m. * Figure 5: Regulation of radiation-induced arachidonic acid release and PGE2 production by caspase 3. () Arachidonic acid (AA) release. *P < 0.02 (t test, n = 3). () PGE2 secretion. *P < 0.05 (t test, n = 3). () PGE2-stimulated tumor growth from 1,000 4T1Fluc tumor cells injected subcutaneously into nude mice. The difference between the two groups are statistical significant from day 17 (P < 0.05, n = 5, one-way ANOVA). Error bars represent s.e.m. * Figure 6: Caspase 3 status correlated with tumor response to therapy in mice as well as in human subjects. () Result of radiation therapy in tumors established from MCF-7 and MFC-7CASP3 cells.The differences between the two groups are highly significant after radiotherapy (P < 1 × 10−6, n = 10, one-way ANOVA). () Results of radiation therapy on B16F10 mouse melanoma tumors grown in wild-type C57BL6 and Casp3−/− mice. The difference between the two groups are significant after radiotherapy from day 11 (P < 0.04, n = 5, one-way ANOVA). Error bars in and represent s.e.m. (,) Kaplan-Meier analyses of cancer recurrence in a cohort of subjects with head and neck squamous cell carcinoma (HNSCC) treated at Princess Margaret Hospital in Toronto () and of survival in a cohort of subjects with advanced breast cancer treated at Shanghai No.1 People's Hospital (). Log-rank tests were used for analysis of statistical significance. For , P = 0.0114, hazard ratio = 3.44, 95% confidence interval: 1.35–8.75. For , P = 0.0006, hazard ratio = 5.29, 95% confidence interval: 1.70–16.46. () ! A schematic representation of the proposed pathway for cell death–mediated tumor cell repopulation. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this study. * Qian Huang & * Fang Li Affiliations * Experimental Research Center, First People's Hospital, Shanghai Jiao Tong University, Shanghai, China. * Qian Huang * Department of Radiation Oncology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Qian Huang, * Fang Li, * Xinjian Liu, * Wenrong Li, * Zhimin He, * Jingping Shen, * David Raben & * Chuan-Yuan Li * National Laboratory of Oncogenes and Related Genes Research, Cancer Institute, Shanghai Jiao Tong University, Shanghai, China. * Qian Huang * Department of Radiation Oncology and Ontario Cancer Institute, Princess Margaret Hospital, University of Toronto, Toronto, Ontario, Canada. * Wei Shi, * Fei-Fei Liu & * Brian O'Sullivan * Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, USA. * Yuanlin Peng & * Joel S Bedford * Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA. * Aik-Choon Tan & * Antonio Jimeno * Department of Surgery, Shanghai First People's Branch Hospital, Shanghai, China. * Ling Zhou * Department of Pathology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Gangwen Han & * Xiao-Jing Wang * Head and Neck Cancer Research Program, University of Colorado Cancer Center, Aurora, Colorado, USA. * Xiao-Jing Wang, * Antonio Jimeno & * David Raben * Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Xiao-Jing Wang, * Antonio Jimeno & * Chuan-Yuan Li * Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Jackie Thorburn, * Andrew Thorburn & * Chuan-Yuan Li Contributions Q.H. and F.L. designed and conducted most of the experiments, analyzed data and wrote the manuscript. X.L. and W.L. carried out analyses on modes of cell death in irradiated cells; W.S. carried out immunohistochemical analysis of human head and neck tumor samples; F.-F.L. and B.O. provided human head and neck cancer samples and analyzed data from the samples; Z.H. conducted some of the caspase reporter experiments; Y.P. carried out arachidonic acid release experiments and J.S.B. analyzed relevant data of arachidonic acid release; A.-C.T. carried out data analyses of human clinical data; G.H. and X.-J.W. helped with immunohistochemical analysis of mouse tumor samples; J.S. constructed some of the plasmids used; A.J. and D.R. provided human head and neck tumor samples; L.Z. carried out immunohistochemical analysis of human breast cancer samples; J.T. and A.T. helped to conduct experiments on autophagy and necrosis; C.-Y.L. conceived of the study, analyzed data and wrote the ma! nuscript. All authors read and agreed on the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Chuan-Yuan Li Author Details * Qian Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Fang Li Search for this author in: * NPG journals * PubMed * Google Scholar * Xinjian Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Wenrong Li Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Shi Search for this author in: * NPG journals * PubMed * Google Scholar * Fei-Fei Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Brian O'Sullivan Search for this author in: * NPG journals * PubMed * Google Scholar * Zhimin He Search for this author in: * NPG journals * PubMed * Google Scholar * Yuanlin Peng Search for this author in: * NPG journals * PubMed * Google Scholar * Aik-Choon Tan Search for this author in: * NPG journals * PubMed * Google Scholar * Ling Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Jingping Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Gangwen Han Search for this author in: * NPG journals * PubMed * Google Scholar * Xiao-Jing Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Jackie Thorburn Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Thorburn Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Jimeno Search for this author in: * NPG journals * PubMed * Google Scholar * David Raben Search for this author in: * NPG journals * PubMed * Google Scholar * Joel S Bedford Search for this author in: * NPG journals * PubMed * Google Scholar * Chuan-Yuan Li Contact Chuan-Yuan Li 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 1–23, Supplementary Note, Supplementary Methods and Supplementary Tables 1–3 Additional data - The brain splits obesity and hypertension
- Nat Med 17(7):782-783 (2011)
Article preview View full access options Nature Medicine | Letter Uncoupling the mechanisms of obesity and hypertension by targeting hypothalamic IKK-β and NF-κB * Sudarshana Purkayastha1, 2, 3 * Guo Zhang1, 2, 3 * Dongsheng Cai1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:883–887Year published:(2011)DOI:doi:10.1038/nm.2372Received14 January 2011Accepted05 April 2011Published online05 June 2011 Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Obesity-related hypertension has become an epidemic health problem and a major risk factor for the development of cardiovascular disease (CVD). Recent research on the pathophysiology of obesity has implicated a role for the hypothalamus in the pathogenesis of this condition1, 2, 3. However, it remains unknown whether the often-seen coupling of hypertension with obesity can also be explained by hypothalamic dysfunction, despite the emerging appreciation that many forms of hypertension are neurogenic in origin4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Our studies here revealed that acute activation of the proinflammatory protein nuclear factor κB (NF-κB) and its upstream activator IκB kinase-β (IKK-β, encoded by Ikbkb) in the mediobasal hypothalamus rapidly elevated blood pressure in mice independently of obesity. This form of hypothalamic inflammation-induced hypertension involved the sympathetic upregulation of hemodynamics and was reversed by sympathetic suppression. Loss-of-fu! nction studies further showed that NF-κB inhibition in the mediobasal hypothalamus counteracted obesity-related hypertension in a manner that was dissociable from changes in body weight. In addition, we found that pro-opiomelanocortin (POMC) neurons were crucial for the hypertensive effects of the activation of hypothalamic IKK-β and NF-κB, which underlie obesity-related hypertension. In conclusion, obesity-associated activation of IKK-β and NF-κB in the mediobasal hypothalamus—particularly in the hypothalamic POMC neurons—is a primary pathogenic link between obesity and hypertension. Breaking this pathogenic link may represent an avenue for controlling obesity-related hypertension and CVD without requiring obesity control. Figures at a glance * Figure 1: Effects of manipulating hypothalamic IKK-β and NF-κB on blood pressure in C57BL/6 mice. (–) Representative 30-min telemetric blood pressure (BP) tracings during the light phase () versus the dark phase (); average values of mean blood pressure (MBP) in the indicated mice during the light phase () versus the dark phase (); and LF-BPV () and LF/HF-HRV () in chow-fed or HFD-fed mice at 1 week after hypothalamic injection of IKK-βCA–, IκBαDN-, or GFP-expressing adenoviruses. *P < 0.05, **P < 0.01; n = 3 or 4 mice per group. Error bars show means ± s.e.m. * Figure 2: Effects of TNF-α injection via the third ventricle on blood pressure in C57BL/6 mice. (–) Longitudinal profiles of systolic blood pressure (), diastolic blood pressure () and mean blood pressure (); dose-dependent increases (Δ) in mean blood pressure averaged over a 15-min peak change period (); LF-BPV () and LF/HF-HRV () over the same15-min period; and blood norepinephrine (NE) concentrations () in C57BL/6 mice after injection of TNF-α or vehicle. () Systolic (), diastolic () and mean () blood pressure levels over a 15-min peak change period in adult C57BL/6 mice that received TNF-α or vehicle injection in the presence (+) or absence (−) of prior i.p. injection of an adrenergic blocker, prazosin (Prz). The dose of injected TNF-α in – and – was 0.5 ng. *P < 0.05, **P < 0.01, NS, nonsignificant; n = 4–6 per group. Error bars show means ± s.e.m. SBP, systolic BP; DBP, diastolic blood pressure; MBP, mean blood pressure. * Figure 3: Activation of IKK-β and NF-κB by TNF-α in POMC neurons. (,) Immunostaining of phosphorylated IKK-β (red) () or IκBα (red) () in POMC neurons (green) (,) of Pomc-ROSA mice treated with TNF-α or vehicle via the third ventricle (3V). pIKK-β: phosphorylated IKK-β; scale bar, 50 μm. We counted the cell numbers of pIKK-β–positive (pIKK-β+) POMC neurons () or IκBα-positive (IκBα+) POMC neurons () as well as the total numbers (total) of POMC neurons (,) in the brain sections across the arcuate nucleus (ARC) . Data represent average cell numbers unilaterally in the median ARC. ***P < 0.001; NS, nonsignificant; n = 3 or 4 per group. Error bars show means ± s.e.m. * Figure 4: Hypotensive effect of POMC neuron-specific IKK-β ablation. (–) Average increases (Δ) in systolic blood pressure (), diastolic blood pressure (), mean blood pressure (), LF-BPV () and LF/HF-HRV () of chow-fed Pomc-Ikbkblox/lox mice, Agrp-Ikbkblox/lox mice, and the genotype-matched control Ikbkblox/lox mice in response to a third-ventricle injection of TNF-α or vehicle during a 15-min peak change period. () Mean blood pressure values in chow- vs. HFD-fed Pomc-Ikbkblox/lox mice and control Ikbkblox/lox mice. () Proposed role of hypothalamic IKK-β and NF-κB in obesity-related hypertension. Arrows in boxes indicates upregulation of IKK-β and NF-κB; dashed boxes indicate the parallel pathway described previously. SBP, systolic BP; DBP, diastolic blood pressure; MBP, mean blood pressure. *P < 0.05, n = 3 or 4 per group. Error bars show means ± s.e.m. Article preview Read the full article * Instant access to this article: US$18 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sudarshana Purkayastha & * Guo Zhang Affiliations * Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, New York, USA. * Sudarshana Purkayastha, * Guo Zhang & * Dongsheng Cai * Diabetes Research Center, Albert Einstein College of Medicine, New York, New York, USA. * Sudarshana Purkayastha, * Guo Zhang & * Dongsheng Cai Contributions D.C. conceived and designed the study; S.P. did experiments shown in Figures 1, 2 and 4 with assistance from G.Z. and D.C.; and G.Z. also carried out experiments shown in Figure 3. All authors did data analysis and interpretation. D.C. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dongsheng Cai Author Details * Sudarshana Purkayastha Search for this author in: * NPG journals * PubMed * Google Scholar * Guo Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Dongsheng Cai Contact Dongsheng Cai Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (778K) Supplementary Figures 1–8 Additional data - A viral strategy to ambush tumors
- Nat Med 17(7):784-785 (2011)
Article preview View full access options Nature Medicine | Article Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors * Timothy Kottke1 * Fiona Errington2 * Jose Pulido1, 3 * Feorillo Galivo1 * Jill Thompson1 * Phonphimon Wongthida1 * Rosa Maria Diaz1 * Heung Chong4 * Elizabeth Ilett2 * John Chester2 * Hardev Pandha5 * Kevin Harrington6 * Peter Selby2 * Alan Melcher2, 8 * Richard Vile1, 2, 7, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:854–859Year published:(2011)DOI:doi:10.1038/nm.2390Received04 January 2011Accepted29 April 2011Published online19 June 2011 Abstract * Abstract * Author information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm! for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date. Figures at a glance * Figure 1: Construction and characterization of VSV-expressed cDNA libraries. () The ASEL VSV-expressed cDNA library contains cDNA from normal human prostate cloned into VSV in direct or reverse orientation. () Lanes 1 and 2, human prostate-specific genes (PSMA, STEAP, TRPA1 and PSCA)37, 38, 39, 40, but not the melanocyte-specific TYRP2 (ref. 11), detected by PCR in the original human prostate plasmid library (lane 1) and in the VSV-cDNA plasmid library (lane 2). Lanes 3 and 4, PCR from cDNA of HT1080 cells infected with ASEL (MOI = 0.1; lane 3) compared to uninfected cells (lane 4). Arrow indicates predicted size for PSCA. GAPDH was used as a loading control. () Western blot analysis of human PSA in BHK cells infected with ASEL direct (lane 1) or ASEL reverse (lane 2), or with control viruses (lanes 3 and 4; MOI ~10). Lane 5, uninfected BHK cells; lane 6, 1 × 104 human prostate LnCap cells. () BHK cells infected with tenfold dilutions of ASEL virus (lanes 1–6) assayed by RT-PCR for PSA or human GAPDH. No PSA-specific signal was detected at dilutio! ns lower than 1:100 of the original virus stock (expression of GFP from 100 PFU of VSV-GFP could be detected by this assay). Plus sign, positive signal for the target RNA was detected by PCR upon nested PCR. Asterisk, no PCR signal for the target RNA was detected upon nested PCR. () IFN-γ assay (ref. 28) of splenocytes infected with VSV-GFP (MOI = 0.1), with VSV-cDNA libraries (direct or reversed) derived from B16 cells that did or did not express OVA (labeled B16-OVA or B16, respectively; MOI = 0.1) or with VSV-OVA at indicated MOIs. Where indicated, splenocytes were cocultured with naive OT-I T cells or, as a control, with irrelevant T cells (pmel)41. Column 9, OT-I activated by SIINFEKL peptide; column 14, OT-I alone (no splenocytes, no VSV). * Figure 2: Intraprostatic injection of ASEL induces autoimmunity. () Prostate weights of mice injected intraprostatically with PBS, VSV-GFP or ASEL, 2 or 10 d after injection (n = 3). (–) Histology of prostates 10 d after intraprostatic injection of PBS () or ASEL (,). Scale bars, 100 μm. (,) IFN-γ () and IL-17 () production from splenocytes 10 d after intraprostatic injection of PBS or of 1 × 107 PFU VSV-GFP or ASEL (six mice per group, splenocytes from each mouse shown), cocultured with lysates of TC2 cells. Mean values of cytokine from two ELISA wells per sample are shown for each mouse. TC2 lysate, ELISA with lysate alone (no splenocytes); C57BL/6, splenocytes alone. * Figure 3: Intravenous injection of ASEL has anti-tumor efficacy. () Prostate weights 60 d after 1 × 107 PFU intravenous injection of ASEL (n = 5) or VSV-GFP (n = 7). () Mean levels of IL-17 secreted by splenocytes from the infected mice, cocultured with lysates of B16 melanoma, TC2 prostate, normal mouse prostate (Pros) or pancreas (Panc). Error bars, s.d. of three separate wells per sample. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8), injected intratumorally (i.t.) or intravenously (i.v.) on days 7, 9 and 11 with 1 × 107 PFU of VSV-GFP, ASEL or heat-inactivated (HI) VSV-GFP. (,) Survival of mice bearing 7-day-old TC2 () or B16 () tumors injected intravenously on days 7, 9 and 11 with ASEL or with a VSV-cDNA library from human melanoma cells (altered-self melanoma epitope library, ASMEL). () Cumulative percentages of mice cured of 7d TC2 tumors when administered three, six or nine injections of ASEL or VSV-GFP intratumorally or intravenously every other day. () IL-17 secreted by splenocytes from the three mice cured of! TC2 tumors by nine intratumoral injections of ASEL in , as well as from three mice treated similarly with VSV-GFP. Splenocytes were cocultured with lysates of B16, TC2, normal mouse prostate or pancreas. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8), either mock-depleted or depleted of CD4+, CD8+ or NK cells, injected intravenously with ASEL on days 7, 9, 11, 14, 16, 18, 21, 23 and 25. * Figure 4: Suboptimal vaccination induces immune escape variants. (–) H&E staining of tumors (1.0 cm diameter) from mice bearing 7-day-old TC2 tumors treated intravenously on days 7, 9 and 11 with PBS (,) or ASEL (,) (typically tumors reached 1.0 cm in diameter at day 20 for PBS (TC2) or day 50 for ASEL (TC2R)). Scale bars, 100 μm. (,) Three TC2R tumors from mice treated with ASEL (lanes 1–3) as in , one TC2 tumor from a mouse treated with PBS (lane 4) as in , and in vitro–cultured TC2 cells (lane 5), analyzed by RT-PCR for expression of mouse prostate-specific factors PSCA, PSMA and STEAP42 () or of N-cadherin, SLUG or SNAIL29, 30, 31, 32 (). GAPDH RNA was assayed as a loading control. Cultured TC2 cells were positive for N-cadherin by nested PCR and weakly positive by western blot at lower levels than detected in TCR2 tumors. * Figure 5: TC2R tumors can be treated with a second vaccination. () The IEEL contained cDNA from three TCR2 tumors cloned into VSV. () Western blot for mouse N-cadherin from BHK cells infected with VSV (lane 1), IEEL reverse (lane 2) or IEEL direct (lane 3) (MOI ~10). Equal loading was confirmed by β-actin probing (data not shown). () Survival of mock-vaccinated (non–VSV immune) or VSV-GFP–vaccinated (VSV-immune) mice bearing 7-day-old TC2 tumors treated with three intravenous injections of VSV-GFP or ASEL, either as viral supernatant (1 × 107 PFU) or as preloaded T(ASEL). () Survival of mice bearing 7-day-old TC2 tumors treated intravenously with VSV-GFP or ASEL on days 7, 9 and 11. On days 25, 27 and 29, mice initially treated with ASEL received intravenous preloaded T(IEEL). () Survival of VSV-vaccinated mice bearing 7-day-old TC2 tumors injected intravenously on days 7, 9 and 11 with VSV-GFP, ASEL or IEEL (1 × 107 PFU), or with T(ASEL) or T(IEEL). On days 20, 22 and 24, surviving mice were treated intravenously with T(IEEL) (T(! ASEL)-T(IEEL) treatment) or ASEL (T(ASEL)-T(ASEL) treatment). (,) IL-17 () and IFN-γ () in splenocytes from mice that rejected TC2 and TC2R tumors after intravenous ASEL-T(IEEL) or T(ASEL)-T(IEEL) treatment as in ,, cocultured with lysates of TC2, TC2R, B16, normal mouse prostate (Pros) or pancreas (Panc) cells. Results are from three survivor mice. (,) IL-17 () and IFN-γ () in splenocytes from mice that did not reject tumors in ,, cocultured with the indicated lysates. Results are from two mice that succumbed to TC2R tumors. Error bars, s.d. of three separate wells per sample. * Figure 6: Immunogenicity of altered-self and self libraries. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8) injected intravenously with 1 × 107 PFU VSV-GFP, ASEL or SEL (days 7, 9 and 11). (–) IL-17 secreted from lymph node cells or splenocytes infected with VSV-GFP, no virus, ASEL or SEL (MOI = 1) for 2 weeks and cocultured with lysates of TC2, B16, normal mouse prostate (Pros), pancreas (Panc) or PBS. Error bars, s.d. of three separate wells per sample. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information Primary authors * These authors contributed equally to this work. * Alan Melcher & * Richard Vile Affiliations * Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA. * Timothy Kottke, * Jose Pulido, * Feorillo Galivo, * Jill Thompson, * Phonphimon Wongthida, * Rosa Maria Diaz & * Richard Vile * Leeds Institute of Molecular Medicine and Cancer Research UK Clinical Centre, St. James' University Hospital, Leeds, UK. * Fiona Errington, * Elizabeth Ilett, * John Chester, * Peter Selby, * Alan Melcher & * Richard Vile * Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, Minnesota, USA. * Jose Pulido * St. George's Hospital Medical School, London, UK. * Heung Chong * University of Surrey, Guildford, UK. * Hardev Pandha * The Institute of Cancer Research, London, UK. * Kevin Harrington * Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA. * Richard Vile Contributions T.K., F.E., J.P., F.G., J.T., P.W., R.M.D., H.C. and E.I. performed experiments; J.P. J.C., H.P., K.H., P.S., A.M. and R.V. conceived the experimental approach, directed the experiments and interpreted the data. H.P., K.H., P.S., A.M. and R.V. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard Vile Author Details * Timothy Kottke Search for this author in: * NPG journals * PubMed * Google Scholar * Fiona Errington Search for this author in: * NPG journals * PubMed * Google Scholar * Jose Pulido Search for this author in: * NPG journals * PubMed * Google Scholar * Feorillo Galivo Search for this author in: * NPG journals * PubMed * Google Scholar * Jill Thompson Search for this author in: * NPG journals * PubMed * Google Scholar * Phonphimon Wongthida Search for this author in: * NPG journals * PubMed * Google Scholar * Rosa Maria Diaz Search for this author in: * NPG journals * PubMed * Google Scholar * Heung Chong Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Ilett Search for this author in: * NPG journals * PubMed * Google Scholar * John Chester Search for this author in: * NPG journals * PubMed * Google Scholar * Hardev Pandha Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Harrington Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Selby Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Melcher Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Vile Contact Richard Vile Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - A Nod toward understanding Crohn's pathology
- Nat Med 17(7):785-787 (2011)
Article preview View full access options Nature Medicine | Article Identification of an innate T helper type 17 response to intestinal bacterial pathogens * Kaoru Geddes1, 5 * Stephen J Rubino2, 5 * Joao G Magalhaes1 * Catherine Streutker3 * Lionel Le Bourhis1 * Joon Ho Cho1 * Susan J Robertson1 * Connie J Kim4 * Rupert Kaul1, 4 * Dana J Philpott1 * Stephen E Girardin2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:837–844Year published:(2011)DOI:doi:10.1038/nm.2391Received23 February 2011Accepted02 May 2011Published online12 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * 日本語要約 * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4+ T helper cells. Enteric innate T helper type 17 (iTH17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103+ dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iTH17 responses. Together, these results identify the Nod-iTH17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iTH17 responses in the developm! ent of inflammatory bowel diseases. Figures at a glance * Figure 1: Nod1 and Nod2 differentially modulate early and late inflammation during C. rodentium-induced colitis. () The degree of colonic histopathology, crypt lengths and bacterial translocation to the spleen assessed in wild-type and Nod1−/−Nod2−/− mice at 7 and 14 d after infection. CFU, colony-forming units. () Representative images (20× magnification) of H&E-stained colon sections of wild-type and Nod1−/−Nod2−/−C. rodentium–infected mice at 7 and 14 d after infection; arrows depict areas of goblet cell depletion and submucosal edema; asterisks depict proximal regions of colon. () Degree of colonic histopathology, crypt length and splenic translocation in lethally irradiated wild-type mice reconstituted with either wild-type (WT→WT) or Nod1−/−Nod2−/− bone marrow (DKO→WT) and Nod1−/−Nod2−/− mice reconstituted with either wild-type (WT→DKO) or Nod1−/−Nod2−/− (DKO→DKO) bone marrow at 12 d after infection. Error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. * Figure 2: Early IL-17 responses during C. rodentium–induced colitis are Nod1 and Nod2 dependent. () Il17a expression in C. rodentium–infected wild-type and Nod1−/−Nod2−/− mice, as quantified by quantitative RT-PCR (qRT-PCR) from the cecum at 4 d (top) and colon at 10 d (bottom) after infection. () Flow cytometry analysis of IL-17A and IL-22 intracellular cytokine staining (ICCS) of cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 4 d), either of all LPLs (left) or CD4+TCRβ+ LPLs (right). () The relative number of CD4+TCRβ+IL-17A+ (TH17) cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 4 d after C. rodentium infection, average of five replicates with three mice pooled per group). () Il22, Lcn2 and Reg3g expression in C. rodentium–infected wild-type and Nod1−/−Nod2−/− mice, as quantified by qRT-PCR in the cecum at 4 d after infection. For qRT-PCR, the average fold change in expression over PBS-treated wild-type mice is shown (n = 10, one representative of two experiments shown). Error bars represent s.e! .m. *P < 0.05. * Figure 3: Acute IL-17 responses during S. typhimurium-induced colitis are dependent on hematopoietic and non-hematopoietic Nod1 and Nod2. () qRT-PCR analysis of Il17a, Il22 and Lcn2 in the cecum of wild-type and Nod1−/−Nod2−/− mice (uninfected or SL1344 infected for 24 h). Bar graphs show average fold change over uninfected controls (n = 6, one representative of three experiments shown). () ICCS analysis of IL-17A and IL-22 in total LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) in cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 24 h after infection with SL1344). () The average relative frequency of all cells, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells in wild-type and Nod1−/−Nod2−/− mice (uninfected or 24 h after infection with SL1344, average of six replicates with three mice pooled per group). () qRT-PCR analysis for Il17a and Il22 on total cells (presort), CD4+ cells, CD11b+CD11c+ cells, and cells remaining after MACS purification (depleted). The bar graphs show fold change in expression over presort cells from uninfected mice (one representative o! f two replicates is shown, six mice pooled per group), and the numbers above the bars represent the fold change between wild-type and Nod1−/−Nod2−/− for each population of cells. () ICCS analysis of IL-17A and IL-22 in CD4+TCRβ+ cecal LPLs from chimeric mice (24 h after infection, one representative of three experiments is shown, three mice pooled per group). Error bars represent s.e.m. *P < 0.05, **P < 0.01. * Figure 4: IL-6 expression during C. rodentium- and S. typhimurium (SL1344)-induced colitis are Nod1 and Nod2 dependent. () Expression of Il6, Il23r and Il23a in the cecum of wild-type and Nod1−/−Nod2−/− mice 4 d after infection with C. rodentium (top) or 24 h after infection with SL1344 (bottom). Average fold change over uninfected controls is shown (n = 6, one representative of three experiments is shown). () Cecal IL-6 amounts in SL1344-infected wild-type and Nod1−/−Nod2−/− mice (24, 48 and 72 h), as measured by ELISA. () Cecal IL-6 amounts in SL1344-infected chimeric mice, as measured by ELISA (n = 6, one representative of three experiments is shown). () qRT-PCR analysis for Il6 on total cells (presort), CD4+ cells, CD11b+CD11c+ cells and cells remaining after MACS purification (depleted). The bar graphs show fold change in expression over presort cells from uninfected mice (one representative of two replicates is shown, six mice pooled per group), and the numbers above the bars represent the fold change between wild type and Nod1−/−Nod2−/− for each population of ce! lls. () Expression of CD103 on either CD11b−CD11c+ cells or CD11b+CD11c+ cecal IELs from wild-type (red) and Nod1−/−Nod2−/− (blue) mice uninfected, infected with C. rodentium for 4 d or infected with SL1344 for 24 h. One representative of three experiments, three mice pooled per group. Error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 5: IL-6 expression during the acute phase of infectious colitis is crucial for TH17 development. () ICCS analysis of IL-17A and IL-22 on total cecal LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) from SL1344-infected wild-type mice (uninfected or 24 h after infection) treated with either control IgG or IL-6–neutralizing antibody (anti–IL-6). (,) Average relative frequency of all IL-17A+, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells from control IgG– or IL-6–neutralizing antibody–treated, SL1344–infected or uninfected wild-type mice () and SL1344-infected or uninfected wild-type and IL-6–knockout mice () (24 h after infection, average of three replicates with three mice pooled per group). () ICCS analysis for IL-17A and IL-22 expression in TCRβ+CD4+ cecal LPLs from C. rodentium–infected (4 d) and SL1344-infected (24 h) chimeric mice that were generated by reconstituting irradiated wild-type mice with either wild-type (WT→WT) or Il6−/− (Il6−/−→WT) bone marrow. Dot plots depict one representative of three experiments with two mi! ce pooled per group. Error bars represent s.e.m. *P < 0.05, **P < 0.01. * Figure 6: Early TH17 cells express memory surface markers and require microbiota for activation. () Expression of CD44, CD62L, CD69 and CCR6 on either all CD4+TCRβ+ cells or CD4+TCRβ+IL-17A+ cells in cecal LPLs from SL1344-infected mice (top) or expression of these cell surface markers on CD4+TCRβ+IL-17A+ cells from the LPLs of uninfected and SL1344-infected mice (24 h after infection) (bottom). () Mean fluorescence intensity (MFI) of CD69 expression on LPLs from uninfected or SL1344-infected mice (average of three replicates with three mice pooled per group). () ICCS analysis of IL-17A and IL-22 in total cecal LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) from SL1344-infected SPF and germ-free mice (uninfected or 24 h after infection). () Average relative frequency of all IL-17A+, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells from SL1344-infected SPF and germ-free mice (uninfected or 24 h after infection) (uninfected group had two replicates, infected group had three replicates, three mice pooled per group). Error bars represent s.e.m. *P < 0.05, **P! < 0.01. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Kaoru Geddes & * Stephen J Rubino Affiliations * Department of Immunology, University of Toronto, Toronto, Ontario, Canada. * Kaoru Geddes, * Joao G Magalhaes, * Lionel Le Bourhis, * Joon Ho Cho, * Susan J Robertson, * Rupert Kaul & * Dana J Philpott * Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. * Stephen J Rubino & * Stephen E Girardin * Department of Laboratory Medicine, St. Michael's Hospital, Toronto, Ontario, Canada. * Catherine Streutker * Department of Medicine, University of Toronto, Toronto, Ontario, Canada. * Connie J Kim & * Rupert Kaul Contributions K.G. and S.J.R. designed and performed all experiments and wrote the manuscript. J.G.M. designed and performed mouse experiments. C.S. performed pathological scoring analysis. L.L.B. generated the Nod1−/−Nod2−/− mice. J.H.C. and S.J.R. performed microbiota analysis. C.J.K. and R.K. provided human colonic samples. D.J.P. and S.E.G. directed the research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Dana J Philpott or * Stephen E Girardin Author Details * Kaoru Geddes Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen J Rubino Search for this author in: * NPG journals * PubMed * Google Scholar * Joao G Magalhaes Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Streutker Search for this author in: * NPG journals * PubMed * Google Scholar * Lionel Le Bourhis Search for this author in: * NPG journals * PubMed * Google Scholar * Joon Ho Cho Search for this author in: * NPG journals * PubMed * Google Scholar * Susan J Robertson Search for this author in: * NPG journals * PubMed * Google Scholar * Connie J Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Rupert Kaul Search for this author in: * NPG journals * PubMed * Google Scholar * Dana J Philpott Contact Dana J Philpott Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen E Girardin Contact Stephen E Girardin Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 and Supplementary Methods Additional data - Lung stem cells: looking beyond the hype
- Nat Med 17(7):788-789 (2011)
Article preview View full access options Nature Medicine | Community Corner Lung stem cells: looking beyond the hype Journal name:Nature MedicineVolume: 17,Pages:788–789Year published:(2011)DOI:doi:10.1038/nm0711-788Published online07 July 2011 3D4Medical / Photo Researchers, Inc. The human lung is known to contain regional pools of progenitor cells that can give rise to specific lung structures, but a multipotent stem cell that can give rise to both endodermal and mesodermal lineages has not been described before and was thought to not exist. Jan Kajstura et al.1 now provide evidence that might overturn this long-held belief. The investigators identified a set of potential stem cells in human lungs that were self renewing, clonogenic and multipotent in vitro and that could regenerate many different lung components—including bronchioles, alveoli, smooth muscle and pulmonary vessels—when injected into a mouse model of lung injury. We asked three experts to discuss the relevance of these findings to understanding of lung development and the implications for regenerative medicine to treat lung disease. Barry Stripp "This study requires careful scrutiny and rigorous validation before its claims can be accepted." The recent article by Kajstura et al.1 suggests that multipotent stem cells can be cultured from human lung tissue and that these cells have the capacity to differentiate into multiple endodermal and mesodermal lineages in vitro or after grafting into damaged lung tissue of mice. If correct, these potentially paradigm-changing observations have substantial implications for policy decisions that could ultimately affect the care of people with lung disease. For scientists and physicians interested in the fundamentals of lung biology and the development of new cures for debilitating lung diseases, this study requires careful scrutiny and rigorous validation before its claims can be accepted. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Competing financial interests The author declares no competing financial interests. Additional data - Pulling down the plug on atherosclerosis: Cooling down the inflammasome
- Nat Med 17(7):790-791 (2011)
Nature Medicine | Between Bedside and Bench Pulling down the plug on atherosclerosis: Cooling down the inflammasome * Göran K Hansson1 * Lars Klareskog1 * Affiliations * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:790–791Year published:(2011)DOI:doi:10.1038/nm0711-790Published online07 July 2011 Atherosclerotic lesions can result in fatal cardiovascular disease, but what triggers the formation of the atheroma plaques and their progression still begs further investigation. In 'Bench to Bedside', Göran K Hansson and Lars Klareskog peruse how the NLRP3 inflammasome can be activated by cholesterol crystals and worsen atherosclerosis by triggering inflammation through the release of IL-1β from macrophages. But these cells can also die at the lesion site, forming a necrotic core in the atheroma by building up apoptotic cells and debris. In 'Bedside to Bench', Ira Tabas discusses a human study showing that lesional necrosis along with thinning of the fibrotic cap are predictive of culprit lesions involved in fatal disease. Understanding the molecular underpinnings of these two morphological features may lead to new therapies to prevent or decrease the risk for major cardiovascular disease. 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 * Göran K. Hansson and Lars Klareskog are in the Department of Medicine and Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Göran K Hansson or * Lars Klareskog Author Details * Göran K Hansson Contact Göran K Hansson Search for this author in: * NPG journals * PubMed * Google Scholar * Lars Klareskog Contact Lars Klareskog Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Pulling down the plug on atherosclerosis: Finding the culprit in your heart
- Nat Med 17(7):791-793 (2011)
Article preview View full access options Nature Medicine | Between Bedside and Bench Pulling down the plug on atherosclerosis: Finding the culprit in your heart * Ira Tabas1Journal name:Nature MedicineVolume: 17,Pages:791–793Year published:(2011)DOI:doi:10.1038/nm0711-791Published online07 July 2011 Bedside to bench Cardiovascular disease, the leading cause of death worldwide, is caused by atherothrombotic vascular occlusion, which leads to unstable angina (progressive ischemia of heart muscle), heart attacks, sudden cardiac death and stroke1. Atherothrombosis is a focal pathological process in medium-sized arteries, notably those that feed the heart and brain, and comprises two distinct stages. The first involves a decades-long accumulation of lipids, macrophages and other inflammatory cells, and extracellular matrix in the subendothelial space, or intima, of the blood vessel wall. This process alone does not cause acute cardiovascular events, because blood flow is preserved through outward remodeling of the arterial wall or, in the setting of gradual lumenal encroachment, new vessel formation2. However, in a small percentage of lesions, acute, occlusive lumenal thrombosis is triggered, leading to ischemia or death of distal organ tissue. When these disease-causing lesions are identified by imaging techniques or at autopsy, they are termed 'culprit lesions' because they are deemed responsible for causing the arterial occlusion. But what is different about the lesions that progress to the point that they cause these acute events and end-organ damage compared to those that do not? This question was addressed in a unique clinical study by Stone et al.3, which reports findings that have important implications for basic research into culprit lesion formation. Autopsy studies of subjects that die of an acute atherothrombotic vascular event, such as a massive heart attack, have found that culprit lesions are distinguished not by lesion size but rather by several distinct morphological features, including collections of dead cells, often referred to as 'necrotic cores', and erosion or rupture of the scar tissue, or 'fibrous cap', that overlies the lesion4. But these studies are retrospective and sample only one point in time and thus cannot be used to draw firm conclusions on the process of culprit lesion formation. Prospective analysis would require serial 'sampling' of lesions during a period in which new atherothrombotic vascular events occur. Although serial tissue sampling in humans is not possible, serial imaging of coronary arteries is feasible. In this context, Stone et al.3 studied individuals who suffered an acute cardiac event and thus required a coronary artery catheterization procedure to reestablish lumen patency at the site of the culprit lesion. During this therapeutic procedure, the investigators imaged many nonculprit lesions in the coronary artery circulation by combining serial angiography with a technique called intravascular ultrasound (IVUS). After a median follow-up of ~3 years, an acute atherothrombotic clinical event occurred in 135 individuals, who then underwent repeat angiography that identified the sites of the new thrombosis. Through comparisons with the earlier images, the authors could identify the earlier lesions that gave rise to these new culprit lesions. As expected, some of the new events were caused by recurrence at the sites of the original culprit lesions. But many events occurred at sites that several years previously were nonculprit lesions. The authors asked whether any features of these 'preculprit' lesions predicted clinical progression. Consistent with previous data, most lesions that progressed did not show marked lumenal narrowing at the earlier time point, but two features, large necrotic cores and thin fibrous caps—which together characterize 'thin-cap fibroatheroma'—were highly predictive of progression to culprit lesions and the only independent risk factor for major cardiovascular events (Fig. 1). Figure 1: Features of a culprit atherosclerotic lesion. Lesions that are identified post hoc as having caused an acute atherothrombotic event have three key features—a large necrotic core, a ruptured or eroded fibrous cap and an overlying occlusive thrombus. The necrotic core is formed by progressive death of macrophage foam cells, accompanied by defective clearance (efferocytosis) of the dead cells by nearby living macrophages or dendritic cells (DCs). The necrotic core, together with inflammatory macrophages and possibly death of collagen-producing smooth muscle cells, leads to thinning and then rupture or erosion of the fibrous cap. The breach in the cap exposes lesional thrombogenic material to the lumen, which can cause acute, occlusive thrombosis and ischemia or infarction of distal myocardial tissue. Earlier, nonculprit lesions that have two key features—a large necrotic core and thinning of the fibrous cap—have a higher probability of progressing to the culprit stage compared with lesions without these features and ! thus are termed 'vulnerable plaques'. Katie Vicari * Full size image (214 KB) How do these findings open new avenues in basic research in this area and help us identify new molecular targets for potential therapeutic intervention? Atherosclerosis consists of numerous, heterogeneous cell biological processes, and different lesions in the same individual vary in terms of which processes are dominant. For example, most lesions have many cholesterol-loaded macrophage foam cells, others have fibrous tissue as the major feature and still others have the dangerous features identified in the above study. Initial lesion formation and progression of mostly early- to mid-stage lesions can be prevented, or even regressed, by lowering the concentration of plasma atherogenic lipoproteins5. But can more specific therapy be directed at potential time bombs such as the lesions with the highest potential to progress to the culprit stage in high-risk patients? Knowing which lesions among the heterogeneous mix are most likely to progress and the mechanisms involved is cr! ucial, considering that, on average, less than 5% of lesions in individuals at risk progress to the culprit stage4. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Author information Article tools * Print * Email * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Ira Tabas is in the Department of Medicine, the Department of Pathology and Cell Biology and the Department of Physiology and Cellular Biophysics, Columbia University, New York, New York, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Ira Tabas Author Details * Ira Tabas Contact Ira Tabas Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Research Highlights
- Nat Med 17(7):794-795 (2011)
Article preview View full access options Nature Medicine | Research Highlights Research Highlights Journal name:Nature MedicineVolume: 17,Pages:794–795Year published:(2011)DOI:doi:10.1038/nm0711-794Published online07 July 2011 Neuroscience: Dampening drug actions Drugs that are taken together can affect how each drug is metabolized or how efficacious a drug may be. Now, Jennifer L. Warner-Schmidt et al. report that nonsteroidal anti-inflammatory drug (NSAID) use can reduce the efficacy of selective serotonin reuptake inhibitor (SSRI) antidepressant drugs in mice and humans (Proc. Natl. Acad. Sci. USA, 9262–9267). SSRIs induce antidepressant-like behaviors in mice, causing, for example, a reduction of immobility when mice are suspended by their tails. The researchers found that SSRIs are less efficacious in affecting these behaviors when the mice are also treated with NSAIDs. In a human study, the researchers found that depressed subjects taking both SSRIs and NSAIDs were less likely to report relief from depression than subjects off these anti-inflammatory drugs. Warner-Schmidt et al. found that SSRIs increase the abundance of six cytokines, including interferon-γ (IFN-γ), in the cortex of treated mice, and these increases were abolished when mice were also treated with the NSAID ibuprofen. IFN-γ treatment of mice led to antidepressant-like effects, and SSRIs were not as capable of inducing antidepressant effects in mice in which IFN-γ signaling was abolished. These findings suggest that cytokines may mediate the antidepressant effects of SSRIs and that depressed individuals should probably avoid taking SSRIs and NSAIDs at the same time. —EC Immunology: Treg cell bonanza Three studies in Science Translational Medicine report new methods to generate ex vivo regulatory T cells (Treg cells), immune cells capable of suppressing immune responses. Because of their ability to control other immune cells, Treg cells may have therapeutic potential against conditions characterized by inappropriate immune responses, such as graft-versus-host disease (GVHD), transplant rejection and autoimmune diseases. But their potential is limited because they cannot be isolated in sufficient numbers to be used therapeutically. The three new studies describe different ways to overcome this problem. Gang Feng et al. (Sci. Transl. Med., 83ra40) found that exposing mouse and human CD4+ T cells to dendritic cells and inhibiting cyclic nucleotide phosphodiesterase 3 generated a large number of Treg cells. The authors then used these cells to prevent immune rejection in models of skin and vascular grafts. Keli Hippen et al. (Sci. Transl. Med., 83ra41) had previously used umbilical cord blood to obtain Treg cells, but the yield was low. They now derived Treg cells from peripheral blood and used a cocktail of cytokines to expand the cells. After stimulation with genetically engineered antigen-presenting cells, the resulting Treg cells were therapeutically useful in a model of GVHD. Last, Pervinder Sagoo et al. (Sci. Transl. Med., 83ra42) tackled another problem that hampers the therapeutic use of Treg cells—specificity. In most cases of immune pathology, some immune responses are beneficial, and some are harmful. If Treg cells are to work in the clinic, they should suppress only the harmful ones, which often target specific alloantigens. Sagoo et al. established a method to isolate alloantigenic Treg cells on the basis of the expression of CD69 and CD71. Once pure, the expanded cells reduced rejection in a skin graft model. As early clinical trials using Treg cells are under way, the methods reported in these studies could aid the design of large-scale trials for various immune pathologies. —JCL Infection: Alternative inflammation pathways In response to infection or injury, leukocytes are recruited from the blood into the affected tissue. In type 1 inflammation, these monocytes differentiate into inflammatory macrophages, whereas helminth infection and allergy are associated with type 2 inflammation and differentiation of monocytes into alternatively activated macrophages. Stephen Jenkins et al. (Science, 1284) now show that during type 2 inflammation, the increase in tissue macrophages occurs through proliferation of resident tissue macrophages rather than recruitment of monocytes from the blood. The authors used Litomosoides sigmodontis, a rodent filarial nematode, to model type 2 inflammation in mice. Depletion of circulating monocytes reduced accumulation of tissue macrophages in a model of type 1 inflammation but had no effect on macrophage accumulation after helminth infection. Instead, with helminth infection, macrophage numbers increased through local proliferation in situ and this was driven by interleukin-4 (IL-4). Infection of IL-4–deficient mice with L. sigmodontis decreased the number of proliferating macrophages, whereas exogenous IL-4 increased macrophage proliferation. Indeed, tissue macrophages recruited to the tissue by a type 1 inflammatory insult can also proliferate in response to IL-4, suggesting that both resident and recruited macrophages can be activated to proliferate by this cytokine. Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Medicine for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * Rent this article from DeepDyve * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data - The immunology of stroke: from mechanisms to translation
- Nat Med 17(7):796-808 (2011)
Nature Medicine | Review The immunology of stroke: from mechanisms to translation * Costantino Iadecola1 * Josef Anrather1 * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:796–808Year published:(2011)DOI:doi:10.1038/nm.2399Published online07 July 2011 Immunity and inflammation are key elements of the pathobiology of stroke, a devastating illness second only to cardiac ischemia as a cause of death worldwide. The immune system participates in the brain damage produced by ischemia, and the damaged brain, in turn, exerts an immunosuppressive effect that promotes fatal infections that threaten the survival of people after stroke. Inflammatory signaling is involved in all stages of the ischemic cascade, from the early damaging events triggered by arterial occlusion to the late regenerative processes underlying post-ischemic tissue repair. Recent developments have revealed that stroke engages both innate and adaptive immunity. But adaptive immunity triggered by newly exposed brain antigens does not have an impact on the acute phase of the damage. Nevertheless, modulation of adaptive immunity exerts a remarkable protective effect on the ischemic brain and offers the prospect of new stroke therapies. As immunomodulation is not dev! oid of deleterious side effects, a better understanding of the reciprocal interaction between the immune system and the ischemic brain is essential to harness the full therapeutic potential of the immunology of stroke. View full text Figures at a glance * Figure 1: Early vascular, perivascular and parenchymal events triggered by ischemia and reperfusion. Hypoxia, ROS and changes in shear stress initiate the cellular events induced by ischemia and reperfusion. In the vessels lumen, ischemia and reperfusion lead to blood clotting, platelet aggregation and cytokine (IL-1α) release. Translocation of P-selectin on the surface of platelets and endothelial cells leads to platelet-leukocyte aggregation. Complement is activated, and arachidonic acid metabolites (AA) are released. In the vascular wall, upregulation of E- and P-selectin on endothelial cells provides a platform for low affinity leukocyte binding through interaction with glycoproteins expressed on leukocytes, for example, P-selectin glycoprotein ligand-1. Firm adhesion is obtained after endothelial expression of ICAM-1 interacting with leukocyte β2 integrins (LFA-1 and Mac-1). Loss of NO promotes vasoconstriction and enhances leukocyte and platelet aggregation. MMP activation could lead to BBB breakdown and matrix proteolysis, facilitating leukocyte extravasation. In t! he perivascular space, chemotactic complement subunits (C5a) acting on mast cell complement receptors (CD88) leads to degranulation and release of histamine and proteases, contributing to BBB leakiness. Cytokines (TNF, IL-1β) are produced by mast cells and perivascular macrophages, providing further signals to guide leukocyte migration across the vessel wall. In the brain parenchyma, injured cells release purines (ATP), which act as early proinflammatory signals leading to production of cytokines and chemokines. Disruption of neuronal-microglial interaction (CX3CL1, CD200) and increases in extracellular glutamate (Glu) acting on microglial GluR1 metabotropic receptor27 also contribute to the proinflammatory milieu. * Figure 2: Cell death and activation of pattern recognition receptors set the stage adaptive immunity. Release of nucleotides (ATP, UTP) from injured cells, including neurons, activates purinergic receptors on microglia and macrophages and leads to production of proinflammatory cytokines25. Although most of these cytokines are transcriptionally induced, IL-1β and IL-18 are processed from their propeptides by the activity of interleukin-1–converting enzyme (ICE; caspase 1). ICE is embedded in a multiprotein complex (NLRP3, or inflammasome) and is activated by microglial P2X7 receptors108. Ischemic cell death leads to the formation of DAMPs, which activate TLRs, especially TLR2 and TLR4 (ref. 21). DAMPs released by ischemia include high-mobility group protein B1, an intracellular DNA binding protein released after cellular injury, heat shock protein 60 and β-amyloid (Aβ), among others30. TLRs, in concert with scavenger receptors such as CD36, upregulate proinflammatory gene expression through the transcription factor nuclear factor-KB30, 109. DAMPs are also derived from ma! trix breakdown by lytic enzymes released from dead cells and by the action of ROS on lipids. The cytokine production and complement activation resulting from these events leads to increased leukocyte infiltration and enhances tissue damage, which, in turn, produces more DAMPs. Antigens unveiled by tissue damage are presented to T cells, setting the stage for adaptive immunity. * Figure 3: Deleterious and beneficial roles of T cells in stroke. In the acute phase of cerebral ischemia, unprimed T cells contribute to tissue damage in an antigen-independent manner (innate immunity), possibly through IFN-γ110 and ROS111 (top left). γδT cells, activated by IL-23 released from microglia and macrophages, produce the cytotoxic cytokine IL-17 and contribute to acute ischemic brain injury41. However, T cells can also be protective. TGF-β produced by neurons, glia, or microglia and macrophages promotes the development of Treg cells secreting the protective cytokine IL-10 and inhibits TH1 and TH2 responses. Treg cells are protective in models of cerebral ischemia42. Induction of mucosal tolerance with CNS antigens produces an adaptive response, which leads to the establishment of autoreactive TH2 cells producing IL-10 (ref. 48) and Treg cells producing IL-10 and TGF-β107 is highly protective in experimental stroke (bottom right). Although there is no evidence that adaptive immunity contributes to acute ischemic brain inju! ry, weeks and months after stroke, autoreactive CD4+ and CD8+ T cells targeting CNS antigens could develop (top right). The resulting cell death could play a part in the delayed brain damage and atrophy that occur after stroke83. * Figure 4: Resolution of inflammation and tissue repair. Clearing of dead cells and suppression of inflammation are key events in brain repair. Find-me signals (UTP, ATP) attract microglia and macrophages through P2Y2 receptors. Eat-me signals include UDP, which acts on P2Y6 receptors to stimulate microglial phagocytosis112, and phosphatidylserine (PtdSer), which is translocated to the outer leaflet of the plasma membrane of apoptotic cells112. PtdSer-binding proteins involved in the clearance of dead cells include milk fat globule epidermal growth factor 8 protein on microglia113 and T cell immunoglobulin and mucin domain-containing molecule 4 (TIM4) on macrophages112. Immunoglobulins directed against CNS antigens, which appear after stroke (Table 2), may also promote phagocytosis by engaging Fc receptors on phagocytic cells. Phagocytosis promotes secretion of IL-10 and TGF-β56, which, in turn, suppress antigen presentation, promote Treg formation, inhibit expression of adhesion molecules in endothelial cells and production of p! roinflammatory cytokines61. TGF-β and IL-10 are also neuroprotective114, 115 and may facilitate brain repair processes. In addition, lipoxins, resolvins and protectins, metabolites of arachidonic acid and omega-3 fatty acids that play an active part in the resolution of inflammation in other organs55, could also contribute to suppress post-ischemic inflammation. Growth factors and MMPs produced by endothelial cells, neurons, astrocytes, oligodendrocytes and microglia are key molecules driving tissue reorganization and repair63, 116. Author information * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Division of Neurobiology, Department of Neurology and Neuroscience, Weill Cornell Medical College, New York, New York, USA. * Costantino Iadecola & * Josef Anrather Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Costantino Iadecola Author Details * Costantino Iadecola Contact Costantino Iadecola Search for this author in: * NPG journals * PubMed * Google Scholar * Josef Anrather Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Table 1 Additional data - A pharmacological approach to first aid treatment for snakebite
- Nat Med 17(7):809-811 (2011)
Nature Medicine | Brief Communication A pharmacological approach to first aid treatment for snakebite * Megan E Saul1 * Paul A Thomas2 * Peter J Dosen3 * Geoffrey K Isbister4, 5 * Margaret A O'Leary5 * Ian M Whyte4, 5 * Sally A McFadden6 * Dirk F van Helden3 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:809–811Year published:(2011)DOI:doi:10.1038/nm.2382Received11 February 2011Accepted19 April 2011Published online26 June 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Snake venom toxins first transit the lymphatic system before entering the bloodstream. Ointment containing a nitric oxide donor, which impedes the intrinsic lymphatic pump, prolonged lymph transit time in rats and humans and also increased rat survival time after injection of venom. This pharmacological approach should give snakebite victims more time to obtain medical care and antivenom treatment. View full text Figures at a glance * Figure 1: Effects of topical application of GTNO on lymphatic transit times. Foot-to-groin lymphatic transit times in each of 15 human subjects with and without GTNO treatment and the mean ± s.e.m. transit time (n = 15; left). Foot-to-groin lymphatic transit times in rats (mean ± s.e.m.) treated with GTNO (n = 16) or control base ointment (right; n = 13; right-hand y axis applies to the rat data only). * Figure 2: Effects of GTNO treatment on venom actions in anesthetized rats. () Kaplan-Meier plot showing GTNO effects on the time to respiratory arrest after subcutaneous venom injection in the rat hind foot (n = 14 control; n = 19 GTNO). () Respiratory rate over time (mean ± s.e.m.). () 405-nm optical absorbance (relative units, RU) of plasma over time (mean ± s.e.m.) in control- (n = 6) and GTNO–treated rats (n = 7). Data comparison was made using two-way repeated-measures analysis of variance with Holm-Sidak post hoc tests (*P < 0.05, **P < 0.01, ***P < 0.001). Comparisons between venom + base ointment and venom + GTNO to the right of the dashed lines in and are all significantly different. Author information * Author information * Supplementary information Affiliations * Department of Nuclear Medicine, John Hunter Hospital, New Lambton, New South Wales, Australia. * Megan E Saul * Department of Nuclear Medicine, Royal Brisbane and Women's Hospital, Herston, Queensland, Australia. * Paul A Thomas * School of Biomedical Sciences & Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia. * Peter J Dosen & * Dirk F van Helden * School of Medicine and Public Health, University of Newcastle, Callaghan, New South Wales, Australia. * Geoffrey K Isbister & * Ian M Whyte * Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle, Waratah, New South Wales, Australia. * Geoffrey K Isbister, * Margaret A O'Leary & * Ian M Whyte * School of Psychology, University of Newcastle, Callaghan, New South Wales, Australia. * Sally A McFadden Contributions M.E.S. conducted the human experiments. P.A.T. supervised the human experiments. P.J.D. conducted the rat experiments. G.K.I. provided expertise on rat experiments, supervised assays and assisted in drafting the manuscript and statistical advice. M.A.O. undertook the absorbance assays. I.M.W. provided input on the human experiments. S.A.M. undertook the statistical analysis and figure presentations. D.F.v.H. conceived of and directed the overall project, supervised the rat experiments, analyzed data and drafted the manuscript. Competing financial interests D.F.v.H. has an Australian patent application (no. 2007327538) for the use of nitric oxide donors as a first aid for treatment of venomous bites. Corresponding author Correspondence to: * Dirk F van Helden Author Details * Megan E Saul Search for this author in: * NPG journals * PubMed * Google Scholar * Paul A Thomas Search for this author in: * NPG journals * PubMed * Google Scholar * Peter J Dosen Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey K Isbister Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret A O'Leary Search for this author in: * NPG journals * PubMed * Google Scholar * Ian M Whyte Search for this author in: * NPG journals * PubMed * Google Scholar * Sally A McFadden Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk F van Helden Contact Dirk F van Helden Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (803K) Supplementary Figure 1 and Supplementary Methods Additional data - The transcription factor cyclic AMP–responsive element–binding protein H regulates triglyceride metabolism
- Nat Med 17(7):812-815 (2011)
Nature Medicine | Brief Communication The transcription factor cyclic AMP–responsive element–binding protein H regulates triglyceride metabolism * Jung Hoon Lee1 * Petros Giannikopoulos1 * Stephen A Duncan2 * Jian Wang3 * Christopher T Johansen3 * Jonathan D Brown4 * Jorge Plutzky4 * Robert A Hegele3 * Laurie H Glimcher1, 5, 6 * Ann-Hwee Lee1, 5 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:812–815Year published:(2011)DOI:doi:10.1038/nm.2347Received30 December 2010Accepted07 March 2011Published online12 June 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Here we report that the transcription factor cyclic AMP–responsive element–binding protein H (CREB-H, encoded by CREB3L3) is required for the maintenance of normal plasma triglyceride concentrations. CREB-H–deficient mice showed hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators Apoc2, Apoa4 and Apoa5 (encoding apolipoproteins C2, A4 and A5, respectively) and concurrent augmentation of the Lpl inhibitor Apoc3. We identified multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein in humans with extreme hypertriglyceridemia, implying a crucial role for CREB-H in human triglyceride metabolism. View full text Figures at a glance * Figure 1: Creb3l3−/− mice show hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by Lpl. () Plasma triglyceride (TG), FFA and cholesterol concentrations measured after a 16-h fast. Each dot represents an individual mouse. () We separated lipoproteins by density gradient ultracentrifugation of pooled plasma (n = 3 per each) after a 24-h fast. Depicted are the triglyceride concentrations in and density of fractions (1 ml each) sequentially collected from top to bottom. () Plasma triglyceride concentrations in WT (n = 9) and Creb3l3−/− (n = 7) male mice deprived of food, starting at 8 a.m. () Hepatic triglyceride abundance measured in the fed state or after a 24-h fast. n = 5 per group. () After concentration, fractions from were separated on 4–20% gradient SDS-polyacrylamide gels. The gel was stained by Coomassie Brilliant Blue G-250. Arrowhead indicates Apoc3 identified by mass spectrometry. VLDL, density (d) < 1.006 g ml−1; IDL, d = 1.006–1.019 g ml−1; LDL, d = 1.019–1.063 g ml−1; HDL, d = 1.063–1.21 g ml−1. () Western blot for Apoc3 in VLDL ! fractions. () Plasma triglyceride concentrations measured after a 4-h fast followed by intravenous injection with tyloxapol (500 mg per kg body weight). n = 4 per group. () Plasma triglyceride concentrations measured after a 16-h fasting followed by oral gavage of olive oil (10 ml kg−1). n = 6 per group. () Lpl activity after heparin treatment (n = 6 per group) measured in the presence of heat-inactivated serum pooled from WT or Creb3l3−/− mice (used as Apoc sources). () Recombinant LPL was incubated with triolein substrate in the presence of plasma collected from WT or Creb3l3−/− mice. Values represent FFA concentration released from triolein. n = 8 per group. Values are means ± s.e.m.; *P < 0.05 and ***P < 0.0001 compared to WT mice. * Figure 2: CREB-H controls genes involved in triglyceride metabolism, and mutations of CREB3L3 are associated with human hypertriglyceridemia. () Hepatic mRNA levels determined by qRT-PCR. Mice were fasted for 24 h before killing. n = 3 mice per group. () Apoc2, Apoa4, Apoa5 and Fgf21 mRNA levels at fed state or after a 24-h fast in WT and Creb3l3−/− mice, as determined by qRT-PCR. n = 4 mice per group. () Locations of the nonsynonymous mutations found in individuals with hypertriglyceridemia, and predicted amino acid changes. bZIP, basic leucine zipper; TM, transmembrane domain. () Hepa1.6 cells concurrently transfected with Apoa4, Apoc2 or Fgf21 promoter-driven luciferase reporters and the indicated CREB-H(N) constructs. Values represent fold induction of luciferase activities compared to the reporter-only transfection. Values are means ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.0001. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE29643 Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA. * Jung Hoon Lee, * Petros Giannikopoulos, * Laurie H Glimcher & * Ann-Hwee Lee * Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. * Stephen A Duncan * Robarts Research Institute and Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada. * Jian Wang, * Christopher T Johansen & * Robert A Hegele * Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. * Jonathan D Brown & * Jorge Plutzky * Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. * Laurie H Glimcher & * Ann-Hwee Lee * Ragon Institute of MGH, MIT and Harvard, Harvard Medical School, Boston, Massachusetts, USA. * Laurie H Glimcher Contributions A.-H.L. designed the experiments. J.H.L. performed in vivo experiments in Creb3l3−/− mice and mutational analysis of CREB-H protein. A.-H.L. and P.G. generated and characterized CREB-H(N)-transgenic mice. J.D.B. performed post-heparin LPL assays. J.W. and C.T.J. performed sequencing experiments. S.A.D. provided Creb3l3−/− mice. A.-H.L., L.H.G., R.A.H. and J.P. analyzed the data. A.-H.L., L.H.G. and R.A.H. wrote the manuscript. Competing financial interests L.H.G. holds equity in and is on the corporate board of directors of Bristol-Myers Squibb. Corresponding authors Correspondence to: * Laurie H Glimcher or * Ann-Hwee Lee Author Details * Jung Hoon Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Petros Giannikopoulos Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen A Duncan Search for this author in: * NPG journals * PubMed * Google Scholar * Jian Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher T Johansen Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan D Brown Search for this author in: * NPG journals * PubMed * Google Scholar * Jorge Plutzky Search for this author in: * NPG journals * PubMed * Google Scholar * Robert A Hegele Search for this author in: * NPG journals * PubMed * Google Scholar * Laurie H Glimcher Contact Laurie H Glimcher Search for this author in: * NPG journals * PubMed * Google Scholar * Ann-Hwee Lee Contact Ann-Hwee Lee Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5, Supplementary Tables 1 and 2 and Supplementary Methods Additional data - Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination
- Nat Med 17(7):816-821 (2011)
Nature Medicine | Article Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination * Sha Mi1 * Xinhua Lee1 * Yinghui Hu1 * Benxiu Ji1 * Zhaohui Shao1 * Weixing Yang1 * Guanrong Huang1 * Lee Walus1 * Kenneth Rhodes1 * Bang Jian Gong1 * Robert H Miller2 * R Blake Pepinsky1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:816–821Year published:(2011)DOI:doi:10.1038/nm.2373Received27 December 2010Accepted06 April 2011Published online06 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal r! ole for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis. View full text Figures at a glance * Figure 1: DR6 is expressed in oligodendrocytes. () RT-PCR quantification of relative DR6 mRNA expression in rat brain at different development stages (mRNA level in sample E18 = 1). () Western blot analysis of DR6 protein expression in rat brain at different development ages. () In situ hybridization analysis of DR6 mRNA expression in adult rat corpus callosum sections. Red, probed with DR6 antisense mRNA; green, stained with antibody to O4; yellow, merge of red and green. The arrowheads indicate O4+DR6+ cells, and blue is DAPI staining. Scale bars, 15 μm. () Immunocytochemical analysis of DR6 protein expression in oligodendrocytes. Red, stained with antibody to DR6; green, stained with antibodies to A2B5, PDGFRα and MBP; yellow, merge of red and green. Scale bars, 95 μm. () Western blot analysis of DR6 protein expression in A2B5+, PDGFRα+ and MBP+ oligodendrocyte cultures. β-actin expression was analyzed from the same samples as an internal control. () Quantification of DR6 protein expression from . Data are shown a! s means ± s.e.m. * Figure 2: DR6 antagonists promote A2B5+ OPC survival and differentiation. () Western blot analysis of MBP, MOG, cleaved casp3 and DR6 proteins in oligodendrocytes after treatment with DR6 or control siRNAs. β-actin expression was analyzed from the same samples as an internal control. () Western blot analysis of MBP, cleaved casp3 proteins in DR6 DN and DR6 FL lentivirus-infected A2B5+ OPC cultures. GFP expression was analyzed from the same samples as an internal control for lentivirus infection. () Quantification of percentage cleaved casp3+ oligodendrocytes after treatment with DR6 DN, DR6 FL and control virus. () Quantification of cleaved casp3+ cells in DR6 FL and control oligodendrocytes after treatment with casp3 and casp6 inhibitors. () Western blot analysis of cleaved casp3, MBP and MAG proteins in oligodendrocytes after treatment with N-APP or buffer control. () P2 oligodendrocytes from DR6 WT and DR6-null mice cultures stained with antibody to MBP. Scale bars, 25 μm. () Quantification of MBP+ mature oligodendrocytes in . () Quantificati! on of cleaved casp3+ oligodendrocytes from DR6 WT and null mice in P2 oligodendrocyte cultures. P values in and were determined by one-way analysis of variance (ANOVA followed by Tukey's test), and in and using the unpaired t test. β-actin expression was analyzed from the same samples as an internal control for all western blots. Data are shown as means ± s.e.m. * Figure 3: Inhibition of DR6 promotes oligodendrocyte survival, maturation and myelination. () Western blot analysis of MBP expression after P2 OPC treatment with DR6 antibodies (10 μg ml−1). () Immonocytochemical analysis to visualize MBP+ and cleaved casp3+ apoptotic oligodendrocytes after 5D10 or control isotype antibody treatment. Arrowheads indicate Casp3+ cells. Scale bars, 100 μm. () Quantification of MBP+ oligodendrocytes and cleaved casp3+ oligodendrocytes from . () Western blot analysis of cleaved casp3 and MBP proteins in 5D10- and control IgG–treated OPC cultures. () Immunocytochemical visualization of myelination in 5D10-, DR6 DN- and DR6 siRNA-treated DRG-OPC cocultures and the corresponding control-treated cocultures. Scale bars, 50 μm. Red, MBP staining. () Quantitative analysis of immunocytochemical staining of MBP+ myelinated axon clusters from . () Western blot analysis of MBP and MOG in cocultures treated with 5D10, DR6 FL and DR6 DN. () Western blot analysis of MBP, MAG and DR6 protein from WT and DR6-null mice cocultures. β-actin expre! ssion was analyzed from the same samples in and as an internal control. () Quantification of MBP+ axons from WT and DR6-null cocultures. P values in , and were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 4: Antibody to DR6 promotes functional recovery and remyelination in the rat EAE model. () EAE clinical score measurement after 5D10 and control antibody treatment. Arrows indicate treatment regimen. () Descending nerve conduction velocity measurements after 5D10 and control antibody treatment. () Electron microscopy analysis of remyelination in EAE rat spinal cords after 5D10 and control antibody treatments and in WT and DR6-null EAE mouse spinal cords. Remyelination is denoted by blue asterisks in lower-magnification images and red asterisks in higher-magnification images. () Quantification of myelinated axons from . P values were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 5: A DR6 antagonist promotes remyelination in brain slice cultures and in the LPC-induced spinal cord demyelination model. () Black gold staining (red) of myelinated axons in LPC-treated corpus callosum brain slice cultures. Brain slices were demyelinated by LPC followed by 5D10 and control IgG treatments for 3 d. Remyelination was visualized by light microscopy. Scale bar, 200 μm. () Quantification of myelin protein MAG by MSD analysis. P values were determined using one-way analysis of variance (nonparametric tests). () Toluidine blue staining and electron microscopy (EM) analysis of remyelination in LPC-demyelinated rat spinal cords after 5D10 and control antibody treatments and in LPC-demyelinated WT and DR6-null mouse spinal cords. Arrowheads, remyelinated axons; asterisks, axon pathology. () Quantification of myelinated axons from . P values in and were determined using the unpaired t test. Data are shown as means ± s.e.m. * Figure 6: Oligodendrocyte maturation and myelination in DR6-null mice. () Quantification of immature PDGFRα+ oligodendrocytes from DR6 knockout and WT corpus callosum at age P15 and P30. () Quantification of CC1+ mature oligodendrocytes from DR6-null and WT corpus callosum at age P15 and P30. () Western blot analysis of MAG and MBP protein expression in DR6-null and WT mouse brain at P15 and P30. DR6 expression was used to confirm the DR6-null mice; β-actin expression was analyzed from the same samples as an internal control. () Quantification of MAG and MBP protein expression in P15 mice from . () EM showing visual () and quantitative () analysis of myelinated axon fibers of DR6 knockout and WT corpus callosum from P15 mice. Scale bars, 500 nM. () G ratio of myelinated axons from . () Colocalization of DR6 mRNA (in situ hybridization) and Olig2+ OPCs by immunohistochemical analysis in human normal and multiple sclerosis (MS) brains. Red, probed with DR6 antisense mRNA; green, stained with antibody to Olig2; yellow, merge of red and green. Sc! ale bars, 50 μm. () Quantification of DR6+Olig2+ cells in . P values in , , , , and were determined using the unpaired t test. Data are shown as means ± s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Biogen Idec, Cambridge, Massachusetts, USA. * Sha Mi, * Xinhua Lee, * Yinghui Hu, * Benxiu Ji, * Zhaohui Shao, * Weixing Yang, * Guanrong Huang, * Lee Walus, * Kenneth Rhodes, * Bang Jian Gong & * R Blake Pepinsky * Center for Translational Neuroscience, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. * Robert H Miller Contributions S.M. supervised all experiments and wrote the paper. X.L., Y.H., B.J., Z.S., W.Y., G.H., L.W. and B.J.G. performed experiments. K.R. and R.B.P. provided helpful discussions, and R.H.M. and R.B.P. revised the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Sha Mi Author Details * Sha Mi Contact Sha Mi Search for this author in: * NPG journals * PubMed * Google Scholar * Xinhua Lee Search for this author in: * NPG journals * PubMed * Google Scholar * Yinghui Hu Search for this author in: * NPG journals * PubMed * Google Scholar * Benxiu Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Zhaohui Shao Search for this author in: * NPG journals * PubMed * Google Scholar * Weixing Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Guanrong Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Lee Walus Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth Rhodes Search for this author in: * NPG journals * PubMed * Google Scholar * Bang Jian Gong Search for this author in: * NPG journals * PubMed * Google Scholar * Robert H Miller Search for this author in: * NPG journals * PubMed * Google Scholar * R Blake Pepinsky 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 1–6 Additional data - Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex
- Nat Med 17(7):822-829 (2011)
Nature Medicine | Article Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex * Joel M Brittain1, 11 * Djane B Duarte1, 11 * Sarah M Wilson1 * Weiguo Zhu1, 2 * Carrie Ballard1, 3 * Philip L Johnson4 * Naikui Liu1, 5 * Wenhui Xiong1, 6 * Matthew S Ripsch1, 7 * Yuying Wang1, 2 * Jill C Fehrenbacher1, 2, 7 * Stephanie D Fitz4 * May Khanna3 * Chul-Kyu Park8 * Brian S Schmutzler1, 2 * Bo Myung Cheon1, 7 * Michael R Due1, 7 * Tatiana Brustovetsky2 * Nicole M Ashpole1, 3 * Andy Hudmon1, 2, 3 * Samy O Meroueh1, 3 * Cynthia M Hingtgen1, 2, 9 * Nickolay Brustovetsky1, 2 * Ru-Rong Ji8 * Joyce H Hurley1, 3 * Xiaoming Jin1, 6 * Anantha Shekhar4, 10 * Xiao-Ming Xu1, 6, 9 * Gerry S Oxford1, 2 * Michael R Vasko1, 2, 7 * Fletcher A White1, 7 * Rajesh Khanna1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:822–829Year published:(2011)DOI:doi:10.1038/nm.2345Received14 September 2010Accepted07 March 2011Published online05 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body co! ntortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain. View full text Figures at a glance * Figure 1: A CRMP-2 peptide suppresses the interaction between CaV2.2 and CRMP-2. () Normalized binding of CaV2.2 to 15-mer peptides (overlapping by 12 amino acids) encompassing full-length CRMP-2 overlaid with spinal cord lysates. The sequence of peptide 96, designated CBD3, is shown. () Immunoprecipitation (IP) of CaV2.2 (top), CRMP-2 (middle) and β-tubulin (bottom) with recombinant CRMP-2 or CaV2.2 antibody from spinal cord lysates in the presence of scramble or CBD3 peptides. () Sensorgram of CBD3 (1, 3, 5 μM) or scramble peptide (1, 3, 5 μM traces) binding to CaV2.2 cytosolic loop 1 (L1) and distal C terminus (Ct-dis). Dissociation was monitored for 4 min. RU, resonance units. () Binding of L1-GST and Ct-dis-GST fusion proteins to CRMP-2 in the presence of scramble or CBD3 peptides (10 μM). CRMP-2 binding to L1 and Ct-dis was probed with a CRMP-2–specific antibody. (,) Top, immunocytochemistry of expressed CaV2.2 in CAD cells without () or with () CBD3 overexpression. Scale bars, 10 μm. Bottom, normalized surface intensity (SI) between the arr! ows demarcating surfaces of cells in and . () Percentage of cells showing surface CaV2.2 expression (n > 100). () Immunoblots of biotinylated (surface) fractions of CAD cells expressing vector (scrambled), an N-terminal region of CRMP-2 (CBD1) or CBD3 probed with CaV2.2 antibody (n = 3). () Top, voltage protocol. Bottom, exemplar traces from hippocampal neurons overexpressing vector (EGFP), CRMP-2 or CRMP-2 + CBD3. () Peak current density (pA/pF) at +10 mV for vector- for CRMP-2– or CRMP-2 + CBD3–transfected neurons. *P < 0.05 versus CRMP-2, Student's t test. Error bars represent means ± s.e.m. * Figure 2: TAT-CBD3 reduces Ca2+ currents in DRGs and excitatory synaptic transmission in lamina II neurons from spinal cord slices. () Representative differential interference contrast/fluorescence images showing robust penetration of FITC-TAT-CBD3 into DRGs (arrowheads) but not other cells (arrows). Nuclei are stained with Hoechst dye in the bottom image. Scale bars, 10 μm. () Representative current traces from a DRG incubated for 15 min with TAT-Scramble (10 μM; green) or TAT-CBD3 (10 μM; purple) in response to the voltage steps illustrated at the top. () Current-voltage relationships for the currents shown in fitted to a b-spline line. Peak currents were normalized to the cell capacitance. () Peak current density measured at −10 mV for DRGs incubated with TAT-Scramble, TAT-CBD3 or TAT-CBD3 + 1 μM ω-conotoxin (CTX). The numbers in parentheses represent numbers of cells tested. *P < 0.05 versus TAT-Scramble. () Top, representative traces of spontaneous EPSCs (sEPSCs) in lamina II neurons in spinal cord slices before treatment (left) or after application of 10 μM TAT-Scramble (middle) or 10 μM T! AT-CBD3 (right). Bottom, enlarged traces. Voltage-clamp recordings (holding voltage, −70 mV) were used to record synaptic responses. () Ratio of sEPSC frequency and amplitude. *P < 0.05 compared with baseline. Error bars represent means ± s.e.m. * Figure 3: TAT-CBD3 reduces capsaicin-stimulated release of iCGRP from spinal cord slices. (–) iCGRP release was measured in three 3-min exposures to HEPES buffer alone (white bars), to HEPES buffer with peptides (white), to HEPES buffer containing 500 nM capsaicin (Cap) with peptides (yellow), then to HEPES alone to re-establish baseline (white). Each column represents the mean ± s.e.m. of iCGRP levels in each 3-min perfusate sample, expressed as percentage of total peptide content in the tissues per minute (n = 7 rats). TAT-Scramble () or TAT-CBD3 (), was included as indicated by the horizontal bars. *P < 0.05 versus basal iCGRP release in the absence of capsaicin (analysis of variance (ANOVA), Dunnett's post hoc test). Neither peptide altered basal iCGRP release (not significant, NS). () Basal release is the amount of iCGRP released in the three fractions exposed to HEPES plus peptides. Stimulated release is the amount of iCGRP released in the three fractions exposed to 500 nM capsaicin + peptides. The evoked release was obtained by subtracting iCGRP release! during three basal fractions (12–18 min) from that during the three capsaicin-stimulated fractions (21–27 min) and expressed as percentage of total iCGRP content in each group. *P < 0.05 versus the respective TAT-Scramble using a Student's t test. () Total content of iCGRP (in fmol mg−1) is the sum of CGRP released during perfusion and from spinal cord tissue measured at the end of the release experiments. * Figure 4: TAT-CBD3 reduces changes in meningeal blood flow induced by capsaicin. () Experimental paradigm for the laser Doppler flowmetry measurements. () Representative normalized traces of middle meningeal blood flow changes in response to nasally administered capsaicin (100 nM) in the presence of TAT-Scramble (30 μM, green trace) or TAT-CBD3 pretreatment (30 μM, purple trace, applied durally 15 min before capsaicin administration). Laser Doppler flowmetry measurements were collected at 1 Hz and binned by averaging every 10 samples for graphical representation. The data from each rat were normalized to the first 3 min of basal data and the horizontal dashed line indicates the calculated baseline. The ordinate represents red blood cell flux measurements in arbitrary units (AU). () Summary of blood flow changes after nasal administration of capsaicin in the absence or presence of previous administration of TAT-CBD3 (3, 10 or 30 μM) or TAT-Scramble to the dura. *P < 0.05 versus vehicle (unpaired Student's t test). The number of rats tested for each con! dition is indicated in parentheses. () Concentration-response curve of percentage inhibition (versus averaged TAT-Scramble) of blood flow by TAT-CBD3. Error bars represent means ± s.e.m. * Figure 5: TAT-CBD3 reduces acute, inflammatory and neuropathic pain. () Time course of the number of flinches after subcutaneous (dorsal surface of paw) injection of formalin (2.5% in 50 μl saline) in rats pretreated with peptides (3–100 μM; 20 μl into dorsal surface of paw) 30 min before formalin (n = 4–10). () The effect of peptides on number of flinches on formalin-induced phase 1 (0–10 min) and phase 2 (15–60 min). *P < 0.05 versus formalin-injected rats. () Paw thickness, as measured 1 h after injection of saline, formalin or formalin + peptides (100 μM). *P < 0.05 versus saline-injected rats. () Pretreatment with TAT-CBD3 peptide attenuates capsaicin-evoked nocifensive behavior. Vehicle (0.3% DMSO) or peptides (concentrations as indicated) in saline (40 μl) was instilled corneally and nocifensive behavior noted. Five minutes later, capsaicin (3 μM in 40 μl saline) was applied corneally and nocifensive behavior noted. *P < 0.05 versus 30 or 100 μM TAT-scramble or 3 μM TAT-CBD3; #P < 0.05 versus all conditions except 3 μ! M TAT-CBD3 (ANOVA with Dunnett's post hoc test). () PWT (mN) of rats injected once with ddC and treated with TAT peptides on day 7 after injection (PID7). Response of ddC alone at PID7 is shown in the brown bar. *P < 0.05 versus ddC or TAT-Scramble (ANOVA with Student-Newman-Keuls post hoc test). (–) Fluorescent imaging of DRGs, isolated 15 min after injection of FITC-TAT-CBD3 (, FITC; green) and immunolableled with a NeuN-specific antibody (, NeuN; red). Cells were also stained with Hoechst (, blue), which labels cell nuclei. () Merged image. Scale bars, 100 μm (–). Error bars represent means ± s.e.m. * Figure 6: TAT-CBD3 has no effect on sensorimotor and cognitive functions but has a mild anxiolytic effect. () Latency to fall off a slow (left) or fast (right) rotating rod. There were no significant differences in rotarod performances between groups (ANOVA with Dunnett's post hoc test). () Latency for mice to find a hidden platform in the Morris water maze was not different between groups. () Time spent in target quadrant. There were no significant differences in percentage time spent in target quadrant or path length between groups (Student's t test). (–) Uptake of FITC-TAT-CBD3 into neurons in ventral horn 15 min after intraperitoneal (i.p.) injection (20 mg kg−1). TAT-CBD3 (, green; FITC) accumulated in motor neurons (arrowheads), which co-labeled with NeuN (, red). Nuclei are stained with Hoechst (blue). Merged images show co-labeling of FITC-TAT-CBD3–containing neurons with NeuN and Hoescht at low () and high magnifications (). Scale bars, 100 μm (–); 40 μm (). () Elevated plus maze test to evaluate anxiety-associated behaviors. () Light dark box test for anxiety-! associated behaviors. (*P < 0.05 versus TAT-Scramble, one-way ANOVA with Dunnett's post hoc test.) () Tail suspension test of depression- or despair-associated behaviors. Error bars represent means ± s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Joel M Brittain & * Djane B Duarte Affiliations * Program in Medical Neurosciences, Paul and Carole Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Joel M Brittain, * Djane B Duarte, * Sarah M Wilson, * Weiguo Zhu, * Carrie Ballard, * Naikui Liu, * Wenhui Xiong, * Matthew S Ripsch, * Yuying Wang, * Jill C Fehrenbacher, * Brian S Schmutzler, * Bo Myung Cheon, * Michael R Due, * Nicole M Ashpole, * Andy Hudmon, * Samy O Meroueh, * Cynthia M Hingtgen, * Nickolay Brustovetsky, * Joyce H Hurley, * Xiaoming Jin, * Xiao-Ming Xu, * Gerry S Oxford, * Michael R Vasko, * Fletcher A White & * Rajesh Khanna * Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Weiguo Zhu, * Yuying Wang, * Jill C Fehrenbacher, * Brian S Schmutzler, * Tatiana Brustovetsky, * Andy Hudmon, * Cynthia M Hingtgen, * Nickolay Brustovetsky, * Gerry S Oxford, * Michael R Vasko & * Rajesh Khanna * Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Carrie Ballard, * May Khanna, * Nicole M Ashpole, * Andy Hudmon, * Samy O Meroueh & * Joyce H Hurley * Department of Psychiatry, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Philip L Johnson, * Stephanie D Fitz & * Anantha Shekhar * Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Naikui Liu * Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Wenhui Xiong, * Xiaoming Jin & * Xiao-Ming Xu * Department of Anesthesia, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Matthew S Ripsch, * Jill C Fehrenbacher, * Bo Myung Cheon, * Michael R Due, * Michael R Vasko & * Fletcher A White * Sensory Plasticity Laboratory, Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Chul-Kyu Park & * Ru-Rong Ji * Department of Neurology, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Cynthia M Hingtgen & * Xiao-Ming Xu * Department of Indiana Clinical and Translational Sciences Institute, Indiana University School of Medicine, Indianapolis, Indiana, USA. * Anantha Shekhar Contributions J.M.B. performed molecular biology, biochemistry and calcium imaging experiments and analyzed the data. D.B.D. carried out the spinal cord slice release and formalin behavior experiments and helped to write the manuscript. S.M.W. performed immunocytochemistry and wrote the manuscript. C.B. carried out the laser Doppler blood flowmetry. J.H.H. analyzed the blood flow data. P.L.J. and S.D.F. performed anxiety and despair behavior experiments. W.Z. and Y.W. performed DRG and hippocampal patching. C.-K.P. conducted electrophysiology in spinal cord slices. W.X. and X.J. performed electrophysiology on brain slices. B.S.S. carried out the DRG release assays. T.B., N.B. and J.M.B. performed and analyzed the calcium imaging experiments. B.M.C., M.R.D. and M.S.R. performed DRG immunocytochemistry and ddC behavior experiments. M.K. and S.O.M. performed the surface plasmon resonance experiments and analyzed the data. N.L. performed the rotarod and water maze experiments. J.C.F. performe! d the nocifensive behavior experiments and editing of the manuscript. N.M.A. and A.H. synthesized the peptide blot. X.-M.X., C.M.H., M.R.V., G.S.O. and A.S. contributed to editing of the manuscript. R.-R.J contributed to electrophysiology of spinal cord slices and editing of the manuscript. F.A.W. analyzed the ddC behavior data and contributed to writing and editing the manuscript. R.K. identified the peptide, conceived the study, designed and supervised the overall project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Rajesh Khanna Author Details * Joel M Brittain Search for this author in: * NPG journals * PubMed * Google Scholar * Djane B Duarte Search for this author in: * NPG journals * PubMed * Google Scholar * Sarah M Wilson Search for this author in: * NPG journals * PubMed * Google Scholar * Weiguo Zhu Search for this author in: * NPG journals * PubMed * Google Scholar * Carrie Ballard Search for this author in: * NPG journals * PubMed * Google Scholar * Philip L Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Naikui Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Wenhui Xiong Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew S Ripsch Search for this author in: * NPG journals * PubMed * Google Scholar * Yuying Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Jill C Fehrenbacher Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie D Fitz Search for this author in: * NPG journals * PubMed * Google Scholar * May Khanna Search for this author in: * NPG journals * PubMed * Google Scholar * Chul-Kyu Park Search for this author in: * NPG journals * PubMed * Google Scholar * Brian S Schmutzler Search for this author in: * NPG journals * PubMed * Google Scholar * Bo Myung Cheon Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Due Search for this author in: * NPG journals * PubMed * Google Scholar * Tatiana Brustovetsky Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole M Ashpole Search for this author in: * NPG journals * PubMed * Google Scholar * Andy Hudmon Search for this author in: * NPG journals * PubMed * Google Scholar * Samy O Meroueh Search for this author in: * NPG journals * PubMed * Google Scholar * Cynthia M Hingtgen Search for this author in: * NPG journals * PubMed * Google Scholar * Nickolay Brustovetsky Search for this author in: * NPG journals * PubMed * Google Scholar * Ru-Rong Ji Search for this author in: * NPG journals * PubMed * Google Scholar * Joyce H Hurley Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoming Jin Search for this author in: * NPG journals * PubMed * Google Scholar * Anantha Shekhar Search for this author in: * NPG journals * PubMed * Google Scholar * Xiao-Ming Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Gerry S Oxford Search for this author in: * NPG journals * PubMed * Google Scholar * Michael R Vasko Search for this author in: * NPG journals * PubMed * Google Scholar * Fletcher A White Search for this author in: * NPG journals * PubMed * Google Scholar * Rajesh Khanna Contact Rajesh Khanna Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods Additional data - Low levels of SIV infection in sooty mangabey central memory CD4+ T cells are associated with limited CCR5 expression
- Nat Med 17(7):830-836 (2011)
Nature Medicine | Article Low levels of SIV infection in sooty mangabey central memory CD4+ T cells are associated with limited CCR5 expression * Mirko Paiardini1, 2, 9 * Barbara Cervasi1, 2, 9 * Elane Reyes-Aviles2 * Luca Micci1, 3 * Alexandra M Ortiz1, 2 * Ann Chahroudi1 * Carol Vinton4 * Shari N Gordon1, 2 * Steven E Bosinger1 * Nicholas Francella2 * Paul L Hallberg2 * Elizabeth Cramer2 * Timothy Schlub5 * Ming Liang Chan5 * Nadeene E Riddick6 * Ronald G Collman6 * Cristian Apetrei7 * Ivona Pandrea7 * James Else1 * Jan Munch8 * Frank Kirchhoff8 * Miles P Davenport5 * Jason M Brenchley4 * Guido Silvestri1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:830–836Year published:(2011)DOI:doi:10.1038/nm.2395Received30 March 2011Accepted09 May 2011Published online26 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4+CCR5+ T cells is lower in sooty mangabeys compared to humans and macaques. Here we found that, after in vitro stimulation, sooty mangabey CD4+ T cells fail to upregulate CCR5 and that this phenomenon is more pronounced in CD4+ central memory T cells (TCM cells). CD4+ T cell activation was similarly uncoupled from CCR5 expression in sooty mangabeys in vivo during acute SIV infection and the homeostatic proliferation that follows antibody-mediated CD4+ T cell depletion. Sooty mangabey CD4+ TCM cells that express low amounts of CCR5 showed reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4+ TCM cells of rhesus macaques. These data suggest that low CCR5 expression on sooty mangabey CD4+ T cells favors the preservation of CD4+ T cell homeostasis and promotes an AIDS-fr! ee status by protecting CD4+ TCM cells from direct virus infection. View full text Figures at a glance * Figure 1: The fraction of CCR5+ cells ex vivo is significantly lower in all subsets of CD4+ T cells of sooty mangabeys as compared to rhesus macaques. The fraction of CCR5+ cells was determined in the TN, TCM and TEM subsets of CD4+ and CD8+ T cells of 18 SIV uninfected sooty mangabeys (SMs) and 30 SIV uninfected rhesus macaques (RMs). () Expression of the surface markers CD28, CD95 and CD62L on CD4+ T cells in a representative sooty mangabey. () Staining of CCR5 on TCM and TEM subsets of CD4+ and CD8+ cells in a representative sooty mangabey and a representative rhesus macaque. SSC, side scatter. Numbers in the quadrants indicate the percentage of CD4+ or CD8+ T cell subsets expressing CCR5. () Fraction of CD4+ (left) or CD8+ (right) T cells that express CCR5 in 18 uninfected sooty mangabeys and 30 uninfected rhesus macaques. Statistical analyses were performed to compare, in sooty mangabeys versus rhesus macaques, the fraction of CCR5+ cells within each CD4+ and CD8+ T cell subset. * Figure 2: CCR5 expression upon in vitro activation and proliferation is significantly lower in CD4+ T cells of sooty mangabeys than rhesus macaques. Fractions of CD4+CCR5+ T cells were determined in PBMCs from uninfected sooty mangabeys and rhesus macaques after in vitro stimulations. (,) Fraction of CD4+Ki-67+ () and CD4+CCR5+ () T cells after stimulation with ConA and IL-2. Asterisks indicate time points when the fraction of CD4+CCR5+ T cells was significantly lower in sooty mangabeys than rhesus macaques (P values are detailed in the Results). () Representative dot plots showing the fraction of CD4+CCR5+ T cells after stimulation in rhesus macaques (top) and sooty mangabeys (bottom). () Flow cytometry dot plots showing Ki-67 and CCR5 double staining in a representative rhesus macaque and sooty mangabey at 120 h after stimulation with ConA and IL-2. () Flow cytometry analysis of cell proliferation by CFSE dilution in PBMCs isolated from rhesus macaques and sooty mangabeys; levels of CCR5 were analyzed on cells at 120 h after stimulation. (,) PBMCs isolated from sooty mangabeys and rhesus macaques were stimulated with r! ecombinant IL-7, and the fraction of CD4+Ki-67+ () and CD4+CCR5+ () T cells determined after stimulation. Asterisks indicate time points where, in rhesus macaques, the IL-7–induced increase in CD4+CCR5+ T cells (as compared to baseline) was statistically significant (P values are detailed in the Results). () Levels of CCR5 mRNA (expressed as CCR5/ glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ratio), as determined on purified CD4+ T cells at 0, 24, 72 and 120 h after stimulation with ConA/IL-2. Statistical analyses were performed to compare CCR5/GAPDH ratio between sooty mangabeys and rhesus macaques. In all graphs, error bars represent s.e.m. * Figure 3: The fraction of CD4+CCR5+ T cells upon in vivo activation is significantly lower in sooty mangabeys as compared to rhesus macaques. We determined the fraction of CD4+CCR5+ T cells from sooty mangabeys and rhesus macaques in two in vivo experimental conditions associated with activation of the CD4+ T cell compartment, that is, acute SIV-infection and antibody-mediated CD4+ T cell depletion. (,) Fraction () and fold change versus preinfection (day 0) () of CD4+CCR5+ T cells at different time points during pathogenic SIVmac239 infection of five rhesus macaques and nonprogressive experimental SIVsmm infection of four sooty mangabeys. (,) Fraction () and fold change versus predepletion (day −14) () of CD4+CCR5+ T cells at different time points after antibody-mediated CD4+ T cell depletion in three uninfected rhesus macaques and three uninfected sooty mangabeys. The AUC of the fraction of CD4+CCR5+ T cells is significantly higher in rhesus macaques than sooty mangabeys. The dotted lines in and indicate the 10-d period in which the antibody to CD4 was administered. In all graphs, error bars represent s.e.m. * Figure 4: Lower fraction of CCR5+ cells after in vitro activation of sorted CD4+ TCM cells of sooty mangabeys as compared to rhesus macaques. The fractions of sorted CD4+ TCM and TEM cells that express CCR5 were longitudinally determined after in vitro mitogen activation (ConA and IL-2) of TCM (CD95+CD62L+) and TEM (CD95+CD62L−) cells isolated from eight SIV-uninfected sooty mangabeys and eight SIV-uninfected rhesus macaques. (,) The fraction of CD4+CCR5+ TEM () and TCM () cells at different time points after stimulation. Asterisks indicate time points where values are significantly higher in rhesus macaques as compared to sooty mangabeys. In all graphs, error bars represent s.e.m. * Figure 5: CD4+ TCM of sooty mangabeys are relatively resistant to SIV infection in vivo. The fraction of SIV-infected CD4+, CD4+ TCM and TEM cells, as determined measuring by quantitative PCR the number of SIV gag DNA copies per cell equivalent, in 18 naturally SIVsmm-infected sooty mangabeys and 7 experimentally SIVmac239-infected rhesus macaques. In all graphs, error bars represent s.e.m. * Figure 6: CD4+ TCM of sooty mangabeys are relatively resistant to SIV infection in vitro. (–) CD4+ TCM and TEM cells purified from 6 sooty mangabeys and 11 rhesus macaques were infected in vitro with SIVsmm M949 (six sooty mangabeys and five rhesus macaques), or with SIVmac (six rhesus macaques). The levels of SIV replication were determined by measuring p27 in the supernatants. () Levels of SIVsmm replication in CD4+ TCM and CD4+ TEM cells of sooty mangabeys at days 3, 6, 9, 12 and 15 after infection (significant differences are indicated by asterisks; P values are detailed in the Results). () Levels of SIVmac replication in CD4+ TCM and CD4+ TEM cells of rhesus macaques at days 3, 6, 9, 12 and 15 after infection. () Levels of SIVsmm replication in CD4+ TCM and CD4+ TEM cells of rhesus macaques at days 3, 6, 9, 12 and 15 after infection. () Ratios of the levels of SIV replication between CD4+ TCM and CD4+ TEM of rhesus macaques and sooty mangabeys at all tested time points (significant differences are indicated by single (P < 0.02) or double (P < 0.01) asteris! ks). () Flow plots of eGFP expression in CD4+ TCM and CD4+ TEM cells defined by flow cytometry of CD28, CD95 and CD62L expression in unfractionated PBMCs derived from two representative sooty mangabeys and infected in vitro with replication competent eGFP-expressing SIVsmm. () Ratio of the percentage of eGFP+ cells between CD4+ TEM and CD4+ TCM of sooty mangabeys (P = 0.0006 indicates that the levels of eGFP+ cells are significantly higher in CD4+ TEM than CD4+ TCM). In all graphs, error bars represent s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Mirko Paiardini & * Barbara Cervasi Affiliations * Yerkes National Primate Research Center, Emory Vaccine Center and Department of Pathology, Emory University, Atlanta, Georgia, USA. * Mirko Paiardini, * Barbara Cervasi, * Luca Micci, * Alexandra M Ortiz, * Ann Chahroudi, * Shari N Gordon, * Steven E Bosinger, * James Else & * Guido Silvestri * Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Mirko Paiardini, * Barbara Cervasi, * Elane Reyes-Aviles, * Alexandra M Ortiz, * Shari N Gordon, * Nicholas Francella, * Paul L Hallberg, * Elizabeth Cramer & * Guido Silvestri * University of Urbino, Urbino, Italy. * Luca Micci * Laboratory of Molecular Microbiology, US National Institutes of Health, Bethesda, Maryland, USA. * Carol Vinton & * Jason M Brenchley * Complex Systems in Biology Group, Centre for Vascular Research, University of New South Wales, Kensington, New South Wales, Australia. * Timothy Schlub, * Ming Liang Chan & * Miles P Davenport * Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Nadeene E Riddick & * Ronald G Collman * Center for Vaccine Research, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. * Cristian Apetrei & * Ivona Pandrea * Institute of Molecular Virology, University Hospital Ulm, Ulm, Germany. * Jan Munch & * Frank Kirchhoff Contributions M.P., B.C. and G.S. designed the study and wrote the paper, with contributions from the other authors as appropriate; M.P., B.C. and E.R.-A. performed the immunophenotypic analyses, analyzed results and prepared the figures; L.M., A.M.O., E.C. and A.C. helped in preparing the reagents, processing the samples and analyzing the data; C.V. and J.M.B. performed the quantitative PCR for SIV gag DNA; S.N.G. provided the data on experimentally SIV-infected sooty mangabeys and rhesus macaques; S.E.B. and N.F. determined CCR5 mRNA levels; P.L.H. performed the sorting experiments; T.S., M.L.C. and M.P.D. contributed to the design of the study and statistical analyses; J.E. supervised the housing and care of the primates and contributed to the design of the study and sample collection; J.M. and F.K. provided the eGFP-expressing SIV reporter virus; C.A., I.P., N.E.R., F.K. and R.G.C. contributed to the study design and preparation of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Guido Silvestri or * Mirko Paiardini Author Details * Mirko Paiardini Contact Mirko Paiardini Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Cervasi Search for this author in: * NPG journals * PubMed * Google Scholar * Elane Reyes-Aviles Search for this author in: * NPG journals * PubMed * Google Scholar * Luca Micci Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandra M Ortiz Search for this author in: * NPG journals * PubMed * Google Scholar * Ann Chahroudi Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Vinton Search for this author in: * NPG journals * PubMed * Google Scholar * Shari N Gordon Search for this author in: * NPG journals * PubMed * Google Scholar * Steven E Bosinger Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas Francella Search for this author in: * NPG journals * PubMed * Google Scholar * Paul L Hallberg Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Cramer Search for this author in: * NPG journals * PubMed * Google Scholar * Timothy Schlub Search for this author in: * NPG journals * PubMed * Google Scholar * Ming Liang Chan Search for this author in: * NPG journals * PubMed * Google Scholar * Nadeene E Riddick Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald G Collman Search for this author in: * NPG journals * PubMed * Google Scholar * Cristian Apetrei Search for this author in: * NPG journals * PubMed * Google Scholar * Ivona Pandrea Search for this author in: * NPG journals * PubMed * Google Scholar * James Else Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Munch Search for this author in: * NPG journals * PubMed * Google Scholar * Frank Kirchhoff Search for this author in: * NPG journals * PubMed * Google Scholar * Miles P Davenport Search for this author in: * NPG journals * PubMed * Google Scholar * Jason M Brenchley Search for this author in: * NPG journals * PubMed * Google Scholar * Guido Silvestri Contact Guido Silvestri 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 and 2 Additional data - Identification of an innate T helper type 17 response to intestinal bacterial pathogens
- Nat Med 17(7):837-844 (2011)
Nature Medicine | Article Identification of an innate T helper type 17 response to intestinal bacterial pathogens * Kaoru Geddes1, 5 * Stephen J Rubino2, 5 * Joao G Magalhaes1 * Catherine Streutker3 * Lionel Le Bourhis1 * Joon Ho Cho1 * Susan J Robertson1 * Connie J Kim4 * Rupert Kaul1, 4 * Dana J Philpott1 * Stephen E Girardin2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:837–844Year published:(2011)DOI:doi:10.1038/nm.2391Received23 February 2011Accepted02 May 2011Published online12 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4+ T helper cells. Enteric innate T helper type 17 (iTH17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103+ dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iTH17 responses. Together, these results identify the Nod-iTH17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iTH17 responses in the developm! ent of inflammatory bowel diseases. View full text Figures at a glance * Figure 1: Nod1 and Nod2 differentially modulate early and late inflammation during C. rodentium-induced colitis. () The degree of colonic histopathology, crypt lengths and bacterial translocation to the spleen assessed in wild-type and Nod1−/−Nod2−/− mice at 7 and 14 d after infection. CFU, colony-forming units. () Representative images (20× magnification) of H&E-stained colon sections of wild-type and Nod1−/−Nod2−/−C. rodentium–infected mice at 7 and 14 d after infection; arrows depict areas of goblet cell depletion and submucosal edema; asterisks depict proximal regions of colon. () Degree of colonic histopathology, crypt length and splenic translocation in lethally irradiated wild-type mice reconstituted with either wild-type (WT→WT) or Nod1−/−Nod2−/− bone marrow (DKO→WT) and Nod1−/−Nod2−/− mice reconstituted with either wild-type (WT→DKO) or Nod1−/−Nod2−/− (DKO→DKO) bone marrow at 12 d after infection. Error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant. * Figure 2: Early IL-17 responses during C. rodentium–induced colitis are Nod1 and Nod2 dependent. () Il17a expression in C. rodentium–infected wild-type and Nod1−/−Nod2−/− mice, as quantified by quantitative RT-PCR (qRT-PCR) from the cecum at 4 d (top) and colon at 10 d (bottom) after infection. () Flow cytometry analysis of IL-17A and IL-22 intracellular cytokine staining (ICCS) of cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 4 d), either of all LPLs (left) or CD4+TCRβ+ LPLs (right). () The relative number of CD4+TCRβ+IL-17A+ (TH17) cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 4 d after C. rodentium infection, average of five replicates with three mice pooled per group). () Il22, Lcn2 and Reg3g expression in C. rodentium–infected wild-type and Nod1−/−Nod2−/− mice, as quantified by qRT-PCR in the cecum at 4 d after infection. For qRT-PCR, the average fold change in expression over PBS-treated wild-type mice is shown (n = 10, one representative of two experiments shown). Error bars represent s.e! .m. *P < 0.05. * Figure 3: Acute IL-17 responses during S. typhimurium-induced colitis are dependent on hematopoietic and non-hematopoietic Nod1 and Nod2. () qRT-PCR analysis of Il17a, Il22 and Lcn2 in the cecum of wild-type and Nod1−/−Nod2−/− mice (uninfected or SL1344 infected for 24 h). Bar graphs show average fold change over uninfected controls (n = 6, one representative of three experiments shown). () ICCS analysis of IL-17A and IL-22 in total LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) in cecal LPLs from wild-type and Nod1−/−Nod2−/− mice (uninfected or 24 h after infection with SL1344). () The average relative frequency of all cells, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells in wild-type and Nod1−/−Nod2−/− mice (uninfected or 24 h after infection with SL1344, average of six replicates with three mice pooled per group). () qRT-PCR analysis for Il17a and Il22 on total cells (presort), CD4+ cells, CD11b+CD11c+ cells, and cells remaining after MACS purification (depleted). The bar graphs show fold change in expression over presort cells from uninfected mice (one representative o! f two replicates is shown, six mice pooled per group), and the numbers above the bars represent the fold change between wild-type and Nod1−/−Nod2−/− for each population of cells. () ICCS analysis of IL-17A and IL-22 in CD4+TCRβ+ cecal LPLs from chimeric mice (24 h after infection, one representative of three experiments is shown, three mice pooled per group). Error bars represent s.e.m. *P < 0.05, **P < 0.01. * Figure 4: IL-6 expression during C. rodentium- and S. typhimurium (SL1344)-induced colitis are Nod1 and Nod2 dependent. () Expression of Il6, Il23r and Il23a in the cecum of wild-type and Nod1−/−Nod2−/− mice 4 d after infection with C. rodentium (top) or 24 h after infection with SL1344 (bottom). Average fold change over uninfected controls is shown (n = 6, one representative of three experiments is shown). () Cecal IL-6 amounts in SL1344-infected wild-type and Nod1−/−Nod2−/− mice (24, 48 and 72 h), as measured by ELISA. () Cecal IL-6 amounts in SL1344-infected chimeric mice, as measured by ELISA (n = 6, one representative of three experiments is shown). () qRT-PCR analysis for Il6 on total cells (presort), CD4+ cells, CD11b+CD11c+ cells and cells remaining after MACS purification (depleted). The bar graphs show fold change in expression over presort cells from uninfected mice (one representative of two replicates is shown, six mice pooled per group), and the numbers above the bars represent the fold change between wild type and Nod1−/−Nod2−/− for each population of ce! lls. () Expression of CD103 on either CD11b−CD11c+ cells or CD11b+CD11c+ cecal IELs from wild-type (red) and Nod1−/−Nod2−/− (blue) mice uninfected, infected with C. rodentium for 4 d or infected with SL1344 for 24 h. One representative of three experiments, three mice pooled per group. Error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. * Figure 5: IL-6 expression during the acute phase of infectious colitis is crucial for TH17 development. () ICCS analysis of IL-17A and IL-22 on total cecal LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) from SL1344-infected wild-type mice (uninfected or 24 h after infection) treated with either control IgG or IL-6–neutralizing antibody (anti–IL-6). (,) Average relative frequency of all IL-17A+, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells from control IgG– or IL-6–neutralizing antibody–treated, SL1344–infected or uninfected wild-type mice () and SL1344-infected or uninfected wild-type and IL-6–knockout mice () (24 h after infection, average of three replicates with three mice pooled per group). () ICCS analysis for IL-17A and IL-22 expression in TCRβ+CD4+ cecal LPLs from C. rodentium–infected (4 d) and SL1344-infected (24 h) chimeric mice that were generated by reconstituting irradiated wild-type mice with either wild-type (WT→WT) or Il6−/− (Il6−/−→WT) bone marrow. Dot plots depict one representative of three experiments with two mi! ce pooled per group. Error bars represent s.e.m. *P < 0.05, **P < 0.01. * Figure 6: Early TH17 cells express memory surface markers and require microbiota for activation. () Expression of CD44, CD62L, CD69 and CCR6 on either all CD4+TCRβ+ cells or CD4+TCRβ+IL-17A+ cells in cecal LPLs from SL1344-infected mice (top) or expression of these cell surface markers on CD4+TCRβ+IL-17A+ cells from the LPLs of uninfected and SL1344-infected mice (24 h after infection) (bottom). () Mean fluorescence intensity (MFI) of CD69 expression on LPLs from uninfected or SL1344-infected mice (average of three replicates with three mice pooled per group). () ICCS analysis of IL-17A and IL-22 in total cecal LPLs (top), CD4+TCRβ+ cells (middle) or TCRγδ+ cells (bottom) from SL1344-infected SPF and germ-free mice (uninfected or 24 h after infection). () Average relative frequency of all IL-17A+, CD4+TCRβ+IL-17A+ or TCRγδ+IL-17A+ cells from SL1344-infected SPF and germ-free mice (uninfected or 24 h after infection) (uninfected group had two replicates, infected group had three replicates, three mice pooled per group). Error bars represent s.e.m. *P < 0.05, **P! < 0.01. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Kaoru Geddes & * Stephen J Rubino Affiliations * Department of Immunology, University of Toronto, Toronto, Ontario, Canada. * Kaoru Geddes, * Joao G Magalhaes, * Lionel Le Bourhis, * Joon Ho Cho, * Susan J Robertson, * Rupert Kaul & * Dana J Philpott * Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. * Stephen J Rubino & * Stephen E Girardin * Department of Laboratory Medicine, St. Michael's Hospital, Toronto, Ontario, Canada. * Catherine Streutker * Department of Medicine, University of Toronto, Toronto, Ontario, Canada. * Connie J Kim & * Rupert Kaul Contributions K.G. and S.J.R. designed and performed all experiments and wrote the manuscript. J.G.M. designed and performed mouse experiments. C.S. performed pathological scoring analysis. L.L.B. generated the Nod1−/−Nod2−/− mice. J.H.C. and S.J.R. performed microbiota analysis. C.J.K. and R.K. provided human colonic samples. D.J.P. and S.E.G. directed the research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Dana J Philpott or * Stephen E Girardin Author Details * Kaoru Geddes Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen J Rubino Search for this author in: * NPG journals * PubMed * Google Scholar * Joao G Magalhaes Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Streutker Search for this author in: * NPG journals * PubMed * Google Scholar * Lionel Le Bourhis Search for this author in: * NPG journals * PubMed * Google Scholar * Joon Ho Cho Search for this author in: * NPG journals * PubMed * Google Scholar * Susan J Robertson Search for this author in: * NPG journals * PubMed * Google Scholar * Connie J Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Rupert Kaul Search for this author in: * NPG journals * PubMed * Google Scholar * Dana J Philpott Contact Dana J Philpott Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen E Girardin Contact Stephen E Girardin Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7 and Supplementary Methods Additional data - Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia
- Nat Med 17(7):845-853 (2011)
Nature Medicine | Article Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia * Ryan C Russell1 * Roxana I Sufan1 * Bing Zhou2 * Pardeep Heir1 * Severa Bunda1 * Stephanie S Sybingco1 * Samantha N Greer1 * Olga Roche1 * Samuel A Heathcote2 * Vinca W K Chow1 * Lukasz M Boba1 * Terri D Richmond3 * Michele M Hickey4 * Dwayne L Barber3 * David A Cheresh5 * M Celeste Simon4, 6 * Meredith S Irwin7 * William Y Kim2 * Michael Ohh1 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:845–853Year published:(2011)DOI:doi:10.1038/nm.2370Received20 December 2010Accepted04 April 2011Published online19 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia–associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in VhlR200W/R200W knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulat! or of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia. View full text Figures at a glance * Figure 1: CP-VHL shows altered binding to ECV components and JAK2. () Lysates from HEK293 cells transfected with the indicated plasmids were immunoprecipitated with antibody to HA and immunoblotted with the indicated antibodies. () Lysates from 35S-radiolabeled 786-O subclones stably expressing the indicated HA-VHL were immunoprecipitated with antibody to HA, resolved by SDS-PAGE and visualized by autoradiography. () HEK293 cells transfected with the indicated combination of plasmids were treated with (+) or without (−) MG132. Equal amounts of cell lysates were immunoblotted with antibody to JAK2 (bottom) or immunoprecipitated with antibody to VHL and immunoblotted with the indicated antibodies. () HEK293 cells transfected with the indicated plasmids were lysed in the absence of MG132. Equal amounts of cell lysates were then immunoblotted with antibody to JAK2 (bottom) or immunoprecipitated with antibody to HA and immunoblotted with the indicated antibodies (top). Asterisk, nonspecific protein bands. WCE, whole-cell extract; IP, immunopre! cipitation; IB, immunoblot. * Figure 2: VHL promotes ubiquitin-mediated destruction of pJAK2. () HEK293 cells transfected with the indicated plasmids were lysed and immunoblotted with the indicated antibodies. () BaF3-EPOR cells infected with retrovirus encoding HA-VHL were lysed and immunoblotted with antibody to VHL (input) or immunoprecipitated with antibody to HA or isotype-matched control antibody and immunoblotted with antibody to VHL (bottom). The same HA-VHL-expressing BaF3-EPOR cells treated with (+) or without (−) erythropoietin (EPO) were immunoprecipitated and immunoblotted with the indicated antibodies (top). Equal amounts of cell lysates (input) were also immunoblotted with the indicated antibodies (middle). () HEK293 cells transfected with the indicated plasmids were treated with (+) or without (−) 20 U ml−1 of EPO and pJAK2 was isolated via immunoprecipitation with antibody to T7 (top), which was then added to an in vitro ubiquitination reaction containing proteasome-depleted S100 fractions with (+) or without (−) VHL (bottom). Reaction mixtur! es were reimmunoprecipitated with antibody to T7 and immunoblotted with the indicated antibodies. () BaF3-EPOR-shVHL or -shScr cells stimulated with EPO for the indicated time periods were lysed and immunoblotted with the indicated antibodies (top). RNA was also extracted and mRNA levels of the STAT5 target genes PIM1 and CISH were measured by quantitative real-time PCR normalized to GAPDH (bottom). The relative level of EPO-stimulated levels of transcripts in BaF3-EPOR-shScr cells was set to 1.0. Values are means ± s.d. of fold changes over three independent experiments. () HEK293 cells transfected with the indicated plasmids were lysed and immunoblotted with the indicated antibodies. () Expression profiles of known genes responsive to HIF (top) and STAT5 (bottom) were generated by analysis of 18 CCRCC samples in comparison to normal matched tissue control (Norm) run on the Affymetrix Human Genome U133B Array (GEO GDS507)44. Intensity indicates the relative mRNA expressio! n level. Median expression, horizontal bar. n = 2. Values are ! means ± s.d. of each sample set. Asterisk, nonspecific protein bands. * Figure 3: VHL binds to and cooperates with SOCS1 to negatively regulate pJAK2. (,) HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated and immunoblotted with the indicated antibodies. Equal amounts of cell lysates were also immunoblotted with the indicated antibodies (bottom). () BaF3-EPOR cells stimulated with (+) or without (−) erythropoietin (EPO) were lysed, immunoprecipitated with antibody to VHL or isotype-matched control antibody and immunoblotted with the indicated antibodies. Equal amounts of cell lysates were also immunoblotted with antibody to SOCS1 (bottom). () Affinity-purified HA-VHL C162F (lane 1) and cell lysates generated from HEK293 cells expressing Flag-SOCS1 or HA-VHL C162F (lanes 2 and 3) were resolved on SDS-PAGE and immunoblotted with antibody to HA. HEK293 cells transfected with empty plasmid (mock) or plasmid encoding Flag-SOCS1 were lysed and immunoblotted with antibody to Flag (lanes 4 and 5) or immunoprecipitated with antibody to Flag and far-western blotted (FB) with purified recombinant HA! -VHL C162F followed by antibody to HA (lanes 6 and 7). IgGH indicates the heavy chain of the antibody. () Whole-cell extracts prepared from HEK293 cells transfected with the indicated plasmids were immunoblotted with the indicated antibodies. () 786-O subclones stably expressing the indicated HA-VHL were lysed and immunoblotted with the indicated antibodies (right) or immunoprecipitated with antibody to HA and immunoblotted with the indicated antibodies (left). Equal amounts of cell lysates were also immunoblotted with the indicated antibodies (bottom). () HEK293 cells transfected with the indicated plasmids in combination with a plasmid encoding HA-SOCS1 were lysed, immunoprecipitated and immunoblotted with antibody to VHL. Equal amounts of cell lysates were also immunoblotted with the indicated antibodies (bottom). () Whole-cell extracts prepared from HEK293 cells transfected with the indicated plasmids and stimulated with EPO were immunoblotted with the indicated antibod! ies. Asterisk, nonspecific protein bands. * Figure 4: CP-VHL mutants are defective in pJAK2 degradation and pharmacologic JAK2 inhibition attenuates VHL-dependent BaF3-EPOR colony formation. () HEK293 cells transfected with the indicated plasmids were lysed, immunoprecipitated and immunoblotted with antibody to HA. Equal amounts of cell lysates were also immunoblotted with antibody to HA (bottom). () HEK293 cells transfected with the indicated plasmids were treated with erythropoietin (EPO) and MG132, lysed, immunoprecipitated and immunoblotted with the indicated antibodies. Equal amounts of cell lysates were also immunoblotted with the indicated antibodies (bottom). () Equal amounts of T7-pJAK2 isolated from EPO-treated HEK293 cells were mixed with lysates of HEK293 cells containing the indicated plasmids. Reaction mixtures were immunoprecipitated with antibody to T7 and immunoblotted with antibody to pJAK2. () BaF3-EPOR-shVHL cells infected with lentivirus encoding HA-VHL (R200W and WT) and treated with EPO for the indicated time periods were lysed and the lysates were immunoblotted with the indicated antibodies (right). Expression of HA-VHL (R200W and WT) was! confirmed via immunoblotting with the indicated antibodies (left). () Polyclonal BaF3-EPOR cells stably expressing shVHL or shScr were plated in 1% vol/vol methylcellulose containing the indicated levels of EPO (top) or IL-3 (bottom). Colony numbers were generated from a single-blind analysis and represent four (top) or three (bottom) independent experiments done in duplicate. Error bars represent s.d. between replicate wells. () BaF3-EPOR cells were plated in 1% vol/vol methylcellulose containing EPO in combination with the indicated concentrations of JAK2 inhibitor TG101209. Colony numbers were generated from a single-blind analysis and represent two independent experiments done in triplicate. Error bars represent s.d. between replicate wells. () BaF3-EPOR-shVHL and -shScr cells were plated in 1% vol/vol methylcellulose containing 100 mU EPO in combination with the indicated concentrations of TG101209. Average colony size is depicted in the representative micrographs. AU! , arbitrary units. Scale bar, 0.2 mm. Colonies extracted from ! methylcellulose were lysed and immunoblotted with indicated antibodies (right). * Figure 5: TG101209 treatment rescues polycythemia features in VhlR/R mice. () Hematocrit of vehicle- or TG101209-treated VhlR/R mice. The normal hematocrit range is highlighted in gray. P < 0.05 after 5 weeks of treatment. n = 5 mice per group. Values are mean ± s.e.m. () Photographs of plantar footpads of vehicle and TG101209-treated VhlR/R mice. Scale bar, 3.0 mm. () Photographs of spleens (scale bar, 4.0 mm) and photomicrographs of H&E-stained sections of spleens (scale bars, 10 μm) from VhlR/R mice treated with vehicle or TG10209. Yellow arrows, megakaryocytes. () Average number of megakaryocytes per high-power field in spleens of vehicle- or TG101209-treated VhlR/R mice. n = 5 mice per group. Values are mean ± s.e.m. () Numbers of CFU-E colonies formed from the splenic hematopoietic precursors of VhlR/R versus WT mice when cultured with 3 U ml−1 erythropoietin (EPO). Spleens from three mice from each genotype were pooled and plated in triplicate. Error bars represent s.e.m. () Single-cell suspensions enriched with erythroid progenitors ge! nerated from spleens of phenylhydrazine-treated VhlR/R or WT mice were washed, starved in cytokine-free medium and treated with increasing concentrations of exogenous EPO for 15 min. Cell lysates were then immunoblotted with the indicated antibodies. () VhlR/R splenic cells were lysed and sonicated with phosphatase inhibitors and MG132. Lysates were equally divided, immunoprecipitated with antibody to VHL or isotype-matched control antibody and immunoblotted with the indicated antibodies. () Numbers of CFU-E colonies formed from the splenic hematopoietic precursors of VhlR/R mice when cultured with vehicle or TG101209. Spleens from three mice from each genotype were pooled and plated in triplicate. Error bars, s.e.m. * Figure 6: The SOCS groove and a revised molecular model of Chuvash polycythemia. () Mutations that influence SOCS1 binding are clustered within the SOCS groove (red) on the VBC crystal structure bound to HIF-1α peptide. () Molecular model of Chuvash polycythemia. See text for details. EPO, erythropoietin; OH, hydroxyl group; P, phosphorylated tyrosine. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada. * Ryan C Russell, * Roxana I Sufan, * Pardeep Heir, * Severa Bunda, * Stephanie S Sybingco, * Samantha N Greer, * Olga Roche, * Vinca W K Chow, * Lukasz M Boba & * Michael Ohh * Department of Hematology Oncology, The Lineberger Comprehensive Cancer Centre, University of North Carolina, Chapel Hill, North Carolina, USA. * Bing Zhou, * Samuel A Heathcote & * William Y Kim * Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada. * Terri D Richmond & * Dwayne L Barber * Abramson Family Cancer Research Institute, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * Michele M Hickey & * M Celeste Simon * University of California, San Diego, Moores Cancer Center, La Jolla, California, USA. * David A Cheresh * Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. * M Celeste Simon * Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada. * Meredith S Irwin Contributions R.C.R. designed and carried out biochemical and mouse experiments. R.I.S. designed and carried out biochemical experiments. B.Z. carried out the mouse experiments. P.H., S.B., S.S.S., S.N.G., O.R., V.W.K.C. and L.M.B. carried out and assisted with the biochemical experiments. S.A.H., T.D.R. and M.M.H. assisted with the mouse experiments. D.L.B., D.A.C. and M.C.S. provided the reagents associated with the mouse experiments. W.Y.K. designed the mouse experiments. M.S.I. conceptualized the model and designed the experiments. M.O. conceptualized the model, designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Ohh Author Details * Ryan C Russell Search for this author in: * NPG journals * PubMed * Google Scholar * Roxana I Sufan Search for this author in: * NPG journals * PubMed * Google Scholar * Bing Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Pardeep Heir Search for this author in: * NPG journals * PubMed * Google Scholar * Severa Bunda Search for this author in: * NPG journals * PubMed * Google Scholar * Stephanie S Sybingco Search for this author in: * NPG journals * PubMed * Google Scholar * Samantha N Greer Search for this author in: * NPG journals * PubMed * Google Scholar * Olga Roche Search for this author in: * NPG journals * PubMed * Google Scholar * Samuel A Heathcote Search for this author in: * NPG journals * PubMed * Google Scholar * Vinca W K Chow Search for this author in: * NPG journals * PubMed * Google Scholar * Lukasz M Boba Search for this author in: * NPG journals * PubMed * Google Scholar * Terri D Richmond Search for this author in: * NPG journals * PubMed * Google Scholar * Michele M Hickey Search for this author in: * NPG journals * PubMed * Google Scholar * Dwayne L Barber Search for this author in: * NPG journals * PubMed * Google Scholar * David A Cheresh Search for this author in: * NPG journals * PubMed * Google Scholar * M Celeste Simon Search for this author in: * NPG journals * PubMed * Google Scholar * Meredith S Irwin Search for this author in: * NPG journals * PubMed * Google Scholar * William Y Kim Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Ohh Contact Michael Ohh 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 1–4 Additional data - Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors
- Nat Med 17(7):854-859 (2011)
Nature Medicine | Article Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors * Timothy Kottke1 * Fiona Errington2 * Jose Pulido1, 3 * Feorillo Galivo1 * Jill Thompson1 * Phonphimon Wongthida1 * Rosa Maria Diaz1 * Heung Chong4 * Elizabeth Ilett2 * John Chester2 * Hardev Pandha5 * Kevin Harrington6 * Peter Selby2 * Alan Melcher2, 8 * Richard Vile1, 2, 7, 8 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:854–859Year published:(2011)DOI:doi:10.1038/nm.2390Received04 January 2011Accepted29 April 2011Published online19 June 2011 Abstract * Abstract * Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm! for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date. View full text Figures at a glance * Figure 1: Construction and characterization of VSV-expressed cDNA libraries. () The ASEL VSV-expressed cDNA library contains cDNA from normal human prostate cloned into VSV in direct or reverse orientation. () Lanes 1 and 2, human prostate-specific genes (PSMA, STEAP, TRPA1 and PSCA)37, 38, 39, 40, but not the melanocyte-specific TYRP2 (ref. 11), detected by PCR in the original human prostate plasmid library (lane 1) and in the VSV-cDNA plasmid library (lane 2). Lanes 3 and 4, PCR from cDNA of HT1080 cells infected with ASEL (MOI = 0.1; lane 3) compared to uninfected cells (lane 4). Arrow indicates predicted size for PSCA. GAPDH was used as a loading control. () Western blot analysis of human PSA in BHK cells infected with ASEL direct (lane 1) or ASEL reverse (lane 2), or with control viruses (lanes 3 and 4; MOI ~10). Lane 5, uninfected BHK cells; lane 6, 1 × 104 human prostate LnCap cells. () BHK cells infected with tenfold dilutions of ASEL virus (lanes 1–6) assayed by RT-PCR for PSA or human GAPDH. No PSA-specific signal was detected at dilutio! ns lower than 1:100 of the original virus stock (expression of GFP from 100 PFU of VSV-GFP could be detected by this assay). Plus sign, positive signal for the target RNA was detected by PCR upon nested PCR. Asterisk, no PCR signal for the target RNA was detected upon nested PCR. () IFN-γ assay (ref. 28) of splenocytes infected with VSV-GFP (MOI = 0.1), with VSV-cDNA libraries (direct or reversed) derived from B16 cells that did or did not express OVA (labeled B16-OVA or B16, respectively; MOI = 0.1) or with VSV-OVA at indicated MOIs. Where indicated, splenocytes were cocultured with naive OT-I T cells or, as a control, with irrelevant T cells (pmel)41. Column 9, OT-I activated by SIINFEKL peptide; column 14, OT-I alone (no splenocytes, no VSV). * Figure 2: Intraprostatic injection of ASEL induces autoimmunity. () Prostate weights of mice injected intraprostatically with PBS, VSV-GFP or ASEL, 2 or 10 d after injection (n = 3). (–) Histology of prostates 10 d after intraprostatic injection of PBS () or ASEL (,). Scale bars, 100 μm. (,) IFN-γ () and IL-17 () production from splenocytes 10 d after intraprostatic injection of PBS or of 1 × 107 PFU VSV-GFP or ASEL (six mice per group, splenocytes from each mouse shown), cocultured with lysates of TC2 cells. Mean values of cytokine from two ELISA wells per sample are shown for each mouse. TC2 lysate, ELISA with lysate alone (no splenocytes); C57BL/6, splenocytes alone. * Figure 3: Intravenous injection of ASEL has anti-tumor efficacy. () Prostate weights 60 d after 1 × 107 PFU intravenous injection of ASEL (n = 5) or VSV-GFP (n = 7). () Mean levels of IL-17 secreted by splenocytes from the infected mice, cocultured with lysates of B16 melanoma, TC2 prostate, normal mouse prostate (Pros) or pancreas (Panc). Error bars, s.d. of three separate wells per sample. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8), injected intratumorally (i.t.) or intravenously (i.v.) on days 7, 9 and 11 with 1 × 107 PFU of VSV-GFP, ASEL or heat-inactivated (HI) VSV-GFP. (,) Survival of mice bearing 7-day-old TC2 () or B16 () tumors injected intravenously on days 7, 9 and 11 with ASEL or with a VSV-cDNA library from human melanoma cells (altered-self melanoma epitope library, ASMEL). () Cumulative percentages of mice cured of 7d TC2 tumors when administered three, six or nine injections of ASEL or VSV-GFP intratumorally or intravenously every other day. () IL-17 secreted by splenocytes from the three mice cured of! TC2 tumors by nine intratumoral injections of ASEL in , as well as from three mice treated similarly with VSV-GFP. Splenocytes were cocultured with lysates of B16, TC2, normal mouse prostate or pancreas. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8), either mock-depleted or depleted of CD4+, CD8+ or NK cells, injected intravenously with ASEL on days 7, 9, 11, 14, 16, 18, 21, 23 and 25. * Figure 4: Suboptimal vaccination induces immune escape variants. (–) H&E staining of tumors (1.0 cm diameter) from mice bearing 7-day-old TC2 tumors treated intravenously on days 7, 9 and 11 with PBS (,) or ASEL (,) (typically tumors reached 1.0 cm in diameter at day 20 for PBS (TC2) or day 50 for ASEL (TC2R)). Scale bars, 100 μm. (,) Three TC2R tumors from mice treated with ASEL (lanes 1–3) as in , one TC2 tumor from a mouse treated with PBS (lane 4) as in , and in vitro–cultured TC2 cells (lane 5), analyzed by RT-PCR for expression of mouse prostate-specific factors PSCA, PSMA and STEAP42 () or of N-cadherin, SLUG or SNAIL29, 30, 31, 32 (). GAPDH RNA was assayed as a loading control. Cultured TC2 cells were positive for N-cadherin by nested PCR and weakly positive by western blot at lower levels than detected in TCR2 tumors. * Figure 5: TC2R tumors can be treated with a second vaccination. () The IEEL contained cDNA from three TCR2 tumors cloned into VSV. () Western blot for mouse N-cadherin from BHK cells infected with VSV (lane 1), IEEL reverse (lane 2) or IEEL direct (lane 3) (MOI ~10). Equal loading was confirmed by β-actin probing (data not shown). () Survival of mock-vaccinated (non–VSV immune) or VSV-GFP–vaccinated (VSV-immune) mice bearing 7-day-old TC2 tumors treated with three intravenous injections of VSV-GFP or ASEL, either as viral supernatant (1 × 107 PFU) or as preloaded T(ASEL). () Survival of mice bearing 7-day-old TC2 tumors treated intravenously with VSV-GFP or ASEL on days 7, 9 and 11. On days 25, 27 and 29, mice initially treated with ASEL received intravenous preloaded T(IEEL). () Survival of VSV-vaccinated mice bearing 7-day-old TC2 tumors injected intravenously on days 7, 9 and 11 with VSV-GFP, ASEL or IEEL (1 × 107 PFU), or with T(ASEL) or T(IEEL). On days 20, 22 and 24, surviving mice were treated intravenously with T(IEEL) (T(! ASEL)-T(IEEL) treatment) or ASEL (T(ASEL)-T(ASEL) treatment). (,) IL-17 () and IFN-γ () in splenocytes from mice that rejected TC2 and TC2R tumors after intravenous ASEL-T(IEEL) or T(ASEL)-T(IEEL) treatment as in ,, cocultured with lysates of TC2, TC2R, B16, normal mouse prostate (Pros) or pancreas (Panc) cells. Results are from three survivor mice. (,) IL-17 () and IFN-γ () in splenocytes from mice that did not reject tumors in ,, cocultured with the indicated lysates. Results are from two mice that succumbed to TC2R tumors. Error bars, s.d. of three separate wells per sample. * Figure 6: Immunogenicity of altered-self and self libraries. () Survival of mice bearing 7-day-old TC2 tumors (n = 7 or 8) injected intravenously with 1 × 107 PFU VSV-GFP, ASEL or SEL (days 7, 9 and 11). (–) IL-17 secreted from lymph node cells or splenocytes infected with VSV-GFP, no virus, ASEL or SEL (MOI = 1) for 2 weeks and cocultured with lysates of TC2, B16, normal mouse prostate (Pros), pancreas (Panc) or PBS. Error bars, s.d. of three separate wells per sample. Author information * Abstract * Author information Primary authors * These authors contributed equally to this work. * Alan Melcher & * Richard Vile Affiliations * Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, USA. * Timothy Kottke, * Jose Pulido, * Feorillo Galivo, * Jill Thompson, * Phonphimon Wongthida, * Rosa Maria Diaz & * Richard Vile * Leeds Institute of Molecular Medicine and Cancer Research UK Clinical Centre, St. James' University Hospital, Leeds, UK. * Fiona Errington, * Elizabeth Ilett, * John Chester, * Peter Selby, * Alan Melcher & * Richard Vile * Department of Ophthalmology and Ocular Oncology, Mayo Clinic, Rochester, Minnesota, USA. * Jose Pulido * St. George's Hospital Medical School, London, UK. * Heung Chong * University of Surrey, Guildford, UK. * Hardev Pandha * The Institute of Cancer Research, London, UK. * Kevin Harrington * Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA. * Richard Vile Contributions T.K., F.E., J.P., F.G., J.T., P.W., R.M.D., H.C. and E.I. performed experiments; J.P. J.C., H.P., K.H., P.S., A.M. and R.V. conceived the experimental approach, directed the experiments and interpreted the data. H.P., K.H., P.S., A.M. and R.V. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Richard Vile Author Details * Timothy Kottke Search for this author in: * NPG journals * PubMed * Google Scholar * Fiona Errington Search for this author in: * NPG journals * PubMed * Google Scholar * Jose Pulido Search for this author in: * NPG journals * PubMed * Google Scholar * Feorillo Galivo Search for this author in: * NPG journals * PubMed * Google Scholar * Jill Thompson Search for this author in: * NPG journals * PubMed * Google Scholar * Phonphimon Wongthida Search for this author in: * NPG journals * PubMed * Google Scholar * Rosa Maria Diaz Search for this author in: * NPG journals * PubMed * Google Scholar * Heung Chong Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth Ilett Search for this author in: * NPG journals * PubMed * Google Scholar * John Chester Search for this author in: * NPG journals * PubMed * Google Scholar * Hardev Pandha Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin Harrington Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Selby Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Melcher Search for this author in: * NPG journals * PubMed * Google Scholar * Richard Vile Contact Richard Vile Search for this author in: * NPG journals * PubMed * Google Scholar Additional data - Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy
- Nat Med 17(7):860-866 (2011)
Nature Medicine | Article Caspase 3–mediated stimulation of tumor cell repopulation during cancer radiotherapy * Qian Huang1, 2, 3, 12 * Fang Li2, 12 * Xinjian Liu2 * Wenrong Li2 * Wei Shi4 * Fei-Fei Liu4 * Brian O'Sullivan4 * Zhimin He2 * Yuanlin Peng5 * Aik-Choon Tan6 * Ling Zhou7 * Jingping Shen2 * Gangwen Han8 * Xiao-Jing Wang8, 9, 10 * Jackie Thorburn11 * Andrew Thorburn11 * Antonio Jimeno6, 9, 10 * David Raben2, 9 * Joel S Bedford5 * Chuan-Yuan Li2, 10, 11 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:860–866Year published:(2011)DOI:doi:10.1038/nm.2385Received17 November 2009Accepted27 April 2011Published online03 July 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg In cancer treatment, apoptosis is a well-recognized cell death mechanism through which cytotoxic agents kill tumor cells. Here we report that dying tumor cells use the apoptotic process to generate potent growth-stimulating signals to stimulate the repopulation of tumors undergoing radiotherapy. Furthermore, activated caspase 3, a key executioner in apoptosis, is involved in the growth stimulation. One downstream effector that caspase 3 regulates is prostaglandin E2 (PGE2), which can potently stimulate growth of surviving tumor cells. Deficiency of caspase 3 either in tumor cells or in tumor stroma caused substantial tumor sensitivity to radiotherapy in xenograft or mouse tumors. In human subjects with cancer, higher amounts of activated caspase 3 in tumor tissues are correlated with markedly increased rate of recurrence and death. We propose the existence of a cell death–induced tumor repopulation pathway in which caspase 3 has a major role. View full text Figures at a glance * Figure 1: In vitro and in vivo evidence for the generation of strong growth-stimulating signals in dying cells. () Stimulation of 4T1Fluc cellular growth in vitro by irradiated 4T1 cells. Top, growth of 4T1Fluc cells as observed by luciferase activities. The difference between each of the higher-dose irradiated groups (8, 10 and 12 Gy) and controls (0 Gy and no feeder) was statistically significant (P < 0.001, t test, n = 4). Bottom, representative images from bioluminescence imaging. () Top, relative growth of MEF-supported tumor cells versus tumor cells seeded alone. *P < 0.001, t test, n = 3. Bottom, representative bioluminescence images. (,) Effect of dying 4T1 () and MEF () cells on 4T1Fluc tumor cellular growth in vivo. Top, quantification of bioluminesent signals. Bottom, representative bioluminescent images of mice. In each of the two experiments, the difference between the two groups was highly significant (P < 0.001 from day 4 for , and from day 1 for , n = 5, one-way analysis of variance (ANOVA) test). In all cases, error bars indicate s.e.m. * Figure 2: The role of caspase 3 in cell death–induced tumor cell proliferation in vitro and in vivo. () Effects of dying wild-type (WT) and Casp3−/− MEF cells on different Fluc-labeled tumor cells. *P < 0.01, t test, n = 3. () The differences between the control groups and the Casp3-kn groups were statistically significant (P < 0.01 between each of the control and each of the caspase 3 knockdown clones, n = 3, t test). Bottom, western blot analysis of caspase 3 levels. () The effect of a dominant-negative caspase 3 (casp3DN) in dying, unlabeled 4T1 cells on 4T1Fluc tumor cell growth. Inset, western blot showing dominant-negative caspase 3 expression (casp3DN was hemagglutinin (HA) labeled). () Western blot analyses of key proteins involved in apoptosis. () The effect of dying wild-type and Casp3−/− MEF cells on growth of 4T1Fluc cells in mice. The difference between the two groups was highly significant statistically (P < 0.001 from day 3 on, n = 4, one-way ANOVA test). () The effect of caspase 3 knockdown in lethally irradiated 4T1 cells on growth of 4T1Fluc cells ! in vivo. The difference between the two groups was statistically significant from day 5 (P < 0.05, n = 5, one-way ANOVA test). In all panels, error bars represent s.e.m. * Figure 3: Relationship between caspase activation and growth of injected tumor cells in the irradiated tumor microenvironment. () Caspase 3 activation in 4T1 tumors as detected by a caspase 3 reporter. Left, schematic of the structure of a proteasome-based caspase 3 reporter (top left) and its mode of action (bottom left). Right, caspase 3 activities in 4T1 tumors transduced with the control as well as Casp3 reporter genes. The difference between the control and caspase 3 reporter groups are significant at days 3, 5 and 7 (P < 0.01, n = 5, t test). CMV, cytomegalovirus promoter; (Ubi)9, polyubiqutin domain consists of nine tandem copies of ubiquitin; pA, polyadenylation signal; DEVD, consensus caspase 3 cleavage site. () Top, growth of 4T1Fluc cells injected into irradiated and nonirradiated established tumors. The difference between the two groups was statistically significant (P < 0.05 from day 7, t test, n = 4). Bottom, representative images of tumor-bearing mice. () Immunofluorescence analysis of growth of intratumorally injected GFP-labeled cells and the indicated protein expression surrounding! the injected cells. SMA, smooth muscle actin, a marker for blood vessels. Scale bars, 100 μm. Error bars represent s.e.m. * Figure 4: A role for caspase 3–activated iPLA2 (Pla2g6) in facilitating cell death–stimulated tumor cell repopulation. () The effect of Pla2g6 levels in dying cells on the growth of 4T1Fluc cells in vitro. P < 0.01, n = 4, t test. () The effect of constitutively active (CA) iPLA2 ΔPla2g6 in Casp3−/− MEF cells in vivo. The differences between the luciferase signals were statistically significant (P < 0.01 on day 7, n = 5, t test). Inset, expression of truncated iPLA2. () The in vivo effect of iPLA2 knockdown in wild-type MEF cells. The difference between the two groups was statistically significant from day 3 (P < 0.02, from day 7, n = 5, one-way ANOVA). Inset, western blot analysis of shRNA knockdown of Pla2g6. Error bars represent s.e.m. * Figure 5: Regulation of radiation-induced arachidonic acid release and PGE2 production by caspase 3. () Arachidonic acid (AA) release. *P < 0.02 (t test, n = 3). () PGE2 secretion. *P < 0.05 (t test, n = 3). () PGE2-stimulated tumor growth from 1,000 4T1Fluc tumor cells injected subcutaneously into nude mice. The difference between the two groups are statistical significant from day 17 (P < 0.05, n = 5, one-way ANOVA). Error bars represent s.e.m. * Figure 6: Caspase 3 status correlated with tumor response to therapy in mice as well as in human subjects. () Result of radiation therapy in tumors established from MCF-7 and MFC-7CASP3 cells.The differences between the two groups are highly significant after radiotherapy (P < 1 × 10−6, n = 10, one-way ANOVA). () Results of radiation therapy on B16F10 mouse melanoma tumors grown in wild-type C57BL6 and Casp3−/− mice. The difference between the two groups are significant after radiotherapy from day 11 (P < 0.04, n = 5, one-way ANOVA). Error bars in and represent s.e.m. (,) Kaplan-Meier analyses of cancer recurrence in a cohort of subjects with head and neck squamous cell carcinoma (HNSCC) treated at Princess Margaret Hospital in Toronto () and of survival in a cohort of subjects with advanced breast cancer treated at Shanghai No.1 People's Hospital (). Log-rank tests were used for analysis of statistical significance. For , P = 0.0114, hazard ratio = 3.44, 95% confidence interval: 1.35–8.75. For , P = 0.0006, hazard ratio = 5.29, 95% confidence interval: 1.70–16.46. () ! A schematic representation of the proposed pathway for cell death–mediated tumor cell repopulation. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this study. * Qian Huang & * Fang Li Affiliations * Experimental Research Center, First People's Hospital, Shanghai Jiao Tong University, Shanghai, China. * Qian Huang * Department of Radiation Oncology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Qian Huang, * Fang Li, * Xinjian Liu, * Wenrong Li, * Zhimin He, * Jingping Shen, * David Raben & * Chuan-Yuan Li * National Laboratory of Oncogenes and Related Genes Research, Cancer Institute, Shanghai Jiao Tong University, Shanghai, China. * Qian Huang * Department of Radiation Oncology and Ontario Cancer Institute, Princess Margaret Hospital, University of Toronto, Toronto, Ontario, Canada. * Wei Shi, * Fei-Fei Liu & * Brian O'Sullivan * Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado, USA. * Yuanlin Peng & * Joel S Bedford * Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA. * Aik-Choon Tan & * Antonio Jimeno * Department of Surgery, Shanghai First People's Branch Hospital, Shanghai, China. * Ling Zhou * Department of Pathology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Gangwen Han & * Xiao-Jing Wang * Head and Neck Cancer Research Program, University of Colorado Cancer Center, Aurora, Colorado, USA. * Xiao-Jing Wang, * Antonio Jimeno & * David Raben * Charles C. Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Xiao-Jing Wang, * Antonio Jimeno & * Chuan-Yuan Li * Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA. * Jackie Thorburn, * Andrew Thorburn & * Chuan-Yuan Li Contributions Q.H. and F.L. designed and conducted most of the experiments, analyzed data and wrote the manuscript. X.L. and W.L. carried out analyses on modes of cell death in irradiated cells; W.S. carried out immunohistochemical analysis of human head and neck tumor samples; F.-F.L. and B.O. provided human head and neck cancer samples and analyzed data from the samples; Z.H. conducted some of the caspase reporter experiments; Y.P. carried out arachidonic acid release experiments and J.S.B. analyzed relevant data of arachidonic acid release; A.-C.T. carried out data analyses of human clinical data; G.H. and X.-J.W. helped with immunohistochemical analysis of mouse tumor samples; J.S. constructed some of the plasmids used; A.J. and D.R. provided human head and neck tumor samples; L.Z. carried out immunohistochemical analysis of human breast cancer samples; J.T. and A.T. helped to conduct experiments on autophagy and necrosis; C.-Y.L. conceived of the study, analyzed data and wrote the ma! nuscript. All authors read and agreed on the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Chuan-Yuan Li Author Details * Qian Huang Search for this author in: * NPG journals * PubMed * Google Scholar * Fang Li Search for this author in: * NPG journals * PubMed * Google Scholar * Xinjian Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Wenrong Li Search for this author in: * NPG journals * PubMed * Google Scholar * Wei Shi Search for this author in: * NPG journals * PubMed * Google Scholar * Fei-Fei Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Brian O'Sullivan Search for this author in: * NPG journals * PubMed * Google Scholar * Zhimin He Search for this author in: * NPG journals * PubMed * Google Scholar * Yuanlin Peng Search for this author in: * NPG journals * PubMed * Google Scholar * Aik-Choon Tan Search for this author in: * NPG journals * PubMed * Google Scholar * Ling Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Jingping Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Gangwen Han Search for this author in: * NPG journals * PubMed * Google Scholar * Xiao-Jing Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Jackie Thorburn Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew Thorburn Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Jimeno Search for this author in: * NPG journals * PubMed * Google Scholar * David Raben Search for this author in: * NPG journals * PubMed * Google Scholar * Joel S Bedford Search for this author in: * NPG journals * PubMed * Google Scholar * Chuan-Yuan Li Contact Chuan-Yuan Li 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 1–23, Supplementary Note, Supplementary Methods and Supplementary Tables 1–3 Additional data - Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs
- Nat Med 17(7):867-874 (2011)
Nature Medicine | Article Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs * Thordur Oskarsson1 * Swarnali Acharyya1 * Xiang H-F Zhang1 * Sakari Vanharanta1 * Sohail F Tavazoie1, 7 * Patrick G Morris2 * Robert J Downey3 * Katia Manova-Todorova4 * Edi Brogi5 * Joan Massagué1, 6 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:867–874Year published:(2011)DOI:doi:10.1038/nm.2379Received08 October 2010Accepted18 April 2011Published online26 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We report that breast cancer cells that infiltrate the lungs support their own metastasis-initiating ability by expressing tenascin C (TNC). We find that the expression of TNC, an extracellular matrix protein of stem cell niches, is associated with the aggressiveness of pulmonary metastasis. Cancer cell–derived TNC promotes the survival and outgrowth of pulmonary micrometastases. TNC enhances the expression of stem cell signaling components, musashi homolog 1 (MSI1) and leucine-rich repeat–containing G protein–coupled receptor 5 (LGR5). MSI1 is a positive regulator of NOTCH signaling, whereas LGR5 is a target gene of the WNT pathway. TNC modulation of stem cell signaling occurs without affecting the expression of transcriptional enforcers of the stem cell phenotype and pluripotency, namely nanog homeobox (NANOG), POU class 5 homeobox 1 (POU5F1), also known as OCT4, and SRY-box 2 (SOX2). TNC protects MSI1-dependent NOTCH signaling from inhibition by signal transducer an! d activator of transcription 5 (STAT5), and selectively enhances the expression of LGR5 as a WNT target gene. Cancer cell–derived TNC remains essential for metastasis outgrowth until the tumor stroma takes over as a source of TNC. These findings link TNC to pathways that support the fitness of metastasis-initiating breast cancer cells and highlight the relevance of TNC as an extracellular matrix component of the metastatic niche. View full text Figures at a glance * Figure 1: TNC expression in lung metastatic foci and association with lung relapse. () Heterogeneous TNC expression in human lung metastasis. TNC immunostaining (arrows) on lung metastasis section from an individual with breast cancer. Scale bar, 50 μm. () Kaplan-Meier analysis of lung metastasis–free survival in subjects with breast cancer, on the basis of TNC expression in dissected lung metastases. TNC immunostaining of tissue sections was used to classify metastases into two groups, TNC low (n = 51) or TNC high (n = 15). P value was determined by log-rank test. () Immunohistochemical analysis of TNC expression in lung metastatic foci of various sizes formed by MDA231-LM2 cells in mice. TNC accumulation at the invasive front in larger metastatic foci. Arrows, TNC expression. Scale bar, 50 μm. () Schematic of orthotopic metastasis assay in mice. MDA231-LM2 or CN34-LM1 cells were injected bilaterally into the fourth mammary fat pads leading to spontaneous lung metastasis. () Schematic of lung colonization assay by intravenous injection of cancer cells.! () Growth curves of mammary tumors arising from MDA231-LM2 or CN34-LM1 cells and their TNC knockdown counterparts. Mammary tumor growth was measured with a digital caliper. Values are means ± s.e.m. () Lung metastasis in mammary tumor–bearing mice determined at week 6 by ex vivo luciferase luminescence. MDA231-LM2 control, n = 15; shTNC(1), n = 15; shTNC(2), n = 14. CN34-LM1 control, n = 10; shTNC(1), n = 8; shTNC(2), n = 10. P values were obtained by two-tailed Student's t test. () Lung colonization, as determined by bioluminescence, in mice injected intravenously with control and shTNC-transduced CN34LM1 cells . Values are mean ± s.e.m. P values were determined by Student's t test. () Representative luminescence images and H&E-stained lung sections from each group. Arrows, metastatic lesions. Scale bar, 100 μm. * Figure 2: Cancer cell–derived TNC mediates resistance to apoptosis in micrometastasis. (,) Apoptosis in early metastatic foci analyzed by immunohistochemistry. Average number () and representative images () of MDA231-LM2 apoptotic foci containing cleaved caspase 3+ cells at day 10 of lung colonization. n = 3 mice per condition; values are mean ± s.e.m. Arrows, apoptotic cells. M, metastatic foci. L, lung parenchyma. Scale bar, 20 μm. () Schematic of conditional TNC knockdown experiment. Lung colonization of MDA231-LM2 cells containing doxycycline (Dox)-inducible TNC shRNA (Tet-shTNC) was allowed for different lengths of time (day x) before TNC knockdown was induced by administration of doxycycline to the mice. Lung colonies were then analyzed (day y). () TNC staining in lung metastatic foci in control or Tet-shTNC MDA231-LM2. Cells were injected intravenously and allowed to colonize the lungs for 25 d. Thereafter, TNC knockdown was induced with doxycycline for 10 d, and lung sections were analyzed by TNC immunohistochemistry (arrows). Scale bar, 20 μm. () A! nalysis of apoptosis in established lung foci. Cleaved caspase 3 was quantified in control and Tet-shTNC (day 25 of lung colonization) mice after doxycycline administration for an additional 10 d. n = 3 mice per condition; values are mean ± s.e.m. () Representative examples of apoptotic cells at the invasive edge of established metastatic foci. Arrows, cleaved caspase 3+ cells. Scale bar, 20 μm. () Bioluminescence analysis of MDA231-LM2–mediated lung colonization in which TNC was conditionally knocked down at different time points. Left, TNC knocked down by 2-week-long doxycycline treatment starting 2 d after tail vein injection. Middle, doxycycline treatment at day 6. Right, doxycycline induction at day 21, maintained for 2 weeks. Two-tailed Student's t test was used to determine all P values. () Immunofluorescence analysis of TNC expression in MDA231-LM2 metastatic foci at different time points during lung colonization. Time points analyzed were days 10 and 36 (D10 an! d D36, respectively) after intravenous inoculation. Antibodies! to stromal derived mouse TNC (mTNC) and tumor cell–derived human TNC (hTNC) were used. DAPI was used for nuclear staining. Scale bar, 20 μm. * Figure 3: Expression of TNC and stem cell markers in oncospheres. () Examples of tumor spheres from various pleural effusion samples from subjects with breast cancer, grown in serum-free medium on nonadhesive plates (scale bar, 20 μm). (–) Gene expression analysis of oncospheres derived from pleural effusion samples compared to monolayer cultures of the same samples. Gene expression was analyzed by quantitative RT-PCR (qRT-PCR). TNC expression () was upregulated in oncospheres together with the expression of embryonic stem cell markers NANOG, OCT4 and SOX2 () and adult stem cell markers MSI1 and LGR5 (). In contrast, the mammary differentiation marker GATA3 and the TNC- and metastasis-suppressor miRNA miR-335 were downregulated in oncospheres (). n = 9–10 pleural fluid–derived cell samples. All P values were calculated by paired Wilcoxon signed rank test (two tailed). Whiskers represent minimum and maximum values. * Figure 4: Regulation of oncosphere growth, MSI1 and LGR5 by TNC. () TNC expression in oncospheres derived from CN34-LM1 cells and CN127 breast cancer cells expressing two independent TNC shRNAs. () Quantitative analysis of oncosphere formation in TNC-deficient CN34-LM1 cells. () Gene expression analysis in TNC-deficient oncospheres. Expression was analyzed by qRT-PCR; values are mean ± s.d. of triplicate experiments. For all genes, the expression in knockdown lines was normalized to control. () Lung colonization of MDA231-LM2 and CN34-LM1 cells transduced with short hairpins against MSI1 and LGR5 individually and in combination. Ctr, control. Student's t test was used to determine P values. n = 5 mice per condition. Whiskers represent minimum and maximum values. * Figure 5: TNC enhances WNT and NOTCH signaling in breast cancer cells. () WNT3A induced LGR5, LEF1 and AXIN2 expression in CN34-LM1 oncospheres. Ctr, control. (,) MSI1 expression induced in TNC knockdown CN34-LM1 oncospheres by treatment with JAK2 inhibitor AG490 () or STAT5 inhibitor nicotinoyl hydrazone (). () Expression of NOTCH target gene HEY2 in TNC knockdown oncospheres. () HEY2 expression in control or TNC knockdown CN34-LM1 and MDA231-LM2 cells transduced with MSI1 expression vector. () Lung luminescence in mice intravenously injected with CN34-LM1 or MDA231-LM2 cells. Control and shTNC knockdown cells were transduced with vector expressing MSI1 or control vector. Normalized photon flux was measured 1 week after tail vein injection. n = 5 mice per condition. P values were obtained using a two-tailed Student's t test. Whiskers represent minimum and maximum values. () Representative images of H&E-stained lung sections from experiment in at 6 weeks. () Immunohistochemical analysis of cleaved caspase 3 in metastatic foci 10 d after tail ve! in injection. Representative images from MDA231-LM2 shTNC cells with or without ectopic MSI1 expression. Scale bar, 20 μm. () Number of cleaved caspase 3+ foci formed by MDA231-LM2 cells at day 10 of lung colonization. Control and shTNC cells were analyzed with or without overexpression of MSI1. n = 4 mice per condition. P values were obtained by two-tailed Student's t test. Error bars represent standard deviation. * Figure 6: TNC association with STAT5 and Musashi in breast cancer metastasis. () Proportion of TNCS+ cases in primary breast tumors and metastatic nodules from different organ sites. Primary breast tumors (n = 344), lung metastases (n = 18), bone metastasis (n = 16), brain metastasis (n = 19) and other metastasis (5 liver, 7 ovary, 1 chest wall and 1 duodenum). *P = 0.0033, **P = 0.01, ***P = 0.008, calculated by Fisher's exact tests. () Kaplan-Meier analysis of bone metastatic free survival (left) and brain metastatic survival (right) in a xenograft mouse model. MDA231-BoM1 and MDA231-BrM2 transduced with shTNC were injected intracardially and metastasis was determined by bioluminescence. Bone metastasis experiment: control, n = 9; shTNC, n = 15 (pooled samples from two TNC hairpins). Brain metastasis experiment: control, n = 9; shTNC, n = 17 (pooled samples from two TNC hairpins). P values were determined by log-rank test. () Relative levels of MSI1 in TNCS+ and TNCS− human metastasis samples. P value was calculated by two tailed Student's t test.! () GSEA. Analysis of genes either up- or downregulated in the STAT5 signature (STAT5S) and their enrichment within TNCS+ metastases. P values were determined by a random permutation test45. () Relative expression of MSI1 within STAT5+ or STAT5− human breast cancer metastases. () Summary model of the role of TNC in the outgrowth of micrometastasis. Metastasis-initiating breast cancer cells with low expression of the metastasis suppressor miR-335 highly express TNC, supporting the survival and outgrowth of these cells in the pulmonary parenchyma. TNC interaction with cancer cells at the invasive front enhances signaling by the NOTCH and WNT pathways, promoting the viability of these cells and the reinitiation of metastatic outgrowth. Author information * Abstract * Author information * Supplementary information Affiliations * Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, New York, USA. * Thordur Oskarsson, * Swarnali Acharyya, * Xiang H-F Zhang, * Sakari Vanharanta, * Sohail F Tavazoie & * Joan Massagué * Department of Medicine, MSKCC, New York, New York, USA. * Patrick G Morris * Department of Surgery, MSKCC, New York, New York, USA. * Robert J Downey * Molecular Cytology Core Facility, MSKCC, New York, New York, USA. * Katia Manova-Todorova * Department of Pathology, MSKCC, New York, New York, USA. * Edi Brogi * Howard Hughes Medical Institute, MSKCC, New York, New York, USA. * Joan Massagué * Present address: The Rockefeller University, New York, New York, USA. * Sohail F Tavazoie Contributions T.O. and J.M. designed experiments, analyzed data and wrote the manuscript. J.M. supervised research. T.O. carried out experiments. S.A. carried out immunostaining and helped with pathway analyses. X.H.-F.Z. carried out bioinformatics analyses. S.V. carried out intracardiac injections and assisted with bone and brain metastasis assays. S.F.T. helped with miRNA analysis. P.G.M. and R.J.D. oversaw collection of clinical samples. K.M.-T. supervised histological staining and analysis. E.B. obtained and evaluated human breast cancer tissue sections. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joan Massagué Author Details * Thordur Oskarsson Search for this author in: * NPG journals * PubMed * Google Scholar * Swarnali Acharyya Search for this author in: * NPG journals * PubMed * Google Scholar * Xiang H-F Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Sakari Vanharanta Search for this author in: * NPG journals * PubMed * Google Scholar * Sohail F Tavazoie Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick G Morris Search for this author in: * NPG journals * PubMed * Google Scholar * Robert J Downey Search for this author in: * NPG journals * PubMed * Google Scholar * Katia Manova-Todorova Search for this author in: * NPG journals * PubMed * Google Scholar * Edi Brogi Search for this author in: * NPG journals * PubMed * Google Scholar * Joan Massagué Contact Joan Massagué Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–22, Supplementary Tables 1–4 and Supplementary Methods Additional data - Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition
- Nat Med 17(7):875-882 (2011)
Nature Medicine | Article Compromised CDK1 activity sensitizes BRCA-proficient cancers to PARP inhibition * Neil Johnson1, 2 * Yu-Chen Li1 * Zandra E Walton1 * Katherine A Cheng1 * Danan Li1 * Scott J Rodig3, 4 * Lisa A Moreau5, 6 * Christine Unitt3, 4 * Roderick T Bronson4 * Huw D Thomas7 * David R Newell7 * Alan D D'Andrea5, 6 * Nicola J Curtin7 * Kwok-Kin Wong1, 2, 8 * Geoffrey I Shapiro1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:875–882Year published:(2011)DOI:doi:10.1038/nm.2377Received19 August 2010Accepted13 April 2011Published online26 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Cells that are deficient in homologous recombination, such as those that lack functional breast cancer–associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA–wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function a! nd consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers. View full text Figures at a glance * Figure 1: Cdk1 depletion or inhibition reduces the formation of Rad51 foci and homologous recombination. () Detection of BRCA1, Rad51 and DAPI by immunofluorescence after γ irradiation in empty vector (V), wild-type (WT) or S1189A S1191A S1497A mutant, HA-tagged BRCA1–expressing MDA-MB-436 cells. Left, representative foci-containing cells; Right, mean ± s.e.m. number of BRCA1-expressing cells with ≥5 Rad51 foci over three experiments. (,) Top, representative foci-containing cells. Bottom, mean ± s.e.m. number of cells containing ≥5 Rad51 and γ-H2AX foci over three experiments. () Detection of Rad51, γ-H2AX and DAPI by immunofluorescence in NCI-H1299 cells inducibly expressing shRNA targeting Cdk1, untreated or treated with γ irradiation with or without doxycycline. Western blots show Cdk1 knockdown. () NCI-H1299 cells, untreated (No Rx) or treated with γ irradiation (IR) with DMSO or RO-3306 and stained as in . () Detection and quantification of GFP-positive U2OS pDR-GFP cells after treatment with scrambled siRNA (Scr) or siRNAs targeting BRCA1 (BR1) or Cdk1 (1–4! individual siRNAs and 1–4 pooled). Western blots show Cdk1 knockdown. () Quantification of GFP-positive U2OS pDR-GFP cells expressing empty vector (V) or Cdk1 containing a silent mutation (SM) after treatment with scrambled siRNA (Scr) or Cdk1 siRNA. Western blots show protein knockdown. () Detection and quantification of GFP-positive U2OS pDR-GFP cells after treatment with DMSO, RO-3306 or AG024322. For –, mean ± s.e.m. number of GFP-positive cells is expressed as a percentage of scrambled siRNA or DMSO-treated controls over three experiments. *P < 0.05. Scale bars, 10 μm. * Figure 2: Cdk1 depletion results in reduced Rad51 foci, multiple chromosome aberrations, G2-M accumulation and cell death after PARP inhibition. () Detection of BRCA1, Rad51, γ-H2AX and DAPI by immunofluorescence in NCI-H1299 cells inducibly expressing shRNA targeting Cdk2 or Cdk1 treated with AG14361 with or without doxycycline. Top, representative foci-containing cells. Bottom, mean ± s.e.m. percentage of cells containing more than five foci over three experiments. () Metaphase spread analyses of NCI-H1299-Cdk1 cells analyzed for chromosomal breaks after 24 h of treatment with AG14361. Top, representative metaphase spreads; arrows indicate chromosomal aberrations. Bottom, mean ± s.e.m. number of chromosome aberrations per cell over three experiments. *P < 0.05. () Cell cycle profiles (left) and detection of TUNEL- positive (middle) NCI-H1299-Cdk2 cells treated with DMSO or AG14361 with or without doxycycline. Vertical lines, TUNEL-positive threshold. Right, mean ± s.e.m. percentage of TUNEL-positive cells in G1, S and G2-M over three experiments. () NCI-H1299-Cdk1 cells treated and analyzed as in . Scale bars, ! 10 μm. * Figure 3: Cdk1-depleted or Cdk1-inhibited cells are highly sensitive to PARP inhibition. () Colony formation of NCI-H1299 and A549 cells inducibly expressing shRNAs targeting Cdk2 (left) or Cdk1 (right) treated with AG14361 with or without doxycycline. Survival is expressed as a percentage ± s.e.m. of colonies formed relative to DMSO-treated cells in the absence or presence or doxycycline. () Colony formation of NCI-H1299-Cdk2 or NCI-H1299-Cdk1 cells treated with shRNA targeting luciferase or PARP-1 with or without doxycycline, expressed as a percentage ± s.e.m. of colonies formed relative to cells treated with shRNA targeting luciferase in the absence or presence of doxycycline. Western blot shows PARP-1 knockdown. *P ≤ 0.0011. () Colony formation of NCI-H1299-Cdk1 cells expressing empty vector (V) or Cdk1 containing a silent mutation (SM) treated with AG014699 with or without doxycycline. Survival is expressed as a percentage ± s.e.m. of colonies formed relative to DMSO-treated cells in the absence or presence of doxycycline. Western blot shows Cdk1 knock! down. () Colony formation of NCI-H1299 cells treated with DMSO or RO-3306 and AG14361 or AG014699. Survival is expressed as a percentage ± s.e.m. of colonies formed relative to DMSO-treated cells in the presence of DMSO or RO-3306. () Colony formation of NCI-H1299 cells treated with DMSO or AG024322 and AG014699. Survival is expressed as a percentage ± s.e.m. of colonies formed relative to DMSO-treated cells in the presence of DMSO or AG024322. () Colony formation of MDA-MB-436 cells expressing empty vector (V), wild-type (WT) or S1189A S1191A S1497A (TM) BRCA1 treated with AG014699. Survival is expressed as a percentage ± s.e.m. of colonies formed relative to cells treated with DMSO in the presence of the respective constructs. Western blot shows BRCA1 protein expression. () Colony formation of MDA-MB-436 cells expressing WT or S1189A S1191A S1497A mutant BRCA1 treated with DMSO plus AG014699 (D) or RO-3306 plus AG014699 (RO). Survival is expressed as a percentage ± s.! e.m. of colonies formed compared to the corresponding DMSO or ! RO-3306-treated control. *P = 0.0094, comparing RO-3306 plus AG014699 with DMSO plus AG014699 in wild-type cells. () Colony formation of NCI-H1299-Cdk1 cells transfected with either scrambled (Scr) or BRCA1 siRNA, treated with vehicle or AG014699 with or without doxycycline. Survival is expressed as a percentage ± s.e.m. of colonies formed compared to the corresponding vehicle-treated control. *P = 0.0154 for scrambled siRNA, comparing the presence and absence of doxycycline. * Figure 4: Cdk1 depletion or inhibition protects nontransformed cells from PARP inhibitor treatment. () Colony formation of cell lines treated with DMSO or RO-3306 and AG014699. Mean ± s.e.m. LC50 values for AG014699 from DMSO (D)- or RO-3306 (R)-treated cells. *P ≤ 0.003. () Colony formation of RPE and NCI-H1299 cells treated with scrambled siRNA (Scr) or Cdk1 siRNA before treatment with AG014699 for 72 h followed by replating for colony formation, expressed as a percentage ± s.e.m. over three experiments of colonies formed in DMSO-treated cells in the presence of scrambled or Cdk1 siRNA. *P = 0.018. () Cell cycle profiles (left) and TUNEL-positive (middle) NCI-H1299 cells transfected with scrambled siRNA (Scr) or Cdk1 siRNA for 48 h, and subsequently treated with DMSO or AG014699 for the indicated times. Right, mean ± s.e.m. percentage of TUNEL-positive cells at 120 h over three experiments. *P = 0.0191. PI, propidium iodide. () Western blot analyses of cells treated as in after 24 h AG014699 treatment. () Viability of Hs578T and Hs578Bst cell lines treated with DMSO! or RO-3306 and increasing concentrations of AG014699, expressed as a percentage ± s.e.m. over three experiments of colonies formed in DMSO-treated cells in the presence of DMSO or RO-3306. () Viability of Hs578T and Hs578TBst cell lines treated with DMSO or AG024322 and AG014699, expressed as percentage ± s.e.m. over three experiments of colonies formed in DMSO-treated cells in the presence of DMSO or AG024322. *P = 0.0023. * Figure 5: Cdk1 inhibition sensitizes cancer cells to inhibition of PARP in vivo. () Growth of NCI-H1299-Cdk1 xenografts in mice given regular or doxycycline-containing diets, treated with vehicle or AG014699 over days 1–23. Mean RTV ± s.e.m. (n = 6), is expressed compared to tumor volumes on day 1. () RTV for individual mice treated in at day 13. () Expression of Cdk1, Cdk2, γ-H2AX and tubulin from representative tumors in mice killed on day 23 measured by western blot. () Growth of NCI-H1299 xenografts over 19 d in mice receiving the indicated treatments. V, vehicle. Mean RTV ± s.e.m. (n = 6) is expressed compared to tumor volumes on day 1. () RTV for individual mice treated in at day 13. () Change in weight of individual mice treated as in at day 18. () Immunohistochemical analysis for BRCA1 (pSer1189), total BRCA1 and γ-H2AX focus formation and Aurora B and TUNEL staining in NCI-H1299 xenografts collected from mice treated for five continuous days with the indicated treatments. Left, representative sections, stained as indicated. Right, mean ± ! s.e.m. foci- or staining-positive cells. *P < 0.05. Scale bars, 10 μm. * Figure 6: Combined inhibition of Cdk1 and PARP causes tumor regression and prolongs survival in the KrasG12DTrp53L/L mutant lung cancer mouse model. () Left, representative MRI images of lung tumor volumes before and after one week of the indicated treatments. Colored arrows show matched lesions in images taken before and after treatment; scale bars, 4.5 mm. Middle, representative H&E stains (scale bars, 500 μm), as well as Ki67 and TUNEL staining of tumors after the indicated treatments (scale bars, 100 μm). Graphs show the mean ± s.e.m. number of positive cells; results for two mice treated with both AG024322 and AG014699 are shown. *P ≤ 0.0002; **P < 0.002 of treatment compared to vehicle. Right, western blot showing BRCA1 expression in mouse normal lung or tumor tissue. () Waterfall plot showing percentage change in tumor volume measured by MRI after 1 week of treatment compared to start of treatment for each mouse. Inset, mean ± s.e.m. relative tumor volume over the first 3 weeks of treatment for mice treated as indicated. At 1 and 3 weeks, each data point represents the average of 4–16 mice; at 2 weeks, we ! analyzed two to four mice in each group. At 3 weeks, the s.e.m. for mice treated with AG014699 was 171.2, on the basis of one tumor that was increased by >900-fold. () Kaplan-Meier analyses showing median survival times from the start of treatment of mice treated with vehicle, AG024322, AG014699 or both of 5.1, 6, 5.6 and 9.5 weeks, respectively. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA. * Neil Johnson, * Yu-Chen Li, * Zandra E Walton, * Katherine A Cheng, * Danan Li, * Kwok-Kin Wong & * Geoffrey I Shapiro * Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Neil Johnson, * Kwok-Kin Wong & * Geoffrey I Shapiro * Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA. * Scott J Rodig & * Christine Unitt * Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA. * Scott J Rodig, * Christine Unitt & * Roderick T Bronson * Department of Radiation Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA. * Lisa A Moreau & * Alan D D'Andrea * Department of Pediatrics, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Lisa A Moreau & * Alan D D'Andrea * Northern Institute for Cancer Research, University of Newcastle, Newcastle upon Tyne, UK. * Huw D Thomas, * David R Newell & * Nicola J Curtin * Ludwig Center at Dana-Farber/Harvard Cancer Center, Boston, Massachusetts, USA. * Kwok-Kin Wong Contributions N.J. and G.I.S. designed this study. N.J., Y-C.L., L.A.M., D.L., Z.E.W., K.A.C., C.U., R.T.B. and H.D.T. performed the experiments. N.J. and G.I.S. analyzed the data. N.J. and G.I.S. communicated with S.J.R., K.-K.W., D.R.N., A.D.d'A. and N.J.C. about the data interpretation and wrote the manuscript. G.I.S. supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Geoffrey I Shapiro Author Details * Neil Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Yu-Chen Li Search for this author in: * NPG journals * PubMed * Google Scholar * Zandra E Walton Search for this author in: * NPG journals * PubMed * Google Scholar * Katherine A Cheng Search for this author in: * NPG journals * PubMed * Google Scholar * Danan Li Search for this author in: * NPG journals * PubMed * Google Scholar * Scott J Rodig Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa A Moreau Search for this author in: * NPG journals * PubMed * Google Scholar * Christine Unitt Search for this author in: * NPG journals * PubMed * Google Scholar * Roderick T Bronson Search for this author in: * NPG journals * PubMed * Google Scholar * Huw D Thomas Search for this author in: * NPG journals * PubMed * Google Scholar * David R Newell Search for this author in: * NPG journals * PubMed * Google Scholar * Alan D D'Andrea Search for this author in: * NPG journals * PubMed * Google Scholar * Nicola J Curtin Search for this author in: * NPG journals * PubMed * Google Scholar * Kwok-Kin Wong Search for this author in: * NPG journals * PubMed * Google Scholar * Geoffrey I Shapiro Contact Geoffrey I Shapiro Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (967K) Supplementary Figures 1–8 and Supplementary Methods Additional data - Uncoupling the mechanisms of obesity and hypertension by targeting hypothalamic IKK-β and NF-κB
- Nat Med 17(7):883-887 (2011)
Nature Medicine | Letter Uncoupling the mechanisms of obesity and hypertension by targeting hypothalamic IKK-β and NF-κB * Sudarshana Purkayastha1, 2, 3 * Guo Zhang1, 2, 3 * Dongsheng Cai1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 17,Pages:883–887Year published:(2011)DOI:doi:10.1038/nm.2372Received14 January 2011Accepted05 April 2011Published online05 June 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Obesity-related hypertension has become an epidemic health problem and a major risk factor for the development of cardiovascular disease (CVD). Recent research on the pathophysiology of obesity has implicated a role for the hypothalamus in the pathogenesis of this condition1, 2, 3. However, it remains unknown whether the often-seen coupling of hypertension with obesity can also be explained by hypothalamic dysfunction, despite the emerging appreciation that many forms of hypertension are neurogenic in origin4, 5, 6, 7, 8, 9, 10, 11, 12, 13. Our studies here revealed that acute activation of the proinflammatory protein nuclear factor κB (NF-κB) and its upstream activator IκB kinase-β (IKK-β, encoded by Ikbkb) in the mediobasal hypothalamus rapidly elevated blood pressure in mice independently of obesity. This form of hypothalamic inflammation-induced hypertension involved the sympathetic upregulation of hemodynamics and was reversed by sympathetic suppression. Loss-of-fu! nction studies further showed that NF-κB inhibition in the mediobasal hypothalamus counteracted obesity-related hypertension in a manner that was dissociable from changes in body weight. In addition, we found that pro-opiomelanocortin (POMC) neurons were crucial for the hypertensive effects of the activation of hypothalamic IKK-β and NF-κB, which underlie obesity-related hypertension. In conclusion, obesity-associated activation of IKK-β and NF-κB in the mediobasal hypothalamus—particularly in the hypothalamic POMC neurons—is a primary pathogenic link between obesity and hypertension. Breaking this pathogenic link may represent an avenue for controlling obesity-related hypertension and CVD without requiring obesity control. View full text Figures at a glance * Figure 1: Effects of manipulating hypothalamic IKK-β and NF-κB on blood pressure in C57BL/6 mice. (–) Representative 30-min telemetric blood pressure (BP) tracings during the light phase () versus the dark phase (); average values of mean blood pressure (MBP) in the indicated mice during the light phase () versus the dark phase (); and LF-BPV () and LF/HF-HRV () in chow-fed or HFD-fed mice at 1 week after hypothalamic injection of IKK-βCA–, IκBαDN-, or GFP-expressing adenoviruses. *P < 0.05, **P < 0.01; n = 3 or 4 mice per group. Error bars show means ± s.e.m. * Figure 2: Effects of TNF-α injection via the third ventricle on blood pressure in C57BL/6 mice. (–) Longitudinal profiles of systolic blood pressure (), diastolic blood pressure () and mean blood pressure (); dose-dependent increases (Δ) in mean blood pressure averaged over a 15-min peak change period (); LF-BPV () and LF/HF-HRV () over the same15-min period; and blood norepinephrine (NE) concentrations () in C57BL/6 mice after injection of TNF-α or vehicle. () Systolic (), diastolic () and mean () blood pressure levels over a 15-min peak change period in adult C57BL/6 mice that received TNF-α or vehicle injection in the presence (+) or absence (−) of prior i.p. injection of an adrenergic blocker, prazosin (Prz). The dose of injected TNF-α in – and – was 0.5 ng. *P < 0.05, **P < 0.01, NS, nonsignificant; n = 4–6 per group. Error bars show means ± s.e.m. SBP, systolic BP; DBP, diastolic blood pressure; MBP, mean blood pressure. * Figure 3: Activation of IKK-β and NF-κB by TNF-α in POMC neurons. (,) Immunostaining of phosphorylated IKK-β (red) () or IκBα (red) () in POMC neurons (green) (,) of Pomc-ROSA mice treated with TNF-α or vehicle via the third ventricle (3V). pIKK-β: phosphorylated IKK-β; scale bar, 50 μm. We counted the cell numbers of pIKK-β–positive (pIKK-β+) POMC neurons () or IκBα-positive (IκBα+) POMC neurons () as well as the total numbers (total) of POMC neurons (,) in the brain sections across the arcuate nucleus (ARC) . Data represent average cell numbers unilaterally in the median ARC. ***P < 0.001; NS, nonsignificant; n = 3 or 4 per group. Error bars show means ± s.e.m. * Figure 4: Hypotensive effect of POMC neuron-specific IKK-β ablation. (–) Average increases (Δ) in systolic blood pressure (), diastolic blood pressure (), mean blood pressure (), LF-BPV () and LF/HF-HRV () of chow-fed Pomc-Ikbkblox/lox mice, Agrp-Ikbkblox/lox mice, and the genotype-matched control Ikbkblox/lox mice in response to a third-ventricle injection of TNF-α or vehicle during a 15-min peak change period. () Mean blood pressure values in chow- vs. HFD-fed Pomc-Ikbkblox/lox mice and control Ikbkblox/lox mice. () Proposed role of hypothalamic IKK-β and NF-κB in obesity-related hypertension. Arrows in boxes indicates upregulation of IKK-β and NF-κB; dashed boxes indicate the parallel pathway described previously. SBP, systolic BP; DBP, diastolic blood pressure; MBP, mean blood pressure. *P < 0.05, n = 3 or 4 per group. Error bars show means ± s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sudarshana Purkayastha & * Guo Zhang Affiliations * Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, New York, USA. * Sudarshana Purkayastha, * Guo Zhang & * Dongsheng Cai * Diabetes Research Center, Albert Einstein College of Medicine, New York, New York, USA. * Sudarshana Purkayastha, * Guo Zhang & * Dongsheng Cai Contributions D.C. conceived and designed the study; S.P. did experiments shown in Figures 1, 2 and 4 with assistance from G.Z. and D.C.; and G.Z. also carried out experiments shown in Figure 3. All authors did data analysis and interpretation. D.C. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dongsheng Cai Author Details * Sudarshana Purkayastha Search for this author in: * NPG journals * PubMed * Google Scholar * Guo Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Dongsheng Cai Contact Dongsheng Cai Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (778K) Supplementary Figures 1–8 Additional data - Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans
- Nat Med 17(7):888-892 (2011)
Nature Medicine | Letter Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans * Rayner Rodriguez-Diaz1, 2, 7 * Robin Dando3, 7 * M Caroline Jacques-Silva1 * Alberto Fachado1 * Judith Molina4 * Midhat H Abdulreda1 * Camillo Ricordi1, 2 * Stephen D Roper3, 5 * Per-Olof Berggren1, 2, 6 * Alejandro Caicedo1, 3, 4, 5 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:888–892Year published:(2011)DOI:doi:10.1038/nm.2371Received29 November 2011Accepted05 April 2011Published online19 June 2011 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell1, 2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1, 3, 4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes! the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. View full text Figures at a glance * Figure 1: Endocrine cells in human pancreatic islets express cholinergic markers. () Z-stack of confocal images of a mouse pancreatic section showing an islet immunostained for vesicular acetylcholine transporter (vAChT, green) and glucagon (red). () Z-stack of confocal images of a human pancreatic section showing vAChT immunostaining in islet cells. Merge of glucagon and vAChT immunostaining appears yellow. () Z-stack of confocal images of a human pancreatic section showing lack of vAChT staining in human islets after preincubation with control peptide. Scale bars, 50 μm (–). () Western blotting analyses of lysates from four separate human islet preparations (HI1–HI4) and human pancreatic exocrine tissue (HP), with mouse brain (MB) as a positive control. Specific bands were seen in human islet lysates for vAChT (~70 kDa; top), for choline acetyltransferase (~63 kDa and ~68 kDa; middle) and for ChT1 (~68 kDa; bottom). A molecular marker was run in parallel (second lane). (–) vAChT (), ChAT () and ChT1 () mRNA expression in brain (B, n = 4), human i! slets (I, n = 12) and human pancreas (P, n = 3). Data represent means ± s.e.m. () vAChT mRNA levels were associated with ChAT mRNA levels (r2 = 0.57; slope significantly different from 0, P < 0.01). * Figure 2: Human alpha cells express vAChT and ChAT. () Confocal images of human pancreatic sections showing vAChT immunostaining (green) co-stained with glucagon immunostaining (red, left), with insulin immunostaining (red, middle) or with somatostatin immunostaining (red, right). Colocalization appears yellow. () Quantification of the percentage of vAChT immunostained cells also labeled for glucagon, insulin or somatostatin (n = 3 human pancreata). Percentages do not add exactly to 100% because analyses were performed on different sections. () Glucagon (red) and vAChT immunostaining (green) in alpha cells at high magnification. Shown are three optical planes through an alpha cell. () Scatter plots of pixel intensities (PI) of glucagon and DAPI immunofluorescence in alpha cells (left, top), insulin and C peptide immunofluorescence in beta cells (left, middle) and glucagon and vAChT immunofluorescence in alpha cells (left, bottom). Bar graph (right) shows the thresholded Pearson's correlation coefficient values for glucagon-DA! PI (left column) insulin–C peptide (middle column) and glucagon-vAChT colocalization (right column; n = 12 cells, analysis of variance (ANOVA) followed by multiple comparison, *P < 0.05). () ChAT immunostaining (green, left) in glucagon-labeled alpha cells (red, middle). Colocalization appears yellow (merge, right). () High magnification confocal image of an alpha cell stained for glucagon (red) and ChAT (green). Scale bars, 50 μm (,) and 5 μm (,). Data represent means ± s.e.m. * Figure 3: Isolated human islets secrete acetylcholine (ACh) in response to alpha cell–specific stimuli. () Photomicrograph of an ACh biosensor (colorized green) apposed to an isolated human islet to monitor ACh secretion evoked by stimulation of islet cells. Responses in the biosensor were recorded by loading biosensors with Fura-2 and imaging cytoplasmic [Ca2+]. Scale bar, 50 μm. () Δ[Ca2+]i (Δ340/380) responses in biosensors, in the absence of human islets, to direct application of ACh (10 μM), kainate (100 μM), KCl (25 mM), changes in glucose concentration (from 3 mM to 16 mM (high glucose) or from 16 mM to 3 mM (low glucose)) or ACh (10 μM) in the presence of the muscarinic antagonist atropine (5 μM). () Trace of the 340/380 Fura-2 ratio in an ACh biosensor positioned against the islet as in shows stimulus-induced secretion of ACh from endocrine cells in a human islet. Horizontal lines denote application of kainate (Kai, 100 μM), a transient increase in glucose concentration from 3 mM to 16 mM (16G) or application of atropine (red, 5 μM). () Summary of data from e! xperiments conducted as those shown in . Bars show means ± s.e.m. for ACh biosensor signals (Δ340/380) in response to stimulation of islets with KCl (25 mM) depolarization (n = 8 experiments), kainate (100 μM, n = 11), increases in glucose concentration (from 3 mM to 16 mM, 3G–16G) or decreases in glucose concentration (from 16 mM to 3 mM, 16G–3G, n = 4). Biosensor responses were blocked by atropine (5 μM). () ACh release in response to increasing the glucose concentration from 3 mM to 11 mM (3G–11G), lowering the glucose concentration from 11 mM to 3 mM (11G–3G) or to depolarization with KCl (25 mM), as determined with a fluorescent enzymatic assay (see Online Methods; n = 6 islet preparations; ANOVA followed by multiple comparison, *P < 0.05). Data represent means ± s.e.m. * Figure 4: Endogenously released ACh amplifies glucose-induced insulin secretion in human islets. () Insulin release from human islets elicited by ACh, the muscarinic agonist oxotremorine and nicotine. Horizontal lines denote stimulus application. Representative traces of n = 3 islet preparations. () Summary of data from experiments similar to those shown in but conducted in the presence of low (3 mM) and high (11 mM) glucose (n = 3 preparations). () Insulin secretion elicited by the acetylcholinesterase inhibitor physostigmine (30 μM) at 3 mM glucose (n = 5 human islet preparations). (–) Physostigmine-induced increases in insulin secretion (ΔInsulin) in the absence (−) or presence (+) of the vAChT blocker vesamicol (10 μM, ), the M3 receptor antagonist J-104129 (50 nM, ) or when the experiment was performed at different glucose concentrations (3 mM, 3G; 11 mM, 11G, ; Student's t test, P < 0.05). () Insulin secretion induced by repeatedly raising glucose from 3 mM to 11 mM in the presence of physostigmine (30 μM) or in the presence of J-104129 (50 nM); representa! tive traces of four experiments. A control experiment with untreated islets was run in parallel (gray symbols). Horizontal lines at bottom denote drug application. 11G indicates 15 min of elevated glucose (11 mM). Islets were stimulated four times with glucose followed by 25 mM KCl. () Summary of data from experiments such as those shown in . Responses are expressed as percentage of the respective insulin response of control islets (100%, gray dashed line; n = 4 preparations for J-104129 treatment; n = 5 preparations for physostigmine treatment). One-sample t tests were used to compare the actual mean to a theoretical mean of 100% (control; *P < 0.05). Data represent means ± s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Rayner Rodriguez-Diaz & * Robin Dando Affiliations * Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA. * Rayner Rodriguez-Diaz, * M Caroline Jacques-Silva, * Alberto Fachado, * Midhat H Abdulreda, * Camillo Ricordi, * Per-Olof Berggren & * Alejandro Caicedo * The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden. * Rayner Rodriguez-Diaz, * Camillo Ricordi & * Per-Olof Berggren * Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida, USA. * Robin Dando, * Stephen D Roper & * Alejandro Caicedo * Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, Miller School of Medicine, University of Miami, Miami, Florida, USA. * Judith Molina & * Alejandro Caicedo * Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, Florida, USA. * Stephen D Roper & * Alejandro Caicedo * Division of Integrative Biosciences and Biotechnology, World Class University Program, University of Science and Technology, Pohang, Korea. * Per-Olof Berggren Contributions R.R.-D., M.C.J.-S., A.F. and J.M. performed hormone assay experiments and ELISAs; R.D. performed experiments with biosensor cells to detect acetylcholine secretion; R.R.-D. and M.C.J.-S. conducted Amplex assays to measure acetylcholine secretion; R.R.-D. and M.H.A. collected, analyzed and quantified immunohistochemical data, and R.R.-D. performed RT-PCR and western blotting. R.R.-D., R.D., M.H.A., C.R., S.D.R., P.-O.B. and A.C. designed the study, analyzed data and wrote the paper. R.R.-D. and R.D. contributed equally to the study. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Per-Olof Berggren or * Alejandro Caicedo Author Details * Rayner Rodriguez-Diaz Search for this author in: * NPG journals * PubMed * Google Scholar * Robin Dando Search for this author in: * NPG journals * PubMed * Google Scholar * M Caroline Jacques-Silva Search for this author in: * NPG journals * PubMed * Google Scholar * Alberto Fachado Search for this author in: * NPG journals * PubMed * Google Scholar * Judith Molina Search for this author in: * NPG journals * PubMed * Google Scholar * Midhat H Abdulreda Search for this author in: * NPG journals * PubMed * Google Scholar * Camillo Ricordi Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen D Roper Search for this author in: * NPG journals * PubMed * Google Scholar * Per-Olof Berggren Contact Per-Olof Berggren Search for this author in: * NPG journals * PubMed * Google Scholar * Alejandro Caicedo Contact Alejandro Caicedo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Author information * Supplementary information Movies * Supplementary Video 1 (92M) Three-dimensional rendering of the vAChT immunostaining pattern in a mouse islet. Nerve fibers immunostained for vAChT are visualized as they enter the core of the islet to innervate beta cells. Reconstruction based on 45 confocal images (z step = 0.3 µm) was performed using Volocity Visualization software. * Supplementary Video 2 (102M) Three-dimensional rendering of the vAChT immunostaining pattern in a human islet. Strong immunostaining is present in cells expressing glucagon. Immunostained nerve fibers cannot be seen in the islet but are visible in the exocrine tissue. Reconstruction based on 45 confocal images (z step = 0.3 µm) was performed using Volocity Visualization software. PDF files * Supplementary Text and Figures (680K) Supplementary Figures 1–3 Additional data - Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels
- Nat Med 17(7):893-898 (2011)
Nature Medicine | Technical Report Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels * Jérôme Lecoq1, 2, 3, 5, 6 * Alexandre Parpaleix1, 2, 3, 6 * Emmanuel Roussakis4, 6 * Mathieu Ducros1, 2, 3 * Yannick Goulam Houssen1, 2, 3 * Sergei A Vinogradov4 * Serge Charpak1, 2, 3 * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 17,Pages:893–898Year published:(2011)DOI:doi:10.1038/nm.2394Received23 July 2010Accepted27 January 2011Published online05 June 2011 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO2) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO2 can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO2 in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO2 in capillaries and by a biphasic PO2 response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO2 opens new avenues for studies of brain metabolism and blood flow regulation. View full text Figures at a glance * Figure 1: Measurements of PO2 in deep cerebral vessels using probe PtP-C343. () Experimental setup. An AOM is placed in the excitation path of a standard two-photon microscope, enabling fast repetitive on-off switching of the laser excitation. () The probe is excited by a brief gate (2.5 μs) of femtosecond pulses from a Ti:sapphire laser (λex = 850 nm, <250 fs, 76 MHz), followed by a phosphorescence detection period (~250 μs). The fluorescence emitted by PtP-C343 is detected by a photomultiplier tube (PMT) in the green channel (PMT1); the phosphorescence is detected by PMT2 in the red channel. () Top, intravenous (i.v.) injection of PtP-C343 and fast scanning with detection of fluorescence reveals capillary in the rat olfactory bulb. Phosphorescence is measured at selected spots (red in the capillary lumen). Scale bar, 10 μm. Bottom, successive phosphorescence acquisition cycles displayed as an image. Each horizontal line is one complete cycle, whereas the vertical axis shows successive cycles. () Phosphorescence decay obtained by averaging of 40! ,000 excitation gates, fitted to a single exponential (red). () Respiratory arrest (RA) induced by air injection into the femoral vein. () Effect of changes in oxygen content in the inhaled air from 21% to 100% or 10% on PO2. Error bars indicate s.e.m. * Figure 2: Temporal and spatial resolution of two-photon phosphorescence measurement. () Log-log plots of the phosphorescence integrated intensity (area under the decay curve after subtraction of the baseline) versus average excitation power. Left, AOM gate 2.5 μs. Maximal power shown: 275 mW after the objective, or 2.7 mW, taking into account 1% duty cycle; focal spot depth 80 μm. Red line (slope = 2) corresponds to pure quadratic dependence. The black arrow indicates the region of quadratic dependence (up to 50–60 mW). Right, AOM gate 25 μs (average laser power ~0.5–0.6 mW, 10% duty cycle). The phosphorescence emission is outside the quadratic regime throughout the entire power range. AU, arbitrary units. () Precision of the lifetime (τ) measurement as a function of the number of averages, their duration and PtP-C343 concentration (injected i.v.). Left, AOM gate: 2.5 μs (PtP-C343 concentration: ~10 μM, average laser power ~0.5–0.6 mW). Lifetime measurements were performed in eight capillaries at ~100 μm depth. 12,000 averages (~3 s) are suffici! ent to reach the desired precision. Right, AOM gate 25 μs (PtP-C343 concentration ~50 μM, average laser power ~7 mW). 3,000 gates (~0.80 s) are sufficient to achieve the same precision as in left. () Spatial confinement of phosphorescence measurements under different excitation regimes (left, AOM gate 2.5 μs; right, AOM gate 25 μs). Point measurements were performed perpendicularly to the longitudinal axis of two capillaries. Phosphorescence decays were observed in the capillary lumen (red dots) but not in the neuropil (blue dots). Measurements at capillary boundaries produced weak signal (purple dots). Scale bars, 5 μm. * Figure 3: Simultaneous measurements of RBC flow and PO2 using fluorescence and phosphorescence of PtP-C343. () RBC detection. The fluorescence of PtP-C343 in the plasma during the excitation gate is shadowed by passing RBCs (white arrows on right pictures corresponding to individual schematics on the left). (,) Automatic detection of RBC transients () allows extraction of local RBC flow rates (, top) as well as the estimation of instantaneous hematocrit (, bottom). () Consecutive measurements of RBC flow using the line-scan approach and our pulse-shading method (n = 13 vessels). (–) Erythrocyte-associated transients (EATs) in single capillaries. () Two RBC fluorescence transients are used as time markers to determine locations of the phosphorescence measurements relative to each RBC. () Change in the PO2 near a single RBC. Decays were acquired during 200 s (20 recordings, 10 s each) in eleven capillaries. Each decay is an average of 40,000 gates. () PO2 values measured in the plasma and in the close vicinity of RBCs. () Consecutive line scans acquired in five capillaries to meas! ure the corresponding distance. Error bars represent s.e.m. * Figure 4: Diffusional shunt of oxygen between arterial and venous compartments in the olfactory nerve layer. () PO2 measurements along a venule approaching an arteriole. Left, three-dimensional reconstruction of the two vessels (extracted from a two-photon fluorescence stack of images). Venule is in blue and arteriole in red. Right, seven successive PO2 measurements (three to six acquisitions at each point) in the venule. Scale bars, 40 μm. () Diffusional shunt of oxygen is independent of vascular fluctuations. The venule lies in the xy plane. Measurements of PO2 were performed repetitively in five points (2.5 s per point). () Venous PO2 as a function of the distance, measured in four animals. () Diffusional shunt of oxygen at complete arteriolar-venular crossings. Error bars represent s.e.m. * Figure 5: Functional hyperemia, vascular and neuropil PO2 dynamics in response to odor stimulation. () Neuronal and vascular responses during odor stimulation. Inset, glomerulus with boundaries that were outlined by olfactory receptor neuron terminals labeled with Calcium Green-1 dextran, and capillaries labeled with fluorescein-dextran. The presynaptic Ca2+ response was recorded over the rectangle. RBC velocity was obtained from line scans drawn in the capillary segment, indicated by the arrow. Scale bar, 20 μm. () RBC and PO2 responses in the same capillary. I.v. injection of PtP-C343 allows detection of the concomitant increases in the RBC flow and PO2 (point measurements of PtP-C343 fluorescence and phosphorescence, respectively) in response to the odorant inhalation (two trials). () Mean RBC flow and PO2 responses (nine trials). () Summary of vascular responses in five rats. () PO2 dynamics in the glomerular neuropil. Left, schematic of the experiment. Middle, three successive measurements of PO2 in neuropil in response to odor stimulation. Right, superposition of pr! esynaptic Ca2+, RBC and PO2 responses in the same glomerulus. Error bars indicate s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Jérôme Lecoq, * Alexandre Parpaleix & * Emmanuel Roussakis Affiliations * Institut National de la Santé et de la Recherche Médicale (INSERM), U603, Paris, France. * Jérôme Lecoq, * Alexandre Parpaleix, * Mathieu Ducros, * Yannick Goulam Houssen & * Serge Charpak * Centre National de la Recherche Scientifique (CNRS), UMR 8154, Paris, France. * Jérôme Lecoq, * Alexandre Parpaleix, * Mathieu Ducros, * Yannick Goulam Houssen & * Serge Charpak * Laboratory of Neurophysiology and New Microscopies, Université Paris Descartes, Paris, France. * Jérôme Lecoq, * Alexandre Parpaleix, * Mathieu Ducros, * Yannick Goulam Houssen & * Serge Charpak * Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * Emmanuel Roussakis & * Sergei A Vinogradov * Current address: James H. Clark Center for Biomedical Engineering and Sciences, Stanford University, Stanford, California, USA. * Jérôme Lecoq Contributions E.R. and S.A.V. designed and synthesized the oxygen probe. J.L. and M.D. designed and built the optical setup. J.L. and M.D. wrote the LabVIEW program controlling the system and analyzing the data. J.L., A.P., Y.G.H. and S.C. conducted the experiments and analyzed the data. J.L. and S.C. initiated the project. All authors edited the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Serge Charpak or * Sergei A Vinogradov Author Details * Jérôme Lecoq Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandre Parpaleix Search for this author in: * NPG journals * PubMed * Google Scholar * Emmanuel Roussakis Search for this author in: * NPG journals * PubMed * Google Scholar * Mathieu Ducros Search for this author in: * NPG journals * PubMed * Google Scholar * Yannick Goulam Houssen Search for this author in: * NPG journals * PubMed * Google Scholar * Sergei A Vinogradov Contact Sergei A Vinogradov Search for this author in: * NPG journals * PubMed * Google Scholar * Serge Charpak Contact Serge Charpak Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (725K) Supplementary Figures 1–3 and Supplementary Methods Additional data - CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation
- Nat Med 17(7):899 (2011)
Nature Medicine | Retraction CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation * Veronica Marcos * Zhe Zhou * Ali Önder Yildirim * Alexander Bohla * Andreas Hector * Ljubomir Vitkov * Eva-Maria Wiedenbauer * Wolf Dietrich Krautgartner * Walter Stoiber * Bernd H Belohradsky * Nikolaus Rieber * Michael Kormann * Barbara Koller * Adelbert Roscher * Dirk Roos * Matthias Griese * Oliver Eickelberg * Gerd Döring * Marcus A Mall * Dominik HartlJournal name:Nature MedicineVolume: 17,Page:899Year published:(2011)DOI:doi:10.1038/nm0711-899aPublished online07 July 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, 1018–1023 (2010); published online 5 September 2010; retracted 15 June 2011 In the version of this article initially published, we reported that CXCL8 could efficiently induce neutrophil extracellular trap (NET) formation in vitro. When we followed up on the effect of recombinant CXCL8 (IL-8) on NET formation by comparing different cell culture conditions and extending our studies to neutrophils obtained from a larger number of healthy blood donors, we found that the CXCL8 effect was donor dependent and was less robust than we previously thought. In investigating the underlying factors, we observed that the CXCL8 effect that we initially observed was favored by our cell culture conditions (CXCL8-72aa (CXCL8 that is 72 amino acids in length) at 100 nM; RPMI-1640 medium; absence of albumin, buffers or serum; supplementation with L-glutamine; and precoating of culture plates with poly-D-lysine 30–70 kDa). We had initially chosen these conditions because we felt that they resembled the pulmonary microenvironment. On the basis of our recent observation! s, however, we conclude that these culture conditions are unstable and allow nonspecific neutrophil activation and autocrine/paracrine CXCL8 release. In light of these results, we revise our conclusions to state that the effect of recombinant CXCL8 on NET formation is less efficient than we previously reported, donor dependent and less robust compared to the effect of phorbol 12-myristate 13-acetate. Thus, we wish to retract the paper. We did not use this in vitro methodology in the ex vivo and in vivo studies of human and mouse cystic fibrosis lung disease. Accordingly, we continue to endorse our NETosis studies in the context of cystic fibrosis lung disease. Additional data Author Details * Veronica Marcos Search for this author in: * NPG journals * PubMed * Google Scholar * Zhe Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Ali Önder Yildirim Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Bohla Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Hector Search for this author in: * NPG journals * PubMed * Google Scholar * Ljubomir Vitkov Search for this author in: * NPG journals * PubMed * Google Scholar * Eva-Maria Wiedenbauer Search for this author in: * NPG journals * PubMed * Google Scholar * Wolf Dietrich Krautgartner Search for this author in: * NPG journals * PubMed * Google Scholar * Walter Stoiber Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd H Belohradsky Search for this author in: * NPG journals * PubMed * Google Scholar * Nikolaus Rieber Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Kormann Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Koller Search for this author in: * NPG journals * PubMed * Google Scholar * Adelbert Roscher Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Roos Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Griese Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Eickelberg Search for this author in: * NPG journals * PubMed * Google Scholar * Gerd Döring Search for this author in: * NPG journals * PubMed * Google Scholar * Marcus A Mall Search for this author in: * NPG journals * PubMed * Google Scholar * Dominik Hartl Search for this author in: * NPG journals * PubMed * Google Scholar - NET loss of air in cystic fibrosis
- Nat Med 17(7):899 (2011)
Nature Medicine | Retraction NET loss of air in cystic fibrosis * A Murat Kaynar * Steven D ShapiroJournal name:Nature MedicineVolume: 17,Page:899Year published:(2011)DOI:doi:10.1038/nm0711-899bPublished online07 July 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, 967–969 (2010); retracted 15 June 2011 In view of the fact that the authors of "CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation" are retracting their report, we wish to retract our News and Views article, which dealt with the above study and was based on the veracity of its data. Additional data Author Details * A Murat Kaynar Search for this author in: * NPG journals * PubMed * Google Scholar * Steven D Shapiro Search for this author in: * NPG journals * PubMed * Google Scholar
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