Latest Articles Include:
- An experiment in progress
- Nat Med 16(9):935 (2010)
Nature Medicine | Editorial An experiment in progress Journal name:Nature MedicineVolume: 16 ,Page:935Year published:(2010)DOI:doi:10.1038/nm0910-935 Singapore's current investment in science is remarkably strong, but it isn't yet clear if the resulting knowledge and innovation will go on to fuel economic growth, as the country expects. View full text Additional data - Lack of uptake threatens promise of HIV vaccines
- Nat Med 16(9):937 (2010)
The news out of Thailand last year that an experimental vaccine reduced the risk of contracting HIV by as much as a third reinvigorated hopes that a vaccine against the elusive virus was possible. But new research showing that large swaths of the public wouldn't bother getting immunized against HIV, were such a vaccine available, calls into question the effectiveness of the approach in controlling the AIDS epidemic. - Line item cut threatens monitoring of more than West Nile virus
- Nat Med 16(9):938 (2010)
Dengue fever is lapping at America's shores. In early August, Florida's Department of Health confirmed that there had been 96 cases of the mosquito-borne disease in the state since the start of the year. - China to scale up collaborations in infectious disease research
- Nat Med 16(9):938 (2010)
The Chinese Center for Disease Control and Prevention (CCDC) has signed an agreement with the TDR, a joint UN–World Health Organization program, to drive the center's medical research toward better understanding infectious disease in developing nations, particularly in Africa. The memorandum of understanding was signed in Shanghai on 14 June, the closing day of a symposium dedicated to the research and control of infectious diseases of poverty. - Theory is acid test for fructose's blood pressure role
- Nat Med 16(9):939 (2010)
Fructose is under fire. In recent years, the simple sugar has endured close scrutiny from scientists who claim that it increases the risk of obesity more than its cousin glucose does. - Price of misconduct probes can surpass $500,000
- Nat Med 16(9):939 (2010)
Integrity is priceless, but investigating it doesn't come cheap either. According to a new analysis, misconduct probes can cost institutions upwards of half a million dollars. - Amidst growing vaccine concerns, NIH sets up engine for answers
- Nat Med 16(9):940 (2010)
Infectious diseases, and, by extension, the vaccines against them, occupy a lofty spot on the totem pole of public health. In the US, many states have laws requiring certain vaccines. - Correction: 'Parse the salt, please'
- Nat Med 16(9):940 (2010)
In the print version of the August 2010 issue of Nature Medicine, the article entitled 'Parse the salt, please' (Nat. Med. 16, 841, 2010 - Straight talk with ... Rajiv Shah
- Nat Med 16(9):941 (2010)
Nature Medicine | News Straight talk with ... Rajiv Shah * Elie Dolgin Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:941Year published:(2010)DOI:doi:10.1038/nm0910-941 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Rajiv Shah was less than a month into his position at the helm of the US Agency for International Development (USAID) when the catastrophic earthquake that hit Haiti propelled him to the frontline of an international relief effort. To meet the challenge, Shah relied on his previous experience in leadership roles at the Bill & Melinda Gates Foundation and at the US Department of Agriculture to help launch the largest urban food distribution effort in history and a vaccination campaign that reached more than a million people. With Haiti now in a state of long-term reconstruction, Shah's focus is shifting toward meeting the Millennium Development Goals (MDGs), the eight major areas of international development that the world agreed to address by 2015. Ahead of the MDG Review Summit this month in New York, spoke to Shah about how he plans to make progress toward reaching the targets. View full text Additional data - Focus on Biomedical Art
- Nat Med 16(9):942 (2010)
Science is an art, but that doesn't mean that all scientists are artists. So, researchers often collaborate with skilled designers to place scientific ideas and results within the social framework in which we live. - Fresh as a daisy: Alexandra Daisy Ginsberg
- Nat Med 16(9):942 (2010)
When I was looking for new work to feature in my upcoming show Talk to Me, an exhibition on the communication between people and objects that is slated to open at The Museum of Modern Art (MoMA) in New York next summer, I immediately thought of Alexandra Daisy Ginsberg, a remarkable recent graduate of the Design Interactions program at the Royal College of Art (RCA) in London. The clarity, aesthetic and intellectual elegance conveyed in Ginsberg's art marries an undeniable design talent with a deep passion for science. - Selling point: David Goodsell
- Nat Med 16(9):943 (2010)
The covers of most major scientific journals are plastered with beautiful, realistic pictures taken with the latest advances in microscope technology—and this month's Nature Medicine is no exception. But few of these images have the qualities of David Goodsell's works of art. - Revitalizing design: Revital Cohen
- Nat Med 16(9):944 (2010)
I first met Revital Cohen when she was a student of mine at the Royal College of Art, and I was immediately struck by the maturity of her work. Cohen manages to achieve the delicate balance between the believable and complete fantasy—a talent that usually takes years to master. - Silence is golden: Phil Gamblen and Guy Ben-Ary
- Nat Med 16(9):945 (2010)
On first encounter, "Silent Barrage" is anything but silent. The artwork consists of a forest of robotic poles that oscillate noisily and erratically as visitors explore the mechanical jungle. - News in brief
- Nat Med 16(9):946-947 (2010)
Jul 21Harvard Medical School in Boston adopted a new, stricter conflict-of-interest policy. Professors can no longer be paid speakers for drug or medical device companies and may not accept free travel, meals or other gifts from industry. - The real war on drugs
- Nat Med 16(9):948-952 (2010)
Nature Medicine | News The real war on drugs * Cassandra Willyard1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:948–952Year published:(2010)DOI:doi:10.1038/nm0910-948 The murder of ten aid workers, including an optometrist and a surgeon, in Afghanistan last month refocused the world's attention on the difficulties of providing health care in conflict zones. Beyond the dangers of delivering acute care such as surgery, dispensing medicines for chronic illnesses ranging from HIV to diabetes remains a challenge in areas affected by war. looks at the lessons relief agencies have learned in recent years providing care amidst increasingly complex conflicts. View full text Additional data Affiliations * Cassandra Willyard is a science writer based in Brooklyn, New York. - Personalized investigation
- Nat Med 16(9):953-955 (2010)
Nature Medicine | News Personalized investigation * Elie Dolgin1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:953–955Year published:(2010)DOI:doi:10.1038/nm0910-953 Despite continued doubts about the clinical utility of direct-to-consumer genetic tests, tens of thousands of people have sent away tubes full of their saliva to learn more about their genetic profiles. Armed with such DNA data, a number of early adopters are showing how empowering—and beneficial to science—personal genetic information can be. reports on one company's plans to make medical genetics more participatory. View full text Additional data Affiliations * Elie Dolgin is a news editor with Nature Medicine in New York. - Masterminds behind pharmaceutical fraud deserve prison time
- Nat Med 16(9):956 (2010)
Nature Medicine | News Masterminds behind pharmaceutical fraud deserve prison time * Jeb White1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:956Year published:(2010)DOI:doi:10.1038/nm0910-956 The US legal system has rightly constructed a high barrier to prosecuting individual corporate employees. However, when a person deliberately masterminds massive pharmaceutical fraud, he or she needs to be held accountable. Whistleblowers hold the key to building stronger, more viable cases against those who devise large-scale corporate criminal activity. View full text Additional data Affiliations * Joseph E. B. White is a partner with the national whistleblower law firm of Nolan & Auerbach, P.A. in Philadelphia, Pennsylvania. White is also the former executive director of the Washington, DC–based Taxpayers Against Fraud, a national public interest organization dedicated to supporting whistleblowers. * Jeb White - Striving toward excellence
- Nat Med 16(9):957 (2010)
Pursuing Excellence in Healthcare: Preserving America's Academic Medical Centers is a thoughtful book presenting an overview of the major challenges presently facing Academic Medical Centers (AMCs) and offering recommendations to both institutions and policy makers. In his book, Feldman argues that preserving AMCs is crucial to our society for providing excellent patient care, teaching future physicians and leading scientific research to ultimately improve care. - Pericyte constriction after stroke: the jury is still out
- Nat Med 16(9):959 (2010)
We are writing in response to the paper by Yemisci et al.1. The authors propose that ischemia-reperfusion causes widespread, long-lasting dynamic constriction of pericytes, leading to microvascular failure that contributes to brain injury and behavioral deficits1. - Reply to: "Pericyte constriction after stroke: the jury is still out"
- Nat Med 16(9):960 (2010)
We thank our colleagues1 for the opportunity to clarify the interpretation of some data in our recent paper2.First, we think that the sausage-like appearance of capillaries after ischemia is caused by pericyte contractions and not by decapitation or fixation artifacts. - Being too inclusive about synuclein inclusions
- Nat Med 16(9):960-961 (2010)
We read with interest the recent review by Obeso et al.1, in which they discuss recent studies on the presence of α-synuclein inclusions in tissue transplanted to people with Parkinson's disease. Although the review cites our related study2, we do not think that the work is correctly described; our work, which shows no α-synuclein pathology in the transplant, is listed by the authors together with other studies that show α-synuclein aggregates. - Reply to: "Being too inclusive about synuclein inclusions"
- Nat Med 16(9):961 (2010)
We appreciate the opportunity to clarify a salient point in our recent review1. Drs. - Sodium guidelines should be taken with a grain of salt
- Nat Med 16(9):961-962 (2010)
Stephen Strauss's news feature1 on low-sodium alternatives to table salt is predicated on the notion that salt consumption is currently at unhealthy levels. But the clinical evidence supporting salt intake reduction to the most commonly recommended level of 1. - A cancer fate in the hands of a samurai
- Nat Med 16(9):963-965 (2010)
Recent studies show that Musashi-2 (MSI2), a molecule that binds RNA, increases proliferation of normal and malignant blood stem cells. In humans, increased amounts of MSI2 correlate with poor prognosis of leukemia—indicating that MSI2 may be a target to treat this type of cancer. - Thyroid hormones: igniting brown fat via the brain
- Nat Med 16(9):965-967 (2010)
Thyroid hormones have widespread cellular effects; however it is unclear whether their effects on the central nervous system (CNS) contribute to global energy balance. Here we demonstrate that either whole-body hyperthyroidism or central administration of triiodothyronine (T3) decreases the activity of hypothalamic AMP-activated protein kinase (AMPK), increases sympathetic nervous system (SNS) activity and upregulates thermogenic markers in brown adipose tissue (BAT). Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus (VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism. This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and increases exp! ression of thermogenic markers in BAT. These effects are reversed by pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of whole-body energy homeostasis. - NET loss of air in cystic fibrosis
- Nat Med 16(9):967-969 (2010)
Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs)1, 2, 3. Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases4, 5. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation6, 7. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blocka! de of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease. - Cocaine sobers up
- Nat Med 16(9):969-970 (2010)
There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction1, 2, 3, 4. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoami! ne oxidase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse. - Antidiabetes wars: a new hope
- Nat Med 16(9):972-973 (2010)
A family of agonist drugs for peroxisome proliferator–activated receptor (PPAR-γ) is used in the clinic to battle insulin resistance for the treatment of type 2 diabetes. Their serious side effects, however, remain a challenge for researchers and clinicians. - Finding the tumor copycat: Therapy fails, patients don't
- Nat Med 16(9):974-975 (2010)
Nature Medicine | Between Bedside and Bench Finding the tumor copycat: Therapy fails, patients don't * Lee M Ellis1, 2lellis@mdanderson.org Search for this author in: * NPG journals * PubMed * Google Scholar * Isaiah J Fidler1ifidler@mdanderson.org Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:974–975Year published:(2010)DOI:doi:10.1038/nm0910-974 The complexity of human metastatic cancer is difficult to mimic in mouse models. As a consequence, seemingly successful studies in murine models do not translate into success in late phases of clinical trials, pouring money, time and people's hope down the drain. In 'Bedside to Bench', Isaiah Fidler and Lee Ellis discuss crucial parameters in cancer growth and therapy and emphasize the disparity between studies in humans and mice. In 'Bench to Bedside', Terry Van Dyke shows how pancreatic tumors developed de novo in the organ site in mice can explain therapy failure in people with cancer and serve as a model to test new drugs. 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 * Lee M. Ellis and Isaiah J. Fidler are in Department of Cancer Biology, Houston, Texas, USA. * Lee M. Ellis is in the Department of Surgical Oncology at the University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. Competing financial interests L.M.S. is a consultant for Schering Plough. He performs ad hoc consulting for Genentech/Roche, Astra Zeneca, Regeneron and Pfizer. Corresponding authors Correspondence to: * Lee M Ellis (lellis@mdanderson.org) or * Isaiah J Fidler (ifidler@mdanderson.org) Additional data - Finding the tumor copycat: Approximating a human cancer
- Nat Med 16(9):976-977 (2010)
Years of developing cancer therapies targeted to specific molecular drivers have yielded some crucial advances, yet only 5% of such therapies tested in the clinic prove efficacious, indicating a need for marked improvement in predictive preclinical research1. Given that cancers are complex diseases with diverse molecular etiologies, the most predictive preclinical models require accurate engineering for the induction of cancer in situ. - Research Highlights
- Nat Med 16(9):978-979 (2010)
- The 2010 scientific strategic plan of the Global HIV Vaccine Enterprise
- Nat Med 16(9):981-989 (2010)
Nature Medicine | Commentary The 2010 scientific strategic plan of the Global HIV Vaccine Enterprise * The Council of the Global HIV Vaccine Enterprise1 * Seth Berkley2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth Bertram3 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-François Delfraissy4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ruxandra Draghia-Akli5 Search for this author in: * NPG journals * PubMed * Google Scholar * Anthony Fauci6 Search for this author in: * NPG journals * PubMed * Google Scholar * Cynthia Hallenbeck7 Search for this author in: * NPG journals * PubMed * Google Scholar * Madame Jeannette Kagame8 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Kim9 Search for this author in: * NPG journals * PubMed * Google Scholar * Daisy Mafubelu10 Search for this author in: * NPG journals * PubMed * Google Scholar * Malegapuru W Makgoba11 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Piot12 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark Walport13 Search for this author in: * NPG journals * PubMed * Google Scholar * Mitchell Warren14 Search for this author in: * NPG journals * PubMed * Google Scholar * Tadataka Yamada15 Search for this author in: * NPG journals * PubMed * Google Scholar * for Members of the Enterprise * José Esparza15 Search for this author in: * NPG journals * PubMed * Google Scholar * Catherine Hankins10 Search for this author in: * NPG journals * PubMed * Google Scholar * Margaret I Johnston6 Search for this author in: * NPG journals * PubMed * Google Scholar * Yves Lévy4 Search for this author in: * NPG journals * PubMed * Google Scholar * Manuel Romaris5 Search for this author in: * NPG journals * PubMed * Google Scholar * for Alternate members * Rafi Ahmed16 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan Bernstein17 Search for this author in: * NPG journals * PubMed * Google Scholar * for Ex-officio members * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:981–989Year published:(2010)DOI:doi:10.1038/nm0910-981 An important moment in HIV vaccine research * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg HIV/AIDS remains one of humanity's greatest challenges. Since 1981, it has claimed over 25 million lives and is currently responsible for over 2.5 million new infections worldwide each year1. Although progress has been made in preventing new HIV infections and in lowering the annual number of AIDS-related deaths through comprehensive prevention programs and increased access to antiretroviral therapy, the number of people living with HIV—now over 33 million—continues to grow1. Currently, only two out of five people who need treatment receive it, and even this modest level of progress in treatment is in jeopardy, as the availability of donor funds plateaus or declines2. Whereas universal access to treatment is an ambitious goal, the annual accrual of newly infected individuals who require treatment testifies to the urgent need for more effective prevention strategies. Vaccines are the primary public health intervention for dozens of infectious diseases worldwide; they are ! easy to administer and yield lasting effects. As one of the most powerful tools for preventing infection against other infectious diseases, a safe, effective, accessible HIV vaccine is therefore one of our greatest priorities—and one of science's greatest challenges. The unique ability of HIV to evade and suppress the immune response, its extraordinary genetic diversity, the properties of its envelope glycoprotein and the ability to establish systemic infection within days and to induce dysfunction and death of the immune cells needed to mount a protective response have posed unprecedented challenges for vaccine development3. Nonetheless, although a highly effective HIV vaccine remains elusive, we have never been closer to the target. Among the most visible achievements of the past five years were the results of RV144, the trial conducted in Thailand, that showed that a poxvirus-protein prime-boost combination provided modest (31%) protection against HIV acquisition4. These results represent the first-ever demonstration of any level of efficacy in preventing HIV acquisition in humans by a vaccine. Although many questions remain, the results of the RV144 trial have brought renewed energy to the field and created a new lens through which t! o evaluate future priorities and set strategic directions. There have been other key advances in HIV vaccine research over the past five years. They include a growing understanding of the role of the mucosa as a barrier to sexually transmitted HIV infection5, descriptions of the earliest immunological responses in humans after acute HIV infection6, the demonstration that HIV infection in humans is usually initiated by one or a very small number of founder viruses7, 8, the development of computational algorithms to inform the design of unique mosaic immunogens to address the challenge of viral sequence diversity by achieving maximum epitope coverage while preserving natural antigen expression and processing9, new insights into the immunological and genetic basis for the ability of some people to control the virus or prevent virus acquisition (so-called 'elite controllers' and 'exposed but uninfected persons', respectively)10, 11, the first proof of substantive simian immunodeficiency virus control by CD8+ effector memory T cells indu! ced through vaccination12, the isolation of new antibodies with broadly neutralizing activity from HIV-infected subjects13, 14, 15 and appreciation of the possible role of non-neutralizing antibodies in protection16, 17, 18. Progress in other areas of biomedicine, including the development of faster and cheaper DNA sequencing, high-throughput and computational technologies, will increasingly affect the progress of HIV vaccine research and development. Last, although two large-scale human efficacy trials—STEP and Phambili—failed to confer protection19, further analysis of these trials has influenced current thinking about the direction of HIV vaccine design, development and clinical evaluation20, 21. It is now incumbent upon the field to translate the opportunities created by these developments into a safe and effective HIV vaccine suitable for use in populations with markedly different epidemiological, social, genetic and behavioral characteristics. This next stage in HIV vaccine research requires a strengthened global strategy that incorporates current efforts and encourages new and existing partners from high-, low- and middle-income countries to embark on a shared scientific agenda. The Global HIV Vaccine Enterprise * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information In 2003, recognizing that a more collaborative global approach was needed to address the scientific and public health challenges of HIV vaccine development, a group of 24 leaders in the field proposed the creation of the Global HIV Vaccine Enterprise22, an alliance of independent organizations committed to accelerating the development of an HIV vaccine through a shared scientific strategic plan, increased resources and greater collaboration. The Enterprise Scientific Strategic Plan articulates the commitment of Enterprise partners to work toward aligning relevant aspects of their own strategies and activities with the goal of contributing to the realization of a shared vision. The Plan sets out to define crucial roadblocks and opportunities that would benefit from increased global cooperation, complementing and building on the research efforts and discoveries of individual scientists. In so doing, the Plan puts forward priorities and strategic considerations of the Enterprise Council, informed by discussions among Enterprise partners and by the recommendations of the Enterprise Science Committee and its Working Groups. The Enterprise's first Scientific Strategic Plan23, published in 2005 and supplemented with two updates24, 25, called for increased collaboration and coordination of partners dedicated to HIV vaccine research and development and identified six priority areas for vaccine development. The Plan's overall impact was evaluated in 2009 and progress was reviewed on two levels: progress against the six identified priorities (detailed below) and commitment of resources and development of programs that align with the vision of the 2005 Plan (Table 1). Table 1: Progress of the HIV vaccine field toward the vision set out in the 2005 Scientific Strategic Plan Full table * Figures/tables index The Plan helped encourage dialogue and coordination among funders and scientists and was the impetus for the commitment of new funding for the establishment of collaborative initiatives in priority areas, complementing the essential work of individual investigators (Table 1). Priorities 1 and 2 emphasized the importance of continued investments in discovery research and the need for standardization of laboratory assays. In response, there have been advances in our understanding of virus-host interactions, including characterization of the transmitted viruses derived from recently infected individuals, description of the earliest cellular and humoral responses to HIV infection, isolation of new broadly neutralizing antibodies, greater understanding of the structural motifs of the HIV envelope protein and new insights into mucosal and innate immunity6, 8, 13, 14, 15, 26. There has also been progress in lab standardization, including greater access to clinical trial specimens for immunological analysis, common reagents and validated assays for studying vaccine responses. These advances have been facilitated in large part by the establishment of the Center for HIV-AIDS Vaccine Immunology (CHAVI) by the US National Institute of Allergy and Infectious ! Diseases (NIAID) and the Collaboration for AIDS Vaccine Discovery (CAVD) by the Bill & Melinda Gates Foundation as well as key collaborative research initiatives by International AIDS Vaccine Initiative (IAVI), the French National Agency for Research on AIDS (ANRS) and the European Commission. Priority 3 proposed the establishment of coordinated, dedicated product development and manufacturing capacity to support HIV vaccine trials. This recommendation has largely gone unfulfilled. Production needs have been met through the available global capacity, although no substantive efforts have been made to ensure coordinated access to manufacturing resources. Priority 4 called for increasing the quantity and quality of sustainable clinical research facilities, and expanding access to well-defined populations at risk of HIV infection. Overall, HIV trial capacity in low- and middle-income countries has improved, with clinical sites supported by many agencies, and the development of research capacity at trial sites has been enabled through different training initiatives. Challenges remain in retaining clinical trials staff, ensuring that working at clinical trial sites remains an attractive career choice and strengthening research capacity in low- and middle-income countries to enable substantive contribution to the HIV vaccine research effort. Priority 5 called for regulatory capacity building and greater exchange of information needed to facilitate regulatory decision-making and address institutional review board (IRB) issues. Examples of progress include the establishment of the African Vaccine Regulatory Forum to support national regulatory authorities in assessing clinical trial applications, monitoring trials and evaluating clinical data and the publication and subsequent updates of ethical and good participatory practices for biomedical HIV-prevention trials27, 28. Priority 6 called for an intellectual property framework to stimulate early-stage research by increasing scientific freedom and the sharing of data and reagents. Although the complexity of intellectual property issues necessitates greater effort, progress has been made by NIAID, IAVI and others to support public-private partnerships in HIV vaccine development. Although the 2005 Plan had a positive impact on the field, three key objectives were not fully reached. First, with notable exceptions, the 2005 Plan had limited success in mobilizing funding from new partners for HIV vaccine research. Second, further effort is required to align clinical research efforts with product development to capitalize fully on the contributions of Enterprise partners to clinical trials infrastructure, regulatory innovation, intellectual property, manufacturing expertise and private sector resources. Finally, the success of Enterprise-inspired collaborative initiatives has helped to highlight gaps that persist, particularly between basic and clinical research, and between the HIV vaccine field and other areas of biomedicine. In January 2009, the Enterprise Council initiated a process to update the 2005 Plan to reflect the anticipated challenges and opportunities affecting HIV vaccine research over the next five years. The Enterprise Science Committee identified five key areas for discussion: (i) immunogens and antigen processing, (ii) host genetics and viral diversity, (iii) new approaches to HIV vaccine research and development, (iv) bridging the gaps between fundamental, preclinical and clinical research and (v) challenges faced by young and early-career investigators. The Working Groups formed around each of these five themes prepared reports with recommendations29, 30, 31, 32, 33. The development of the Enterprise 2010 Scientific Strategic Plan (2010 Plan) was then informed by these reports. The 2010 Plan's two scientific priorities * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information Recognizing the importance of pursuing a diverse range of vaccine concepts and approaches, the 2010 Plan prioritizes two main drivers key to the next phase of HIV vaccine research and development that specifically require global collaboration. First, the Plan recognizes that clinical trials and human clinical investigation present an unequalled opportunity to obtain important information about the human immune system and its response to vaccine candidates and that they are pivotal to advancing both vaccine discovery and vaccine development. Human efficacy trials are essential to defining the ability of vaccines to prevent infection or disease and for the discovery of vaccine-induced correlates and signatures of protection, which would ultimately accelerate the development or improvement of HIV vaccines for future licensure and public health use. This scientific imperative—made possible by major advances in laboratory and computational techniques that have opened up complex biological systems, including the human immune system, to rigorous and rapid scientific analysis34—underpins the importance of clinical efficacy trials to advancing vaccine discovery and development. Second, the Plan recognizes that trials must be linked to and build upon the tools and concepts of basic biomedical science, including genomics and computational biology, immunology, virology and model systems, to optimize both vaccine design and information on vaccine biology in humans. A strengthened clinical trials effort must therefore be accompanied by sustained, strong support for fundamental vaccine discovery research. In pursuing an increasingly science-driven clinical trials effort, the field will advance promising candidates toward vaccine licensure and, at the same time, contribute fundamental scientific insights that will improve future vaccine design, product development and clinical trials. The 2010 Plan is therefore predicated on a multidisciplinary approach that bridges the lab and the clinic, entrenching human research as intrinsic to the discovery process, and mobilizing the collaborative efforts of basic, preclinical and clinical scientists in highly iterative vaccine design and testing. To accelerate the development of a highly efficacious vaccine, two interlinked priorities form the core of the 2010 Plan (Fig. 1). Figure 1: The interconnected priorities and cross-cutting considerations of the Enterprise 2010 Plan. * Full size image (148 KB) * Figures/tables index One of the greatest barriers to the design, prioritization and refinement of vaccine concepts is the absence of an established correlate of vaccine-induced protection. Notwithstanding our growing understanding of HIV pathogenesis and immunity, we lack proven immunological markers to guide fully rational vaccine design and predict vaccine protection in humans. Ultimately, the clinical relevance of immunological assays (breadth, depth, kinetics and location) can only be understood in the light of a clear efficacy signal. But beyond the search for a correlate, and beyond the success or failure of a given vaccine candidate, clinical studies produce valuable biological and sociobehavioral data essential to future vaccine development. Trials designed to maximize this learning opportunity will ensure that the contributions and expectations of trial volunteers are effectively translated into improved, more efficient product development efforts. Therefore, Priority 1 takes the view that clinical efficacy trials should not be perceived as the culmination of a series of basic science experiments but rather as an integral part of the discovery process. To that end, teams of investigators, with complementary scientific, clinical, behavioral and ethical interests and technical skills, must form highly integrated teams to address new concepts in vaccine design and product development. The field would benefit from clinical research consortia where hypotheses are generated, debated and tested, new trial designs that accelerate the research effort are optimized and implemented and multidisciplinary teams dedicate themselves to executing trials that simultaneously advance both discovery and product development objectives. Clinical research consortia that fulfill this dual mandate have the potential to transform clinical trials. Speedier execution of clinical trials is essential if we are to capitalize on scientific advances, expedite further clinical and laboratory evaluation of promising candidates and drive future vaccine development. Improvements may be achieved by implementing a series of process efficiencies, including accelerating protocol development and funding, facilitating ethical and regulatory approvals, exploring new approaches to trial design and ensuring timely manufacture and availability of 'good manufacturing practice' material. Reducing the timeframe will also require that the field be more nimble about acquiring, analyzing and rapidly sharing laboratory data through advanced planning and by capitalizing on new technologies and widely accessible databases. Future clinical trials will be shaped by the evolving landscape of the epidemic. Decreasing incidence rates within populations historically at higher risk of HIV exposure will require larger trials, whereas recruitment—which is already a rate-limiting factor in most large-scale trials—may be further slowed by the need to explain increasingly complex trial designs to regulators, policy-makers, communities, potential volunteers and other research stakeholders. Moreover, the results of clinical testing of other prevention strategies, such as microbicides, preexposure prophylaxis or 'test-and-treat' approaches, will become available in the near future (http://www.avac.org/ht/d/sp/i/398/pid/398/). If the results of these trials lead to the implementation of new prevention strategies, vaccine trials may become larger, more complex (for instance, combination prevention studies) and more costly. The vaccine development effort, therefore, must be informed by and informative to ot! her prevention studies to ensure that HIV vaccine research is integrated within the overarching goal of preventing HIV infection and transmission. Social, behavioral and ethical research, as well as a robust community engagement strategy, will increase in importance as the prevention armamentarium embraces new behavioral, biomedical and policy strategies. Implementing Priority 1 would be accelerated by a coherent global effort to maximize the effective use of resources, infrastructure and partnerships throughout all stages of clinical vaccine discovery and development. Six elements should also be considered: trial design, regulatory and operational capacity, community engagement, research platforms, databases for sharing trial data globally and an insistence on pursuing diverse hypotheses. Many of these considerations were identified in the 2005 Plan and remain priorities in the context of a scientifically intensified global HIV vaccine trials endeavor. With a view to advancing iterative, scientifically integrated product development trials that rapidly generate laboratory results and achieve definitive clinical results over a shorter trial horizon, the field should do the following ( Box 1 ): Box 1: Targets for Priority 1 Full box 1. Accelerate the clinical testing of promising vaccine candidates while maximizing opportunities to advance scientific discovery. Scientifically coordinated, multidisciplinary clinical research endeavors, with a particular focus on phase 2b efficacy trials, would consist of teams of clinical, preclinical and basic scientists capable of pursuing a hypothesis-driven scientific program in the context of product development efforts that might lead to future licensure; scientifically justified and IRB-approved clinical sampling, extensive lab work and the availability of specimens to address key questions in vaccine immunology; manufacturing of clinical-grade materials for clinical trials, including process development, production and formulation of immunogens and adjuvants; and access to formulations and adjuvants, including those from the private sector, for the testing of combination vaccine regimens. Because these studies may entail a higher cost per trial, available resourc! es will need to be increased, used more efficiently or both. Multidisciplinary teams will therefore be needed to design and implement the most informative trials. 2. Support a diversity of approaches to vaccine research and trial design. A strengthened clinical trials endeavor is predicated on the development, testing and systematic comparison of a diversity of vaccine concepts that explore different mechanisms to achieve effective and sustained protection against HIV. Diversity is best fostered by encouraging new ideas and new players and introducing process improvements so that more trials can be carried out. To reap the full benefit of a diverse clinical trials portfolio, it is crucial, as noted in point 5 below, that data generated in different trials be comparable, regardless of the sponsor. 3. Strengthen regulatory and clinical trial capacity to expedite the review, approval and execution of trials. The field, including national regulatory authorities, UNAIDS and World Health Organization (WHO), should continue to strengthen regional, national and global regulatory processes for trial design ethical review and data analysis to facilitate innovative trials. Integrating scientific discovery and product development objectives should be clearly presented so that the long-term benefits to volunteers and communities are discussed and understood. The establishment and maintenance of trial sites and cohorts should be optimized, with due consideration to the development of sustainable, versatile sites that can be adapted to other health priorities. 4. Engage concerned communities, including volunteers, advocates and community leaders, in the design and implementation of scientifically robust and ethically sound trials. HIV vaccine trials cannot succeed without substantial community engagement, particularly given the need to involve large numbers of healthy volunteers at risk of acquiring HIV. Therefore, as trial design and trial objectives become more complex, it will be important to engage communities in dialogue about the scientific and clinical value of the increased clinical sampling required by discovery-oriented trials and to set realistic expectations with respect to future licensure pathways and access to prevention alternatives. Strengthening the clinical trials endeavor will therefore require a concerted effort to build on existing community advisory efforts to increase participation from the trial design phase throughout the trial life cycle, eliciting the contributions of affected populations in appraising ! trial conduct and maximizing the value of research for volunteers. It will be especially important to ensure that trial protocols are appropriate, sensitive and well understood, that communities are fully engaged in respectful dialogue about the scientific purpose and expectations of trials, and that communities, nongovernmental organizations, media and policy-makers have the necessary scientific literacy. To this end, we should build on the recent publications containing guidelines and advice on these issues27, 28, 35. Where appropriate, the context-specific frameworks for community engagement that have been developed by WHO and UNAIDS, ANRS, the European & Developing Countries Clinical Trials Partnership (EDCTP), NIAID Human Vaccine Trials Network (HVTN), IAVI, US Military HIV Research Program (MHRP) and others should be adapted. Crucial to community engagement efforts will be the strengthening of local researchers' leadership opportunities and the development of context-! specific skills, expertise and tools at the country and local ! levels. 5. Expand and improve laboratory platforms and assays to analyze immunological responses to vaccination. A robust clinical trials endeavor requires improved methodologies for measuring the human immune response, techniques for assessing mucosal and innate immunity after vaccination, and high-throughput assays to develop and verify signatures of protection. As the repertoire of assays used to measure the immune response to vaccination expands and becomes more sophisticated, assay harmonization and standardization, and broad dissemination of laboratory platforms to countries where trials are being conducted will be increasingly important. Fundamental questions about the mechanisms that underlie pathways to immune protection and govern vaccine efficacy remain unanswered. We lack basic insights into the nature, quality and quantity of immune responses needed for protection and how to induce them through rationally designed vaccine concepts. Moreover, the field is just beginning to understand the window of opportunity during the first few days of infection, when vaccine-induced immunity might arrest systemic dissemination and the establishment of chronic infection. Detailed insights into these events, especially at mucosal barriers5, 24, are essential to drive the design of preventive vaccines that focus on blocking infection. It is also crucial that we deepen our understanding of the genetic underpinning of the interplay between host defenses and viral evolution that leads to such drastically variable phenotypes as, for example, exposed uninfected, long-term nonprogressors and rapid progressors36, 37, 38. Funda! mental research, driven by an appropriate mix of funding to individual and teams of investigators, will continue to play a major part in HIV vaccine research. We now have an opportunity to pursue intensive strategies that exploit the potential of model systems and harness ongoing advances from other disciplines of biomedicine to explain clinical phenomena and advance our understanding of the pathways to immunity. Vaccine development would benefit substantially from having access to the best models, technologies and data to answer fundamental questions about the prevention of HIV infection and transmission. Rapid adoption of new ideas and technologies from different areas of science is central to a science-driven clinical trials endeavor, where the potential of research in humans is, in part, defined by available assays and systems and the ease with which specimens and data can be transferred to researchers with relevant expertise. With this in mind, the field should do the following ( Box 2 ): Box 2: Targets for Priority 2 Full box 1. Continue to develop and adopt new technologies. The field would benefit from looking outward more in its search for new tools and ideas, embracing technologies and approaches arising from other areas of biomedical research. Examples include imaging technologies for studying mucosal immunity and the trafficking of viral or vaccine antigens and immune effectors, genomic technologies to better understand host factors that regulate the immune response, high-throughput screening methodologies for optimizing vaccine components and vaccination regimens, and new immunogen-design and gene-delivery technologies. 2. Foster collaboration with researchers from disciplines that have the potential to transform current approaches to HIV vaccine discovery and development. For example, systems biology provides a promising approach for the integrative analysis and modeling of large data sets that could drive improvements in vaccine design, vaccine delivery strategies and methods for sustaining responses39, 40, 41, 42. Globally accessible databases, coupled with the computational power for the systematic analysis of large data sets and for cross-database queries, would greatly facilitate these approaches. Potential mechanisms to promote increased interaction with researchers from other areas of science include funding strategies to encourage researchers to form new multidisciplinary groups around important scientific questions in vaccine design, organizing joint scientific conferences on topics of mutual interest, inviting new investigators into existing collaborative structures and actively ! encouraging new investigators from other areas of science to enter HIV vaccine research. 3. Seek consensus on the principle of rapid access to data and develop the infrastructure to annotate, deposit and analyze large amounts of data. Data are the foundation of biomedical research. Over the next several years, the amount of data from HIV vaccine research will markedly increase as a result of increases in the number and complexity of trials and the increased application of high-throughput and systems-biology approaches. Realizing the full value of these data requires deployment of the newest computational technologies and rapid data access. Funders, researchers and community representatives need to agree on a shared set of principles for data access and a global approach to develop the necessary infrastructure43. Progress in HIV vaccine research will continue to depend on a variety of preclinical models, in particular nonhuman primate (NHP) and humanized mouse models that can be used to explore new approaches to engineer the immune system and to evaluate vaccine immunogenicity. It is generally accepted that the immune system of NHPs most closely approximates the human immune system and therefore provides an in vivo model in which many aspects of virus infection and immunity can be explored. Recently, the NHP field has recognized that repeated low-dose mucosal challenges more closely approximate the mechanisms of sexual transmission of HIV and hence may increase the relevance of the model for HIV vaccine design and testing44. We identified two areas of focus for NHP research: enhancing our understanding of viral-host interactions, including virus vulnerabilities to immune effector mechanisms triggered by different vaccine concepts and the relationship between the innate and the adaptive immune responses in viral containment, and dissecting the earliest events after infection at the mucosa, an immunological window that cannot be easily addressed in humans. However, the utility of the NHP model as a translational bridge in HIV vaccine design and development has been limited by the lack of standardized methodologies and the continued use of a variety of challenge models that differ from each other and from the human situation, by the high costs associated with NHP research that have underpowered many studies, and by the relative paucity of shared reagents, tools, technologies and infrastructure to optimize the model and enable closer coordination with clinical studies. The NHP model cannot serve as a gatekeeper for the advancement of candidates to clinical trials. Nevertheless, the availability of a uniformly accepted model or models would enable the standardized testing and comparison of vaccine concepts to inform hypothesis-driven clinical trials. To capitalize on the potential of the NHP model, the field should do the following ( Box 2 ): 1. Make better use of global NHP infrastructure and enhance current capacity to support appropriately powered experiments. As a consensus in the field develops around repeated low-dose challenge studies, larger numbers of animals will be required. This will put additional strain on primate facilities and increase the costs of experiments. Therefore, it will be essential to explore ways of lowering the costs of NHP studies by maximizing the use of available resources and by increasing the level of collaboration, for example by the creation of multidisciplinary consortia. 2. Develop, standardize and share tools and technologies to support NHP studies. The field should strive to develop and use comprehensive standardized in vitro and in vivo immunological, virological and genomic assays and reagents, as well as mechanisms for sharing data among investigators. Collaborative, multidisciplinary consortia are one way to encourage the standardization and sharing of reagents, protocols and data. Efforts underway in this regard include the European Primate Network, a recent initiative funded under the EU Framework, which aims to bring together researchers and breeding centers to standardize processes and optimize the use of NHP models, and the Institute for Laboratory Animal Research, which is leading the development of an international primate plan, including characterization of NHP genetics and the development of global informatics to share information on animal tissue and diagnostics. Cross-cutting considerations * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information Developing a framework that better integrates the clinical trials endeavor with discovery research requires strengthening and/or establishing additional enabling structures and processes. The following three cross-cutting considerations are essential to achieve the field's scientific priorities (Fig. 1). Industry expertise and resources, including technologies, research platforms, vaccine components and formulations and production capacity are essential to moving promising vaccine candidates from research and development to licensure and distribution. At the same time, publicly funded academic research has focused on upstream discovery and on developing the infrastructure necessary for trials, including trial sites and relationships with volunteer communities. There is, therefore, a clear complementarity of expertise and a compelling public health imperative for industry and academia to explore innovative approaches to collaboration. Despite this complementarity of strengths and shared interests, it has been challenging for industry to justify sustained, upstream, high-risk investment in HIV vaccine research. As a result, these collaborations typically form on an ad hoc basis, involve a single company with an academic group and have short-term objectives. The field's challen! ge and opportunity now is to explore systematic, strategic approaches that maximize the scientific value of such partnerships, minimize risk for industry, and allow for more open, nonexclusive arrangements, especially for precompetitive areas of research. The urgency and opportunity, together with the risks and cost of HIV vaccine research, require that we explore the following multipronged strategy to strengthen partnerships and engagement with industry ( Box 3 ): Box 3: Targets for industry engagement Full box 1. Explore innovative models for collaborations with pharmaceutical and biotechnology companies, public funders and researchers. The field should explore innovative models of partnership that build on either existing arrangements in the health and other sectors or new collaborative models specific for HIV vaccine development. For example, IAVI, in partnership with the Bill and Melinda Gates Foundation, has established an Innovation Fund that proactively surveys the commercial biotech universe to identify and finance innovative technologies offering the potential for breakthroughs in HIV vaccine discovery and development. 2. Develop working arrangements to protect intellectual property while ensuring maximum public benefit. With the multidisciplinary product development endeavor proposed in the 2010 Plan, there is urgency in developing a globally accepted intellectual property framework that supports open practices and the freedom to operate while judiciously protecting industry interests. As articulated in the 2005 Plan, the field should explore new mechanisms of sharing the results of precompetitive research while safeguarding the intellectual property of industry partners and protecting further downstream discoveries and at the same time working to ensure that future vaccines are available to all populations in need. There is an increasing number of examples of innovative public-private partnerships that could be looked at as starting points for discussion. In addition, Enterprise members should consider committing to principles such as the recent Statement of Principles and Strategies for! the Equitable Dissemination of Medical Technologies (http://www.autm.net/source/Endorsement/) endorsed by the NIH and over a dozen major universities and, where possible, should improve and harmonize material transfer agreements and develop grant terms that mandate the sharing of resources. The path forward will rely heavily on the application of new and diverse approaches to build a vaccine pipeline driven by multidisciplinary clinical and scientific investigation. This long-term effort must be sustained by reinforcing current research and development efforts with fresh talent, including individuals from areas most affected by the epidemic and from early-career investigators who bring fresh perspectives, enthusiasm and creativity. To attract the brightest minds to the field and provide them with the training and support needed to cross disciplines and platforms to navigate basic, preclinical and clinical domains, the field should do the following ( Box 4 ): Box 4: Targets for the cross-cutting consideration on people Full box 1. Establish, support and sustain global research excellence. The global clinical research effort requires a strategic commitment to developing and sustaining talent in low- and middle-income countries by expanding training opportunities, providing mentorship (locally and globally, and in clinical and basic research domains), ensuring protected research time and salary support for basic and preclinical researchers and clinician-scientists pursuing independent research programs, developing attractive career pathways for clinical trial staff, and providing opportunities for meaningful contribution and leadership within the global research effort. To this end, the imminent relocation of the African AIDS Vaccine Partnership offices to Africa provides an opportunity to further mobilize efforts across the African continent. Similarly, the AIDS Vaccine for Asia Network is providing a facilitative mechanism for strengthening and coordinating vaccine research and development activiti! es in Asia. Building on the programs of EDCTP, IAVI, the Wellcome Trust, the NIH Fogarty International Center and individual academic institutions, capacity building would benefit from coupling local financial and organizational contributions from host country governments with sustainable funding commitments from development agencies and international partners. Moreover, emerging scientific and economic powers where HIV is a public health concern have an important stake in the HIV vaccine research effort and should be actively encouraged to contribute their knowledge-based workforces and scientific resources to the goals of the Enterprise. 2. Attract and mentor young and early-career investigators. Despite their importance to progress in science, young and early-career investigators in both developed and developing countries face serious obstacles to establishing their careers, securing independent funding, developing multidisciplinary expertise and achieving visibility and recognition, particularly in the context of large collaborative initiatives. Strategies are needed to strengthen and clarify career paths for early-career investigators through mentorship, training and leadership opportunities and to increase the availability of funds to pursue unique approaches to vaccine research. 3. Develop and sustain strong institutional capacity. Scientific and medical personnel from low- and middle-income countries face special challenges. To retain highly trained professionals and enable them to make maximal use of their expertise, strong scientific and healthcare institutions are essential. Without strong institutions, individual capacity building will result in the emigration of highly qualified personnel from the countries that need them most. Aid and scientific agencies have an opportunity to expand current efforts to build long-term partnerships with countries and institutions in low- and middle-income countries to develop the institutional support that is a necessary element for both HIV vaccine research and capacity development more broadly. The current global level of funding for HIV vaccine research (about $850 million in 2008) is a substantial sum45. However, when placed in the context of the financial response to the global epidemic that has reached tens of billions of dollars annually, it represents a comparatively small investment of global AIDS resources. If we are to bring the epidemic under control and mitigate the burden of this disease, we must sustain and fortify our quest for a vaccine. The 2010 Plan puts forward an ambitious program of research and development. Clinical trials are expensive, especially the large, scientifically rich efficacy trials called for in the 2010 Plan. Moreover, a strengthened clinical trials endeavor that includes a marked increase in the number and complexity of clinical efficacy trials will require a major increase in support. The 2010 Plan recognizes that resources are scarce and that most financial commitments have been made by a handful of organizations45. Therefore, a first priority must be to ensure that existing resources are prioritized, sustained and optimized. However, it is unlikely that more efficient use of current resources will be sufficient to achieve the priorities and targets laid out in the 2010 Plan. Rather, new sources of investment are needed to exploit recent scientific progress and to match the world's investments in HIV vaccine research with the urgency, size and cost of the epidemic. To this end, it will! be important to do the following ( Box 5 ): Box 5: Targets for cross-cutting consideration on funding and resources Full box 1. Use existing resources more efficiently through increased coordination and sharing of global capacity and expertise. Having taken major steps over the past few years toward coordinated HIV vaccine research efforts, the field must now take stock of available resources (facilities, platforms and policies) across the globe to harness their full potential, avoid redundant investments and foster a more coordinated research effort. For instance, the field should ensure that the contributions of volunteers in cohort studies and clinical trials are maximized by the efficient use and sharing of existing resources (for example, clinical trials sites) before developing new ones. Similarly, the sharing of high-quality primate facilities around the world should be maximized. Enterprise partners should work together to identify and facilitate opportunities for cost sharing. 2. Diversify and increase funds. Implementing the Priorities and Cross-cutting Considerations in the 2010 Plan requires new investments. Only a small number of funding organizations provide the majority of funds devoted to HIV vaccine research43. This situation is less than desirable for the following reasons: first, relying so heavily on a small number of funding partners places the global HIV vaccine research agenda at risk if even one of those funders changes priorities or cuts back their investments; second, it compromises a priority articulated in the Plan for a diversity of approaches to vaccine development; and third, it limits the possibility of new funds if the same small number of funders are repeatedly asked to invest more. Put simply, the global HIV/AIDS challenge requires a global effort that is commensurate with the size of the challenge. Therefore, Enterprise partners should articulate an implementation strategy that makes it clear for other organizations wher! e new investment and expertise is needed. Conclusions and next steps * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information The creation of the Global HIV Vaccine Enterprise and its emphasis on a shared Scientific Strategic Plan represents an unprecedented response by the international scientific community to the scientific, public health and humanitarian challenges posed by HIV/AIDS. Enterprise stakeholders have a shared commitment to fulfill three essential functions: conducting regular assessments of scientific priorities and updating them to reflect lessons learned, new opportunities and the influence of new scientific findings and new technologies, establishing global processes to address priority areas and establishing a culture of mutual accountability for effective implementation of the Plan by funders and investigators. These commitments remain imperative to the fulfillment of the 2010 Plan in driving progress in the field. Over the past 18 months, major scientific advances have signaled the beginning of an important new phase in HIV vaccine research. At the same time, there is increasing evidence that the epidemic is in danger of spinning out of control46. It is our collective responsibility to ensure that this moment is not lost. 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Download references Acknowledgments * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information The 2010 Scientific Strategic Plan of the Global HIV Vaccine Enterprise was developed through a broad process of consultation. Thanks are given to the co-chairs of the various Enterprise Working Groups (A. Aderem, B. Autran, D. Barouch, L. Corey, R. King, J. Mascola, F. McCutchan, A. McMichael, T. Ndung'u, L. Picker, B. Pulendran, R. Rappuoli and R. Steinman), to Working Group members and to the Enterprise Science Committee for their invaluable insights and recommendations that have informed the development of this document. R. Lackman, R. Wiley and B. Snow provided much appreciated assistance and advice. Special thanks to A. Manrique and Y. Voronin for their invaluable support, input and suggestions at all stages during the process of developing the Plan. Author information * An important moment in HIV vaccine research * The Global HIV Vaccine Enterprise * The 2010 Plan's two scientific priorities * Cross-cutting considerations * Conclusions and next steps * References * Acknowledgments * Author information Affiliations * Complete lists of authors and affiliations appear at the end of this paper. * The Council of the Global HIV Vaccine Enterprise * IAVI, New York, New York, USA. * Seth Berkley * MHRP, Rockville, Maryland, USA. * Kenneth Bertram * ANRS, Paris, France. * Jean-François Delfraissy & * Yves Lévy * European Commission, Brussels, Belgium. * Ruxandra Draghia-Akli & * Manuel Romaris * NIH-NIAID, Bethesda, Maryland, USA. * Anthony Fauci & * Margaret I Johnston * Enterprise Secretary-Treasurer, New York, New York, USA. * Cynthia Hallenbeck * First Lady of Rwanda, Kigali, Rwanda. * Madame Jeannette Kagame * Merck Research Laboratories, Rahway, New Jersey, USA. * Peter Kim * WHO-UNAIDS, Geneva, Switzerland. * Daisy Mafubelu & * Catherine Hankins * University of KwaZulu-Natal, Durban, South Africa. * Malegapuru W Makgoba * London School of Hygiene and Tropical Medicine, London, UK. * Peter Piot * Wellcome Trust, London, UK. * Mark Walport * AVAC: Global Advocacy for HIV Prevention, New York, New York, USA. * Mitchell Warren * The Bill & Melinda Gates Foundation, Seattle, Washington, USA. * Tadataka Yamada & * José Esparza * Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia, USA. * Rafi Ahmed * Global HIV Vaccine Enterprise, New York, New York, USA * Alan Bernstein Consortia * The Council of the Global HIV Vaccine Enterprise * Members of the Enterprise * Seth Berkley, * Kenneth Bertram, * Jean-François Delfraissy, * Ruxandra Draghia-Akli, * Anthony Fauci, * Cynthia Hallenbeck, * Madame Jeannette Kagame, * Peter Kim, * Daisy Mafubelu, * Malegapuru W Makgoba, * Peter Piot, * Mark Walport, * Mitchell Warren & * Tadataka Yamada * Alternate members * José Esparza, * Catherine Hankins, * Margaret I Johnston, * Yves Lévy & * Manuel Romaris * Ex-officio members * Rafi Ahmed & * Alan Bernstein Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Alan Bernstein (abernstein@vaccineenterprise.org) Note: Opinions reflected in this manuscript do not necessarily reflect the official policies of the agencies represented by Council members. Additional data - Advanced antisense therapies for postexposure protection against lethal filovirus infections
Warren TK Warfield KL Wells J Swenson DL Donner KS Van Tongeren SA Garza NL Dong L Mourich DV Crumley S Nichols DK Iversen PL Bavari S - Nat Med 16(9):991-994 (2010)
Nature Medicine | Brief Communication Advanced antisense therapies for postexposure protection against lethal filovirus infections * Travis K Warren1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Kelly L Warfield1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Jay Wells1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dana L Swenson1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kelly S Donner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Sean A Van Tongeren1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole L Garza1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lian Dong1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dan V Mourich2 Search for this author in: * NPG journals * PubMed * Google Scholar * Stacy Crumley2 Search for this author in: * NPG journals * PubMed * Google Scholar * Donald K Nichols1 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrick L Iversen2piversen@avibio.com Search for this author in: * NPG journals * PubMed * Google Scholar * Sina Bavari1sina.bavari@amedd.army.mil Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:991–994Year published:(2010)DOI:doi:10.1038/nm.2202Received22 March 2010Accepted28 July 2010Published online22 August 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Currently, no vaccines or therapeutics are licensed to counter Ebola or Marburg viruses, highly pathogenic filoviruses that are causative agents of viral hemorrhagic fever. Here we show that administration of positively charged phosphorodiamidate morpholino oligomers (PMOplus), delivered by various dosing strategies initiated 30–60 min after infection, protects >60% of rhesus monkeys against lethal Zaire Ebola virus (ZEBOV) and 100% of cynomolgus monkeys against Lake Victoria Marburg virus (MARV) infection. PMOplus may be useful for treating these and other highly pathogenic viruses in humans. View full text Figures at a glance * Figure 1: Postexposure protection of ZEBOV-infected rhesus monkeys by AVI-6002. In all experiments, monkeys were challenged with approximately 1,000 plaque-forming units of ZEBOV-Kikwit by intramuscular injection, and PMOplus was administered in PBS beginning 30–60 min after challenge. () Combined Kaplan-Meier survival analysis from two proof-of-concept experiments in which monkeys (n = 8) were treated daily with 40 mg per kg body weight AVI-6002. Doses were divided into equal volumes and administered at intraperitoneal and subcutaneous sites. Four monkeys received treatments for 14 d, and four received treatment for 10 d. A single untreated monkey served as an infection control. (–) Multiple-dose postexposure efficacy assessment of AVI-6002 for treatment of ZEBOV infection in rhesus monkeys. Monkeys were randomly assigned to treatment groups, and the in-life portion of the experiment was conducted under single-blind experimental conditions. AVI-6002 was delivered at one of four dose levels: 40 (6002-40; n = 5), 28 (6002-28; n = 5), 16 (6002-16; n =! 5) or 4 (6002-4; n = 5) mg per kg body weight. Four monkeys were treated with 40 mg per kg body weight negative-control PMOplus formulation (AVI-6003; 6003-40), and one monkey was treated with PBS. All treatments were administered by bolus intravenous injection daily through day 14 after infection. Statistically significant differences (*P < 0.05) between means of AVI-6002 treatments and the AVI-6003 treatment are indicated. () Kaplan-Meier survival curves. () Mean platelet counts. () Mean peripheral blood lymphocyte counts. () Mean aspartate aminotransferase (AST) concentration (instrument interference prevented lymphocyte quantification of day 28 6002-16 sample). () Plasma viremia assessed by quantitative real-time PCR; results from samples collected from individual monkeys on day 8 are shown. * Figure 2: Postexposure protection of MARV-infected cynomolgus monkeys by AVI-6003. In all experiments, monkeys were challenged with approximately 1,000 plaque-forming units of MARV-Musoke by subcutaneous injection, and treatments were initiated beginning 30–60 min after challenge and were administered daily through day 14 after infection. PMOplus was formulated in PBS for delivery. () Combined survival and viremia from two independent proof-of-concept evaluations. AVI-6003 was administered at doses of 40 (n = 4) or 30 (n = 3) mg per kg body weight via intraperitoneal and subcutaneous injections or was delivered at 40 mg per kg body weight by subcutaneous (n = 3) or intravenous (n = 3) injection. Treatments delivered at intraperitoneal and subcutaneous sites were administered by injecting equal volumes at each site. Viremia results from day 8, obtained by standard plaque assay (Vero cells), are presented and are depicted as the range and geometric mean. PFU, plaque-forming units. (–) Multiple-dose assessment of AVI-6003 for treatment of MARV infection a! fter exposure in cynomolgus monkeys. In-life study components were conducted under single-blind experimental conditions, and monkeys were randomized to treatments. AVI-6003 was delivered intravenously at one of three doses: 30 (6003-30; n = 5), 15 (6003-15; n = 5) or 7.5 (6003-7.5; n = 5) mg per kg body weight. Four monkeys were treated with 30 mg per kg body weight negative-control PMOplus formulation (AVI-6002; 6002-30), and one monkey was treated with PBS. Statistically significant differences (*P < 0.05) between means of AVI-6003 treatments and the AVI-6002 treatment are indicated. () Kaplan-Meier survival curves. () Mean platelet counts. () Mean peripheral blood lymphocyte counts. () Mean aspartate aminotransferase concentration. () Plasma viremia assessed by standard plaque assay; the maximum viremia value (occurring at either at day 8 or day 10 after infection in all monkeys) obtained during the course of infection is shown for each monkey. This research was conducte! d in compliance with the US Animal Welfare Act and other feder! al statutes and regulations relating to animals and experiments involving animals, and it adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council. The studies were approved by the USAMRIID Institutional Animal Care and Use Committee, and USAMRIID is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Travis K Warren & * Kelly L Warfield Affiliations * United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Maryland, USA. * Travis K Warren, * Kelly L Warfield, * Jay Wells, * Dana L Swenson, * Kelly S Donner, * Sean A Van Tongeren, * Nicole L Garza, * Lian Dong, * Donald K Nichols & * Sina Bavari * AVI Biopharma, Corvallis, Oregon, USA. * Dan V Mourich, * Stacy Crumley & * Patrick L Iversen * Current address: Integrated BioTherapeutics, Germantown, Maryland, USA. * Kelly L Warfield & * Dana L Swenson Contributions T.K.W. designed and supervised multiple-dose primate evaluations, evaluated results and wrote the manuscript. K.L.W. designed, supervised and conducted proof-of-concept nonhuman primate and rodent investigations and evaluated results. J.W., D.L.S., K.S.D., S.A.V.T. and N.L.G. conducted the nonhuman primate and rodent studies and analyzed samples. L.D. conducted quantitative PCR analysis. D.K.N. conducted post-mortem analyses of all nonhuman primate subjects. D.V.M. and S.C. were responsible for synthesis of PMOplus agents. P.L.I. and S.B. designed experiments, evaluated results and provided project oversight. All authors read and approved the final version of the manuscript. Competing financial interests P.L.I. and S.B. claim intellectual property regaring PMOplus technologies for treatment of viral infections. P.L.I., D.V.M. and S.C. are employees of AVI BioPharma. Corresponding authors Correspondence to: * Sina Bavari (sina.bavari@amedd.army.mil) or * Patrick L Iversen (piversen@avibio.com) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (296K) Supplementary Figures 1 and 2, Supplementary Tables 1–3 and Supplementary Methods Additional data - Adaptation of HIV-1 envelope gp120 to humoral immunity at a population level
Bunnik EM Euler Z Welkers MR Boeser-Nunnink BD Grijsen ML Prins JM Schuitemaker H - Nat Med 16(9):995-997 (2010)
Nature Medicine | Brief Communication Adaptation of HIV-1 envelope gp120 to humoral immunity at a population level * Evelien M Bunnik1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zelda Euler1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthijs R A Welkers1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Brigitte D M Boeser-Nunnink1 Search for this author in: * NPG journals * PubMed * Google Scholar * Marlous L Grijsen2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan M Prins2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hanneke Schuitemaker1, 3h.schuitemaker@amc.uva.nl Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:995–997Year published:(2010)DOI:doi:10.1038/nm.2203Received03 June 2010Accepted28 July 2010Published online29 August 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg By comparing HIV-1 variants from people who became infected at the beginning of the epidemic and from people who have recently contracted the virus, we observed an enhanced resistance of the virus to antibody neutralization over time, accompanied by an increase in the length of the variable loops and in the number of potential N-linked glycosylation sites on the HIV-1 envelope gp120 subunit. The enhanced neutralization resistance of HIV-1 in contemporary seroconverters coincided with the poorer elicitation of neutralizing antibody responses, which may have implications for vaccine design. View full text Figures at a glance * Figure 1: Increased neutralization resistance and reduced immunogenicity of contemporary subtype B HIV-1 as compared to historical HIV-1 isolates. (,) Sensitivity to neutralization by HIVIg batch 1 () and HIVIg batch 2 () of clonal HIV-1 variants isolated during primary infection from participants of the Amsterdam Cohort Studies who seroconverted between 1985 and 1989 (historical seroconverters, n = 8) or between 2003 and 2006 (contemporary seroconverters, n = 13), using one to four virus variants per participant. Each data point shows the average 50% inhibitory concentration (IC50) of all virus variants from one seroconverter. Horizontal bars represent the median IC50 of all viruses per group. () Sensitivity of clonal HIV-1 variants isolated from historical seroconverters (n = 14) or contemporary seroconverters (n = 20) to neutralization by individual sera from 22 unrelated contemporary seroconverters. Each data point represents the average IC50 of a single serum sample tested on all viruses from either historical or contemporary seroconverters. Differences between viruses from both groups of seroconverters were evalu! ated with a paired Student's t test. () Sensitivity of same virus panel in to neutralization by serum from contemporary seroconverter M31281. Each data point shows the average IC50 of all virus variants from one seroconverter, and horizontal bars represent the median IC50 of all viruses per group. (–) Sensitivity of the same virus panel used in and to neutralization by monoclonal antibodies b12 (), 2G12 (), 2F5 () and 4E10 (). Each data point shows the average IC50 of all virus variants from one seroconverter. Horizontal bars represent the median IC50 of all viruses per group. For all panels, neutralization sensitivities were determined with a peripheral blood mononuclear cell–based assay. Differences between groups in panels , and – were assessed with a Mann-Whitney U test. () Number of heterologous virus variants from a multiclade virus panel (n = 23) that could be neutralized by sera obtained approximately 3 years after SC from participants of the Amsterdam Cohort ! Studies who seroconverted in the period 1985–1986 (n = 31), ! 1987–1989 (n = 25) or 1990–1996 (n = 25), as determined by a U87-based neutralization assay. Horizontal bars represent the medians. The changing breadth of heterologous neutralizing serum activity over calendar time was evaluated for statistical significance by a Jonckheere-Terpstra test. SC, seroconversion; NS, not significant. * Figure 2: Increased Env length and increased number of PNGSs in the viral envelope in contemporary subtype B HIV-1 as compared to historical isolates. (–) The lengths of gp120 (), the V1 loop of gp120 () and the V4 loop of gp120 () and the number of PNGSs in gp120 (), the variable (V) regions of gp120 () and the V1 loop of gp120 () are shown for viruses isolated during primary infection from participants of the Amsterdam Cohort Studies who seroconverted between 1985 and 1989 (n = 14) or between 2003 and 2006 (n = 20). Each data point represents the average value for all viruses from one seroconverter. (–) Similar comparisons are shown for subtype B envelope sequences from the Los Alamos database, with a documented year of virus isolation between 1985 and 1988 (n = 27) or between 2003 and 2005 (n = 72) for the length of gp120 (), the length of the V1V2 loop of gp120 () and for the number of PNGSs in gp120 () and in the V1 loop of gp120 (). In all panels, horizontal bars represent the median. Differences between groups were evaluated with a Mann-Whitney U test. aa, amino acid residues. Amsterdam Cohort Studies have been ! conducted in accordance with the ethical principles set out in the declaration of Helsinki, and written informed consent was obtained from each cohort participant prior to data and material collection. The study was approved by the Academic Medical Center Institutional Medical Ethics Committee of the University of Amsterdam. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zelda Euler & * Matthijs R A Welkers Affiliations * Department of Experimental Immunology, Sanquin Research, Landsteiner Laboratory, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. * Evelien M Bunnik, * Zelda Euler, * Matthijs R A Welkers, * Brigitte D M Boeser-Nunnink & * Hanneke Schuitemaker * Department of Infectious Diseases, Tropical Medicine and AIDS, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. * Marlous L Grijsen & * Jan M Prins * Present addresses: Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands (M.R.A.W.) and Crucell Holland BV, Leiden, The Netherlands (H.S.). * Matthijs R A Welkers & * Hanneke Schuitemaker Contributions E.M.B. designed the study, performed experiments, analyzed data and wrote the manuscript; Z.E. performed experiments and analyzed data; M.R.A.W. performed part of the sequence analyses; B.D.M.B.-N. performed part of the neutralization experiments; M.L.G. and J.M.P. selected and recruited individuals for the contemporary seroconverter cohort and contributed samples and data of these subjects; and H.S. designed the study, supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hanneke Schuitemaker (h.schuitemaker@amc.uva.nl) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (164K) Supplementary Figures 1–3, Table 1 and Methods Additional data - Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease
Lee BD Shin JH Vankampen J Petrucelli L West AB Ko HS Lee YI Maguire-Zeiss KA Bowers WJ Federoff HJ Dawson VL Dawson TM - Nat Med 16(9):998-1000 (2010)
Nature Medicine | Brief Communication Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease * Byoung Dae Lee1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Joo-Ho Shin1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jackalina VanKampen3 Search for this author in: * NPG journals * PubMed * Google Scholar * Leonard Petrucelli3 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew B West1, 2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Han Seok Ko1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Yun-Il Lee1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kathleen A Maguire-Zeiss4 Search for this author in: * NPG journals * PubMed * Google Scholar * William J Bowers5 Search for this author in: * NPG journals * PubMed * Google Scholar * Howard J Federoff6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Valina L Dawson1, 2, 8, 9, 11vdawson@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Ted M Dawson1, 2, 8, 11tdawson@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:998–1000Year published:(2010)DOI:doi:10.1038/nm.2199Received25 January 2010Accepted21 July 2010Published online22 August 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Leucine-rich repeat kinase-2 (LRRK2) mutations are a common cause of Parkinson's disease. Here we identify inhibitors of LRRK2 kinase that are protective in in vitro and in vivo models of LRRK2-induced neurodegeneration. These results establish that LRRK2-induced degeneration of neurons in vivo is kinase dependent and that LRRK2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of Parkinson's disease. View full text Figures at a glance * Figure 1: Identification of inhibitors of LRRK2 kinase. () LRRK2 autophosphorylation (% of control) in the presence or absence of kinase inhibitors (Supplementary Table 1). Red indicates LRRK2 kinase inhibitors. ***P < 0.001 by analysis of variance (ANOVA) compared to the other groups. Neuman-Keuls post hoc test. Degrees of freedom = 34 (total) and F = 18.4144. () Representative phosphoimage of WT LRRK2 and LRRK2 G2019S autophosphorylation in the presence or absence of LRRK2 kinase inhibitors. LRRK2 kinase-dead (D1994A) and KN-93 are negative controls. IB, immunoblot. (,) Dose-response curves of WT LRRK2 and G2019S LRRK2 autophosphorylation after treatment with LRRK2 kinase inhibitors. (–) Raf kinase inhibitors dose-response curves on WT LRRK2 (), G2019S LRRK2 () and LRRK1 () autophosphorylation. () G2019S LRRK2 autophosphorylation and 4E-BP1 phosphorylation in the presence or absence of LRRK2 kinase inhibitors. The G2019S LRRK2 kinase-dead mutant (G2019S-D1994A), ZM336372 and indirubin are negative controls. () Quantification ! of G2019S LRRK2 autophophorylation and 4E-BP1 phosphorylation in the presence or absence of LRRK2 kinase inhibitors. ***P < 0.001, by ANOVA, Neuman-Keuls post hoc test. Degrees of freedom for LRRK2 = 17 (total) and F = 22.401. Degrees of freedom for 4E-BP1 = 17 (total) and F = 22.453. All data represents the mean ± s.e.m. from three independent experiments. * Figure 2: LRRK2 kinase inhibition protects against LRRK2-induced neuronal toxicity. () Quantification of neuronal injury, normalized to the number of viable neurons transfected with eGFP in three experiments. ***P < 0.001 and *P < 0.05 by ANOVA compared to eGFP control. +++P < 0.001 by ANOVA compared to LRRK2 G2019S. #P < 0.05 by ANOVA compared to LRRK2 D1994A. Tukey-Kramer post hoc test. Degrees of freedom = 21 (total) and F = 42.436. () Quantification of neuronal injury in the presence or absence LRRK2 kinase inhibitors. ***P < 0.001 by ANOVA compared to eGFP control. +++P < 0.01 by ANOVA compared to DMSO control. Tukey-Kramer post hoc test. Degrees of freedom = 28 (total) and F = 47.3152. () Quantification of neuronal cell death via TUNEL. ***P < 0.001 by ANOVA compared to eGFP control. +++P < 0.01 by ANOVA compared to LRRK2 G2019S. Neuman-Keuls post hoc test. Degrees of freedom = 14 (total) and F = 12.4378. () TUNEL quantification in the presence or absence of LRRK2 kinase inhibitors. **P < 0.01 by ANOVA compared to eGFP control. ++P < 0.01 by ANOVA com! pared to DMSO control. Neuman-Keuls post hoc test. Degrees of freedom = 20 (total) and F = 16.6113. () LRRK2 and GFP immunoblots of striatum and substantia nigra (SN) 2 weeks after intrastriatal infusion of HSV-eGFP, WT LRRK2 (HSV-WT LRRK2), G2019S LRRK2 (HSV-G2019S LRRK2) and G2019S-D1994A LRRK2 (HSV-G2019S-D1994A LRRK2). () Substantia nigra tyrosine hydroxylase (TH) immunolabeling 3 weeks after HSV-mediated delivery of eGFP, WT LRRK2, G2019S LRRK2 or G2019S-D1994A LRRK2 in the presence or absence of LRRK2 kinase inhibitors. Scale bar, 500 μm. () Tyrosine hydroxylase–positive and Nissl-positive cell counts comparing eGFP, WT LRRK2, G2019S LRRK2 or G2019S-D1994A LRRK2. Each bar represents the mean number (± s.e.m., n = 8) of tyrosine hydroxylase–positive cells. ***P < 0.001 by ANOVA compared to eGFP control and WT LRRK2. +++P < 0.001 by ANOVA compared to G2019S-D1994A LRRK2. Tukey-Kramer post hoc test. Degrees of freedom = 67 (total) and F = 6.5115 for Nissl staining ! groups. Degrees of freedom = 68 (total) and F = 7.1292 for tyr! osine hydroxylase staining groups. () Tyrosine hydroxylase–positive and Nissl-positive cell counts comparing LRRK2 G2019S in the presence or absence of LRRK2 kinase inhibitors. Each bar represents the mean number (± s.e.m., n = 8) of tyrosine hydroxylase–positive cells. *P < 0.05, **P < 0.01 and ***P < 0.001 by ANOVA compared to DMSO vehicle control. Tukey-Kramer post hoc test. Degrees of freedom = 70 (total) and F = 5.6004 for Nissl staining groups. Degrees of freedom = 88 (total) and F = 5.0678 for tyrosine hydroxylase staining groups. All procedures used in this study involving mice were approved by the Johns Hopkins Medical Institute Animal Care Committee and by the Mayo Foundation Institutional Animal Care and Use Committee. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Valina L Dawson & * Ted M Dawson Affiliations * Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Byoung Dae Lee, * Joo-Ho Shin, * Andrew B West, * Han Seok Ko, * Yun-Il Lee, * Valina L Dawson & * Ted M Dawson * Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Byoung Dae Lee, * Joo-Ho Shin, * Andrew B West, * Han Seok Ko, * Yun-Il Lee, * Valina L Dawson & * Ted M Dawson * Department of Neuroscience, Mayo Clinic College of Medicine, Jacksonville, Florida, USA. * Jackalina VanKampen & * Leonard Petrucelli * Department of Neuroscience, Georgetown University Medical Center, Washington, DC, USA. * Kathleen A Maguire-Zeiss * Center for Neural Development and Disease, Department of Neurology, University of Rochester Medical Center, Rochester, New York, USA. * William J Bowers * Office of the Executive Vice President and Executive Dean, Georgetown University Medical Center, Washington, DC, USA. * Howard J Federoff * Department of Neurology, Georgetown University Medical Center, Washington, DC, USA. * Howard J Federoff * Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Valina L Dawson & * Ted M Dawson * Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Valina L Dawson * Present address: Center for Neurodegeneration and Experimental Therapeutics, University of Alabama School of Medicine, Birmingham, Alabama, USA. * Andrew B West Contributions B.D.L., J.V., L.P., A.B.W., V.L.D. and T.M.D. designed the experiments. B.D.L., J.V., H.S.K., Y.I.L. and J.-H.S. generated data. K.A.M.-Z., W.J.B. and H.J.F. generated HSV-encoded LRRK2s. B.D.L. and J.V. analyzed data. B.D.L., V.L.D. and T.M.D. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing financial interests T.M.D. is a paid consultant to Merck KGAA. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies. Corresponding authors Correspondence to: * Ted M Dawson (tdawson@jhmi.edu) or * Valina L Dawson (vdawson@jhmi.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–6, Supplementary Tables 1–3 and Supplementary Methods Additional data - Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance
López M Varela L Vázquez MJ Rodríguez-Cuenca S González CR Velagapudi VR Morgan DA Schoenmakers E Agassandian K Lage R de Morentin PB Tovar S Nogueiras R Carling D Lelliott C Gallego R Orešič M Chatterjee K Saha AK Rahmouni K Diéguez C Vidal-Puig A - Nat Med 16(9):1001-1008 (2010)
Nature Medicine | Article Hypothalamic AMPK and fatty acid metabolism mediate thyroid regulation of energy balance * Miguel López1, 2, 3m.lopez@usc.es Search for this author in: * NPG journals * PubMed * Google Scholar * Luis Varela1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * María J Vázquez1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sergio Rodríguez-Cuenca3 Search for this author in: * NPG journals * PubMed * Google Scholar * Carmen R González1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Vidya R Velagapudi4 Search for this author in: * NPG journals * PubMed * Google Scholar * Donald A Morgan5 Search for this author in: * NPG journals * PubMed * Google Scholar * Erik Schoenmakers3 Search for this author in: * NPG journals * PubMed * Google Scholar * Khristofor Agassandian6 Search for this author in: * NPG journals * PubMed * Google Scholar * Ricardo Lage1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Pablo Blanco Martínez de Morentin1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sulay Tovar1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Rubén Nogueiras1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * David Carling7 Search for this author in: * NPG journals * PubMed * Google Scholar * Christopher Lelliott8 Search for this author in: * NPG journals * PubMed * Google Scholar * Rosalía Gallego9, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Matej Orešič4, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Krishna Chatterjee3, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Asish K Saha10, 11 Search for this author in: * NPG journals * PubMed * Google Scholar * Kamal Rahmouni5 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Diéguez1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Antonio Vidal-Puig3ajv22@cam.ac.uk Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1001–1008Year published:(2010)DOI:doi:10.1038/nm.2207Received11 June 2010Accepted06 August 2010Published online29 August 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Thyroid hormones have widespread cellular effects; however it is unclear whether their effects on the central nervous system (CNS) contribute to global energy balance. Here we demonstrate that either whole-body hyperthyroidism or central administration of triiodothyronine (T3) decreases the activity of hypothalamic AMP-activated protein kinase (AMPK), increases sympathetic nervous system (SNS) activity and upregulates thermogenic markers in brown adipose tissue (BAT). Inhibition of the lipogenic pathway in the ventromedial nucleus of the hypothalamus (VMH) prevents CNS-mediated activation of BAT by thyroid hormone and reverses the weight loss associated with hyperthyroidism. Similarly, inhibition of thyroid hormone receptors in the VMH reverses the weight loss associated with hyperthyroidism. This regulatory mechanism depends on AMPK inactivation, as genetic inhibition of this enzyme in the VMH of euthyroid rats induces feeding-independent weight loss and increases expressio! n of thermogenic markers in BAT. These effects are reversed by pharmacological blockade of the SNS. Thus, thyroid hormone–induced modulation of AMPK activity and lipid metabolism in the hypothalamus is a major regulator of whole-body energy homeostasis. View full text Figures at a glance * Figure 1: Energy balance, AMPK pathway and POMC expression. () Western blotting (left) and quantification (middle) of hypothalamic pAMPKα, AMPKα1, AMPKα2, total AMPK (tAMPKα), pACCα, ACCα (bottom band in the total ACC (tACC) blot), ACCβ (top band in the tACC blot) and FAS and hypothalamic AMPKα1 and AMPKα2 activities (right) in euthyroid and hyperthyroid rats. (–) Hypothalamic levels of Fasn (), malonyl-CoA content () and CPT1 activity () in euthyroid and hyperthyroid rats. (–) Body weight change (), daily food intake (), hypothalamic malonyl-CoA content (), Pomc mRNA levels in the arcuate nucleus (ARC) () and western blotting (left) and quantification (right) of hypothalamic pFoxO1 and pSTAT3 () in euthyroid and hyperthyroid rats treated i.c.v. with vehicle or cerulenin for 4 d. ¶P = 0.1, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle or euthyroid vehicle; #P < 0.05 euthyroid cerulenin versus hyperthyroid cerulenin; ###P < 0.001 hyperthyroid vehicle versus hyperthyroid cerulenin; all data are expressed as means ± ! s.e.m. All experiments were repeated at least twice. * Figure 2: Effects of chronic central T3 administration. (–) Body weight change (,) and daily food intake (,) of euthyroid (,) and hypothyroid (,) rats i.c.v.-treated with T3 for 4 d. (–) Western blotting (left) and quantification (right) of hypothalamic pAMPKα, AMPKα1, AMPKα2, pACCα and ACCα () and mRNA expression profiles in BAT () of euthyroid rats i.c.v.-treated with T3 for 4 d. (–) Body weight change (), daily food intake () and mRNA expression profiles in BAT () of euthyroid rats i.c.v.-treated with T3 and subcutaneously (s.c.) treated with the β3-AR–specific antagonist SR59230A for 4 d. !P = 0.09, $P = 0.08, +P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle; #P < 0.05, ##P < 0.01 T3 i.c.v. versus T3 i.c.v. SR59230A; all data are expressed as means ± s.e.m. * Figure 3: Effects of central T3 on BAT activation via the SNS. () Double immunohistochemistry (top, scale bar, 200 μm; bottom, scale bar, 20 μm) showing pAMPKα and TRα coexpression in the VMH. (–) Western blotting (left) and quantification (right) of hypothalamic pAMPKα, AMPKα1, AMPKα2, pACCα and ACCα (), immunohistochemistry showing c-Fos immunoreactivity in the dorsal motor nucleus of the vagus (DMV) (top, scale bar, 200 μm) and in the raphe pallidus (RPa) and the inferior olive (IO) nuclei (bottom, scale bar, 100 μm) and c-Fos–immunoreactive (c-Fos-IR) cells in those nuclei () and BAT SNA (,) of euthyroid rats 1–3 h (protein and c-Fos) or 6 h (SNA) after i.c.v. treatment with T3. (,) Western blotting (left) and quantification (right) of hypothalamic pAMPKα, AMPKα1, AMPKα2, pACCα and ACCα () and BAT SNA () of euthyroid rats 1 h after VMH microinjection of T3. ¶P = 0.1, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle; #P < 0.05 T3 i.c.v. 2 ng versus T3 i.c.v. 4 ng; all data are expressed as means ± s.e.m. 3V,! third ventricle; CC, central canal; HN, hypoglossal nucleus. * Figure 4: Effects of genetic ablation of thyroid hormone receptor in the VMH. (–) Body weight change (), daily food intake (), plasma T3 () and T4 () abundance and mRNA expression profiles in BAT () of hyperthyroid (and euthyroid when indicated) rats stereotaxically treated with GFP-expressing adenoviruses or GFP plus TR-DN adenoviruses into the VMH. *P < 0.05, **P < 0.01, ***P < 0.001 versus euthyroid GFP or hyperthyroid GFP; all data are expressed as means ± s.e.m. * Figure 5: Effects of inactivation of hypothalamic de novo lipogenesis. (–) Body weight change (left) and daily food intake (right) (), hypothalamic malonyl-CoA content () and Ucp1 and Ucp3 mRNA levels in the BAT () of hyperthyroid (hyperthyr) rats (and euthyroid (euthyr) rats when indicated) treated with vehicle or TOFA. (–) Body weight change (left) and daily food intake (right) (), hypothalamic malonyl-CoA content () and Ucp1 and Ucp3 mRNA levels and in the BAT () of hyperthyroid (and euthyroid when indicated) rats treated with vehicle or AICAR. (–) Malonyl-CoA content in the ventral hypothalamus (), body weight change (left) and food intake (right) () and mRNA expression profiles in BAT () of hyperthyroid (or euthyroid when indicated) rats stereotaxically treated with GFP-expressing adenoviruses or GFP plus AMPK AMPKα-CA adenoviruses into the VMH. ¶P = 0.1, *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle or GFP; ###P < 0.01 hyperthyroid vehicle versus hyperthyroid TOFA or AICAR and hyperthyroid GFP versus hyperthyroid AMPKα-CA; a! ll data are expressed as means ± s.e.m. * Figure 6: Effects of selective inactivation of AMPK in the VMH. (–) Malonyl-CoA content in the ventral hypothalamus (), body weight change (), food intake () and mRNA expression profiles in BAT () of euthyroid rats stereotaxically treated with GFP-expressing adenoviruses or GFP plus AMPKα-DN into the VMH. (–) Body weight change (), food intake () and mRNA expression profiles in BAT () of rats administered GFP-expressing adenoviruses stereotaxically into the VMH plus administered vehicle s.c.; administered GFP plus AMPKα-DN adenoviruses plus s.c.-treated with vehicle; or administered GFP plus AMPKα-DN adenoviruses plus s.c.-treated with the β3-AR specific antagonist SR59230A. () Proposed model of the effect of thyroid hormones excess on hypothalamic fatty acid metabolism. Hyperthyroidism and T3 upregulate de novo lipogenesis in the hypothalamus, which results from decreased activity of AMPK, activation of ACC and increased expression of Fasn. Thyroid hormone–induced changes in the hypothalamic lipid biosynthetic pathway increase! s levels of hypothalamic malonyl-CoA and complex lipids. These changes are associated with the activation of the SNS through the raphe pallidus and the inferior olive nuclei, resulting in increased expression of BAT markers, such as Ucp1, Upc3, Ppargc1a (which encodes PGC1α) and Ppargc1b (which encodes PGC1β), promoting negative energy balance and weight loss. *P < 0.05, **P < 0.01, ***P < 0.001 versus GFP; #P < 0.05, ##P < 0.01 AMPKα-DN vehicle versus AMPKα-DN SR59230A; all data are expressed as means ± s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Rosalía Gallego, * Matej Orešič, * Krishna Chatterjee & * Asish K Saha Affiliations * Department of Physiology, School of Medicine, University of Santiago de Compostela-Instituto de Investigación Sanitaria, Santiago de Compostela (A Coruña), Spain. * Miguel López, * Luis Varela, * María J Vázquez, * Carmen R González, * Ricardo Lage, * Pablo Blanco Martínez de Morentin, * Sulay Tovar, * Rubén Nogueiras & * Carlos Diéguez * Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y Nutrición (CIBERobn) (A Coruña), Spain. * Miguel López, * Luis Varela, * María J Vázquez, * Carmen R González, * Ricardo Lage, * Pablo Blanco Martínez de Morentin, * Sulay Tovar, * Rubén Nogueiras & * Carlos Diéguez * Institute of Metabolic Science, Metabolic Research Laboratories, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK. * Miguel López, * Sergio Rodríguez-Cuenca, * Erik Schoenmakers, * Krishna Chatterjee & * Antonio Vidal-Puig * Technical Research Centre of Finland (VTT), Espoo, Finland. * Vidya R Velagapudi & * Matej Orešič * Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA. * Donald A Morgan & * Kamal Rahmouni * Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA. * Khristofor Agassandian * Cellular Stress Group, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, Imperial College, London, UK. * David Carling * Department of Biosciences, AstraZeneca, Research and Development, Mölndal, Sweden. * Christopher Lelliott * Department of Morphological Sciences, School of Medicine, University of Santiago de Compostela (A Coruña), Spain. * Rosalía Gallego * Diabetes Research Unit, Boston Medical Center, Boston, Massachusetts, USA. * Asish K Saha Contributions M.L., L.V., M.J.V., S.R.-C., C.R.G., R.L., P.B.M.d.M., S.T. and R.N. performed the in vivo experiments, analytical methods (real-time RT-PCR, in situ hybridization, western blotting and enzymatic assays) and collected and analyzed the data. V.R.V. and M.O. developed analytical platforms and performed and analyzed lipidomic experiments. D.A.M., K.A. and K.R. performed and analyzed the sympathetic nerve activity recording studies. D.C. developed AMPK-DN– and AMPK-CA–encoding adenoviruses. E.S. and K.C. generated TR-DN constructs and validated the TR-DN–encoding adenoviruses. R.G. developed and performed immunohistochemistry and immunofluorescence experiments. A.K.S. developed and performed metabolic analyses. M.L., L.V., S.R.-C., C.L., K.C., K.R., C.D. and A.V.-P. designed the experiments. M.L., S.R.-C., R.N., C.L., K.C., K.R., C.D. and A.V.-P. discussed the manuscript. M.L., C.D. and A.V.-P. coordinated and directed the project. M.L. and A.V.-P. developed the hypothesis! and wrote the manuscript. Competing financial interests C.L. is an employee of AstraZeneca Research and Development and holds stock in AstraZeneca Research and Development. Corresponding authors Correspondence to: * Miguel López (m.lopez@usc.es) or * Antonio Vidal-Puig (ajv22@cam.ac.uk) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Table 1, Supplementary Figures 1–7 and Supplementary Methods Additional data - Allosteric inhibition of lysyl oxidase–like-2 impedes the development of a pathologic microenvironment
- Nat Med 16(9):1009-1017 (2010)
Nature Medicine | Article Allosteric inhibition of lysyl oxidase–like-2 impedes the development of a pathologic microenvironment * Vivian Barry-Hamilton1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rhyannon Spangler1 Search for this author in: * NPG journals * PubMed * Google Scholar * Derek Marshall1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott McCauley1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hector M Rodriguez1 Search for this author in: * NPG journals * PubMed * Google Scholar * Miho Oyasu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda Mikels1 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Vaysberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Haben Ghermazien1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Wai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos A Garcia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arleene C Velayo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brett Jorgensen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Donna Biermann1 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Tsai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer Green1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shelly Zaffryar-Eilot2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alison Holzer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott Ogg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dung Thai1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gera Neufeld2 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Van Vlasselaer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Victoria Smith1vsmith@arresto.com Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1009–1017Year published:(2010)DOI:doi:10.1038/nm.2208Received14 December 2009Accepted05 August 2010Published online05 September 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We have identified a new role for the matrix enzyme lysyl oxidase–like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-β (TGF-β) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor β-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in on! cologic and fibrotic diseases. View full text Figures at a glance * Figure 1: LOXL2 localization in the stroma of diverse tumor types. () Quantitative real-time-PCR analysis of LOXL2 transcript in solid tumors compared to non-neoplastic tissues. GIST, gastrointestinal stromal tumor; adeno., adenocarcinoma; serous, serous adenocarcinoma; ovarian endom., ovarian endometrioid carcinoma; sem., seminoma; squam., squamous cell carcinoma; carc., carcinoma; islet, islet cell tumor. () Immunohistochemistry (IHC) analysis showing LOXL2, α-SMA as a marker of activated fibroblasts and collagen I (COL1A) in matched breast adenocarcinoma tumor sections. Tissues were stained with 3,3′- diaminobenzidine (brown) and counterstained with hematoxylin (blue). () Immunofluorescence double staining (yellow) of LOXL2 and α-SMA in colon adenocarcinoma. (−) LOXL2 IHC in lung squamous cell carcinoma (), pancreatic adenocarcinoma () and colon adenocarcinoma () and IHC for LOXL2 and CD31 (), as a marker of vasculature, in renal clear cell carcinoma. (,) LOXL2 IHC in healthy lung () and heart tissue () compared to a CD31 control f! or matched sections. Scale bars, 50 μm. * Figure 2: In vitro characterization of inhibitory monoclonal antibody AB0023. (,) Immunofluorescence of Hs578t cells, transfected with a nontargeting control small interfering RNA, using AB0023 (Alexa fluor; green) and a COL1A-specific antibody (cy3; red) () compared to siLOXL2 knockdown cells stained using the same antibodies (). () Immunofluorescence images showing the effects of AB0023 on the morphological changes in MCF-7 cells induced by LOXL2-containing conditioned medium (MDA-MB-231 CM) (actin cytoskeleton visualized with rhodamine phalloidin; nuclei stained with DAPI). MCF-7 CM, control conditioned medium. Scale bars, 50 μm. () A fibroblast–endothelial cell coculture experiment showing the effects of AB0023 on vessel formation by endothelial cells at concentrations of 0.1, 10 and 50 μg ml−1. () Quantitative analysis of triplicate wells for each of six concentrations of AB0023, revealing dose-dependent inhibition of vessel branching, number and length. Data are represented as mean ± s.d. * Figure 3: Effects of AB0023 on fibroblast activation and growth factor abundance in the tumor microenvironment. () Immunohistochemistry (IHC) analysis of MDA-MB-435 primary tumor model showing LoxL2 expression and α-Sma–positive fibroblasts. Scale bar, 50 μm. () Effects on MDA-MB-435 primary tumor volume by treatment with M64, AB0023 (*P = 0.022) and docetaxel (**P < 0.001). () Quantitative analysis of cross-linked collagen by Sirius red staining and IHC data for the MDA-MB-435 efficacy study from independent tumors and sections showing significant reductions for tumors in the AB0023-treated animals in cross-linked collagen (*P = < 0.001), activated fibroblasts as assessed by α-Sma signal (*P < 0.001), tumor-associated endothelial cells as assessed by CD31 signal (*P < 0.001), and VegfA (*P < 0.001), Cxcl12 (*P = 0.002), Ctgf (*P = 0.003) and Loxl2 (*P < 0.001). Significant reductions were also observed for the number of CD31+ cells in the docetaxel treatment group (**P = 0.005) and for VegfA amounts in the M64 and docetaxel treatment groups (**P < 0.001, ***P = 0.001). Supplemen! tary Data contains details on statistical analyses. (,) Examples of Sirius red staining for cross-linked collagen () and α-Sma signal () of tumors from the vehicle and AB0023 treatment groups. Scale bars, 50 μm. (,) Reduction of Tgf-β1 amounts (*P < 0.001) () and Tgf-β signaling, as assessed by the ratio of p-Smad2 to total Smad-2 amounts (, *P = 0.049), by ELISA of lysates generated from tumors from the AB0023-treated mice compared to vehicle controls. () Quantitative analysis of p-Smad2 by IHC, showing significant reduction of p-Smad2 signal in tumors from AB0023-treated mice compared to other treatment groups (*P = 0.005). () Autophagy marker beclin-1 signal in tumors from different treatment groups in the MDA-MB-435 primary tumor model (*P = 0.014, **P < 0.001). * Figure 4: Comparison of AB0023 and β-APN and effects of AB0023 on metastatic burden in xenograft models. () Effects on MDA-MB-435 primary tumor volume by treatment with AB0023 and β-APN (*P = 0.018). () A quantitative analysis of cross-linked collagen (*P = 0.023), α-Sma (*P = 0.016) and CD31 signal (*P = 0.014) from tumors in this study. (,) Day 24 MDA-MB-231 tumor cell burden, assessed by bioluminescent signal in the femur (, *P = 0.008, **P = 0.032) and total ventral bone (, *P = 0.034, **P = 0.036). () Bioluminescence measurements of tumer cell buden from ex vivo imaging of lung, pancreas (panc) and liver showing the effects of AB0023 treatment (*P = 0.027, **P = 0.031, ***P = 0.039) in an SKOV3 metastasis model. Data are represented as means ± s.e.m. () Immunohistochemistry analysis showing Loxl2, α-Sma, glial fibrillary acidic protein (Gfap), cross-linked collagen and Ki-67 in matched sections from a pancreatic metastatic tumor treated with AB0023. Scale bars, 50 μm. * Figure 5: Effects of AB0023 in a liver fibrosis model. (,) LOXL2 expression in human hepatitis C–associated liver fibrosis (), and LOXL2 and LOX in steatohepatitic liver (). Scale bars, 50 μm. () Kaplan-Meier survival curve showing survival benefit of AB0023 treatment in a CCl4-induced model of liver fibrosis (100% survival, *P = 0.006). (–) Percentage fibrotic area determined by METAVIR scoring, reflecting the number of portal triads for each METAVIR score divided by the total number of portal triads in the region analyzed in each treatment group, showing effects of AB0023 on bridging fibrosis (portoportal or portocentral) (, *P = 0.011 or **P = 0.013). Examples of α-Sma signal in livers from vehicle and AB0023 treatment groups (). Scale bars, 50 μm. Analysis of α-Sma signal showing decrease of myofibroblast activation in the portoportal regions of the liver in vehicle and AB0023 treatment groups (, *P = 0.008). () Quantitative analysis of p-Smad3 signal by immunohistochemistry showing significant reduction of p-Smad3 s! ignal in livers from AB0023-treated mice (*P < 0.001) versus livers from vehicle-treated mice, compared to increased p-Smad3 levels in livers from M64-treated mice (**P < 0.001). Data are represented as means ± s.e.m. * Figure 6: Effects of AB0023 in bleomycin-induced lung fibrosis. () Immunohistochemistry (IHC) analysis showing LOXL2 and α-SMA (as a marker of activated fibroblasts) in fibroblastic foci in serial human IPF sections. () IHC analysis showing Loxl2 expression associated with fibroblasts and reactive pneumocytes in lungs of vehicle-treated mice. Scale bars, 50 μm. () (*P < 0.001). () H&E stainings of lungs from vehicle-treated and AB0023-treated mice exposed to bleomycin. Scale bar, 50 μm. () Ashcroft scores representing the fibrotic changes in the lungs of saline-exposed control mice, and vehicle and AB0023 treatment groups for bleomycin-exposed mice (*P = 0.003). () Quantitative analysis of α-Sma (*P = 0.009), Tgf-β1 (*P < 0.001), endothelin-1 (*P = 0.005), Loxl2 (*P < 0.001) and Cxcl12 (*P < 0.001) signal after AB0023 treatment versus vehicle treatment. () Lung weights from mice exposed to saline (Saline, collected at day 22), or mice exposed to bleomycin and analyzed at initiation of antibody treatment (day 6, Collect Rx) or after ! antibody treatment from day 6 to day 22 (Bleo + AB0023 or Bleo + AC1) (*P = 0.008). () H&E and Masson's trichrome stainings showing honeycomb lung in the AC1-treated mice compared to the less fibrotic lungs from AB0023-treated mice. Scale bars, 50 μm. () Average Ashcroft score with AB0023 treatment versus AC1 treatment (*P = 0.027). () Quantitative analysis of cross-linked collagen (*P < 0.001, **P < 0.001), α-Sma (*P < 0.001, **P < 0.001) and Loxl2 (*P < 0.001, **P < 0.001) in lungs from saline-exposed, Collect Rx, AC1 and AB0023 treatment groups. Data are represented as means ± s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Arresto BioSciences, Palo Alto, California, USA. * Vivian Barry-Hamilton, * Rhyannon Spangler, * Derek Marshall, * Scott McCauley, * Hector M Rodriguez, * Miho Oyasu, * Amanda Mikels, * Maria Vaysberg, * Haben Ghermazien, * Carol Wai, * Carlos A Garcia, * Arleene C Velayo, * Brett Jorgensen, * Donna Biermann, * Daniel Tsai, * Jennifer Green, * Alison Holzer, * Scott Ogg, * Dung Thai, * Peter Van Vlasselaer & * Victoria Smith * Cancer Research and Vascular Biology Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel. * Shelly Zaffryar-Eilot & * Gera Neufeld Contributions V.B.-H. and R.S. performed immunohistochemistry and histology on human and mouse tissues and all associated analyses, with V.B.-H. leading the immunohistochemistry group, and they both participated in manuscript preparation. V.B.-H. performed EMT studies, D.M. performed transcript analyses, managed the tissue collection, designed the wound-healing model and participated in manuscript preparation, S.M. performed cloning and expression and participated in EMT experiments and manuscript preparation, H.M.R. performed data analysis, IC50 studies and antibody characterization, M.O. performed immunohistochemistry and analysis for the liver fibrosis study, A.M. and M.V. performed tension experiments and antibody characterization, A.M. participated in manuscript preparation, H.G. performed immunohistochemistry analysis for the SKOV3 study and participated in manuscript preparation, C.W. performed transcript analysis, C.A.G., A.C.V., B.J., D.B. and D.T. generated, characterized and qu! ality controlled all antibodies and proteins under the leadership of C.A.G., J.G. performed tissue ELISA, S.Z.-E. contributed to antibody characterization and performed the microvessel density analysis, A.H. supervised the MDA-MB-435 mouse studies, S.O. assisted with the management of contract research groups, D.T. participated in toxicology studies and manuscript preparation, G.N. developed the Y698F mutant and contributed to oncology studies and manuscript preparation, P.V.V. participated in experimental design and manuscript preparation and V.S. designed the metastasis, fibrosis and toxicology animal studies and analyses, supervised the experimental work and wrote the paper. Competing financial interests As current or former employees of Arresto BioSciences, V.B.-H., R.S., D.M., S.M., H.M.R., M.O., A.M., M.V., H.G., C.W., C.A.G., A.C.V., B.J., D.B., D.T., A.H., S.O., D.T., P.V.V. & V.S. have an equity stake in the company. Corresponding author Correspondence to: * Victoria Smith (vsmith@arresto.com) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Data, methods and figures 1–7 Additional data - CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation
- Nat Med 16(9):1018-1023 (2010)
Nature Medicine | Letter CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation * Veronica Marcos1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhe Zhou2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Ali Önder Yildirim3, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Bohla3 Search for this author in: * NPG journals * PubMed * Google Scholar * Andreas Hector1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ljubomir Vitkov5, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Eva-Maria Wiedenbauer1 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolf Dietrich Krautgartner6 Search for this author in: * NPG journals * PubMed * Google Scholar * Walter Stoiber6 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd H Belohradsky1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nikolaus Rieber1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Kormann1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara Koller7 Search for this author in: * NPG journals * PubMed * Google Scholar * Adelbert Roscher1 Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Roos8 Search for this author in: * NPG journals * PubMed * Google Scholar * Matthias Griese1 Search for this author in: * NPG journals * PubMed * Google Scholar * Oliver Eickelberg3 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerd Döring9 Search for this author in: * NPG journals * PubMed * Google Scholar * Marcus A Mall2 Search for this author in: * NPG journals * PubMed * Google Scholar * Dominik Hartl1, 4dominik.hartl@med.uni-tuebingen.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1018–1023Year published:(2010)DOI:doi:10.1038/nm.2209Received07 June 2010Accepted10 August 2010Published online05 September 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs)1, 2, 3. Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases4, 5. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation6, 7. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of ! CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease. View full text Figures at a glance * Figure 1: CXCR2 mediates NETosis. () NET formation was induced by stimulation of primary neutrophils isolated from healthy control subjects (left and right) or differentiated neutrophilic-like HL-60 cells (dHL-60) (middle) with recombinant human CXCL8 (100 nM) or recombinant human CXCL2 (100 nM) at 37 °C for 3 h. Left, peripheral blood–isolated neutrophils were pretreated for 30 min with control IgG2a (IgG), CXCR1-specific IgG2a (anti-CXCR1) or CXCR2-specific IgG2a (anti-CXCR2) blocking antibodies (all 20 μg ml−1) before chemokine stimulation. Middle, dHL-60 cells transfected with a mock control, CXCR1 or CXCR2 were stimulated with chemokines as described above. Right, peripheral blood–isolated neutrophils were pretreated for 30 min with the CXCR2 inhibitors SB225002 (100 nM), SB265610 (100 nM) or vehicle only and were then stimulated with chemokines as described above. White bars represent untreated cells (top graphs) or detergent-lysed cells (bottom graphs). Error bars represent means ± s.e.m. of ! five independent experiments. *P < 0.05; **P < 0.01. () Representative images of CXCR2-mediated NETosis. Isolated neutrophils from healthy control individuals were stimulated for 3 h at 37 °C with CXCL8 (100 nM) with or without pretreatment with CXCR1-specific IgG2a or CXCR2-specific IgG2a blocking antibodies (all 20 μg ml−1). Blue, DAPI staining of DNA. Scale bar, 10 μm. * Figure 2: CXCR2-mediated NET formation is NADPH oxidase independent. () Neutrophils from healthy controls were pretreated for 30 min with the NADPH oxidase inhibitors diphenylene iodonium (DPI, 10 μM) or apocynin (10 μM) or the ROS scavenger N-acetyl cysteine (NAC) (10 μM) and were then stimulated for 3 h with PMA (20 nM) or the recombinant human chemokines CXCL8 (100 nM) or CXCL2 (100 nM). Where indicated, neutrophils from individuals with CGD were used instead of healthy control neutrophils. White bars indicate PMA-treated cells. () Representative images of NETosis using neutrophils from individuals with CGD. Isolated neutrophils from individuals with CGD were stimulated for 3 h at 37 °C with PMA (20 nM), CXCL8 (100 nM), CXCL2 (100 nM) or CXCL2 (100 nM) and P. aeruginosa (PAO1) bacteria (1 × 106 bacteria per ml, bottom right image). Representative images out of five independent experiments are shown. Top and bottom right, scanning electron microscopy (SEM); bottom left and middle, confocal laser scanning microscopy (CLSM). Blue, DAPI (! DNA) stain; red, citrullinated histones. PMA-stimulated CGD neutrophils showed no fluorescence for citrullinated histones. Scale bar for CLSM images, 10 μm; scale bar for top left and middle SEM images, 100 μm; scale bar for top right SEM image, 5 μm; scale bar for bottom right SEM image, 2 μm. () Peripheral blood–isolated neutrophils from healthy controls (left) or individuals with CGD (middle) were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), the extracellular signal–regulated kinase (ERK)-MAPK inhibitor PD98059 (10 μM), the p38-MAPK inhibitor SB203580 (10 μM), the Src family kinase inhibitor PP2 (10 μM) or an inhibitor vehicle and were then stimulated for 3 h with recombinant human CXCL8 (100 nM), recombinant human CXCL2 (100 nM) or PMA (20 nM). Right, CXCR2-overexpressing dHL-60 cells were treated with siRNA specific for PI3K, ERK, p38 or Src kinases or control siRNA. NET formation was analyzed by quantification of free DNA and CLSM. The! percentage of NET DNA is depicted. () Peripheral blood–isol! ated neutrophils from healthy controls were pretreated for 30 min with the PI3K inhibitor wortmannin (100 nM), the ERK-MAPK inhibitor PD98059 (10 μM), the p38-MAPK inhibitor SB203580 (10 μM), the Src family kinase inhibitor PP2 (10 μM), the PAD4 inhibitor chloride amidine (200 μM) or an inhibitor vehicle and were then stimulated for 3 h with recombinant human CXCL8 (100 nM), recombinant human CXCL2 (100 nM), P. aeruginosa (PAO1) bacteria (1 × 106 bacteria per ml) or PMA (20 nM). Relative luminosity of citrullinated histone staining (CitrH3) was quantified with a Zeiss LSM software application. *P < 0.05; **P < 0.01. Error bars represent means ± s.e.m. of five independent experiments. * Figure 3: NET formation in cystic fibrosis lung disease. () Immunological characterization of NETs in cystic fibrosis airway fluids by CLSM. Top two rows, NETs in induced cystic fibrosis sputum. Blue, DAPI staining; red, elastase. Bottom two rows, blue, DAPI; red, citrullinated histones. Scale bar, 20 μm. () Ultrastructure of NETs. Left and middle, SEM images of NETosis in cystic fibrosis airway fluids. The arrow indicates bacteria entrapped in NETs. Scale bar, 2 μm. Right, transmission electron microscopy staining of citrullinated histones in cystic fibrosis airway fluids (sputa). () NETs in and cystic fibrosis lung function. Sputum was collected from healthy controls and individuals with cystic fibrosis, and NETs were analyzed. Representative CLSM images of airway fluids from one healthy individual and three individuals with cystic fibrosis, stratified for lung function, are depicted. Scale bar, 10 μm. FEV1, forced expiratory volume in 1 s (% of predicted). DAPI staining of nuclei and extracellular DNA strands in cystic fibro! sis sputa. () Stratification of individuals with cystic fibrosis. Top left, correlation of free DNA levels with FEV1 in sputum supernatants from 50 individuals with cystic fibrosis (CF). Bottom left, free DNA levels in sputum supernatants from healthy controls and individuals with cystic fibrosis, stratified by FEV1. Right, individuals with cystic fibrosis stratified into low (<10 ng ml−1 free DNA in sputum), intermediate (10–15 ng ml−1 free DNA in sputum) and high (>15 ng ml−1 free DNA in sputum) groups of NET producers and their lung function (FEV1 and MEF75/25), P. aeruginosa colony-forming units (CFU) in sputum and free elastase activity in sputum. () Neutrophils were incubated for 3 h at 37 °C with Hanks' balanced salt solution (HBSS) or cell-free sputum supernatants from five healthy controls or five individuals with cystic fibrosis. Where indicated, cystic fibrosis sputum supernatants were pretreated with antibodies to neutralize CXCR2 ligands (anti-CXCL2, a! nti-CXCL1 and anti-CXCL8; all 20 μg ml−1) or nonspecific an! tibodies to IgG (20 μg ml−1). Where indicated, neutrophils were pretreated with the CXCR2 antagonist SB225002 (anti-CXCR2, 100 nM), the Src family kinase inhibitor PP2 (10 μM) or a vehicle control (DMSO) for 30 min. Neutrophils from individuals with CGD were used instead of healthy control neutrophils where indicated. NETosis was analyzed by quantifying free DNA as described in the Online Methods. *P < 0.05. Error bars represent means ± s.e.m. * Figure 4: CXCR2 mediates increased NET formation in mouse cystic fibrosis lung disease in vivo. () Amount of CXCR2 chemokines in BALF from βENaC-Tg (n = 19) and wild-type (WT, n = 11) mice. () NETs in BALF from βENaC-Tg (n = 19) and WT (n = 11) mice. () CXCR2 chemokines (left) and pulmonary obstruction (right) correlate with NETosis in lungs of βENaC-Tg mice. () CXCR2 mediates NETosis in mouse cystic fibrosis–like lung disease in vivo. βENaC-Tg and WT mice were treated once daily for a period of 3 d with the small-molecule CXCR2 antagonist SB225002 (WT mice, n = 6; βENaC-Tg mice, n = 7) or vehicle control (WT mice, n = 9; βENaC-Tg mice, n = 7). The top left graph shows NETosis in BALF, the top right graph shows total lung neutrophils, the bottom left graph shows respiratory burst by BALF neutrophils and the bottom right graph shows phagocytotic capacity by BALF neutrophils. MFI, mean fluorescence intensity. () Representative SEM of BALF from βENaC-Tg mice treated with SB225002 or with vehicle control. Scale bar, 5 μm. () CXCR2 inhibition improves lung functio! n in βENaC-Tg mice. Resistance (left) and forced expiratory volume at 100 ms (FEV100) (right) were analyzed in vehicle-treated (WT mice, n = 9; βENaC-Tg mice, n = 7) or CXCR2 inhibitor–treated (WT mice, n = 6; βENaC-Tg mice, n = 7) βENaC-Tg or WT mice as described in detail in the Online Methods. *P < 0.01. Error bars represent means ± s.e.m. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zhe Zhou & * Ali Önder Yildirim Affiliations * Research Center, Children's Hospital, Ludwig-Maximilians-University, Munich, Germany. * Veronica Marcos, * Andreas Hector, * Eva-Maria Wiedenbauer, * Bernd H Belohradsky, * Nikolaus Rieber, * Michael Kormann, * Adelbert Roscher, * Matthias Griese & * Dominik Hartl * Division of Pediatric Pulmonology and Cystic Fibrosis Center, Department of Pediatrics III, University of Heidelberg, Heidelberg, Germany. * Zhe Zhou & * Marcus A Mall * Comprehensive Pneumology Center, Institute of Lung Biology and Disease, University Hospital, Ludwig-Maximilians-University and Helmholtz Zentrum München, Munich, Germany. * Ali Önder Yildirim, * Alexander Bohla & * Oliver Eickelberg * Children's Hospital and Interdisciplinary Center for Infectious Diseases, University of Tübingen, Tübingen, Germany. * Andreas Hector, * Michael Kormann & * Dominik Hartl * Department of Operative Dentistry & Periodontology, Saarland University, Homburg, Germany. * Ljubomir Vitkov * Division of Light and Electron Microscopy, Department of Organismic Biology, University of Salzburg, Salzburg, Austria. * Ljubomir Vitkov, * Wolf Dietrich Krautgartner & * Walter Stoiber * Department of Dermatology and Allergy, Ludwig-Maximilians-University, Munich, Germany. * Barbara Koller * Sanquin Research, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. * Dirk Roos * Institute of Medical Microbiology and Hygiene, University of Tübingen, Tübingen, Germany. * Gerd Döring Contributions V.M. and E.M.W. performed the in vitro and ex vivo experiments. Z.Z. performed mouse experiments. A.Ö.Y. and A.B. performed pulmonary function tests. L.V. contributed to data analysis and discussion. A.H. provided patient material and clinical data and contributed to data analysis and discussion. W.D.K., G.D. and W. S. contributed to data analysis and discussion. B.H.B. and N.R. provided material from human subjects and clinical data. B.K. performed CXCL2 assays and contributed to data analysis and discussion. D.R. contributed to data analysis and discussion. G.D., M.K. and A.R. contributed to data analysis, discussion and manuscript preparation. M.G. provided material from human subjects and clinical data and contributed to discussion. O.E. designed pulmonary function experiments, contributed to discussion and wrote the manuscript. M.A.M. designed animal experiments, contributed to discussion and wrote the manuscript. D.H. designed and supervised the study, analyzed data, ! provided funding and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dominik Hartl (dominik.hartl@med.uni-tuebingen.de) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figs. 1–10, Supplementary Tables 1–3 and Supplementary Methods Additional data - Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use–dependent inhibitor of dopamine synthesis
Yao L Fan P Arolfo M Jiang Z Olive MF Zablocki J Sun HL Chu N Lee J Kim HY Leung K Shryock J Blackburn B Diamond I - Nat Med 16(9):1024-1028 (2010)
Nature Medicine | Letter Inhibition of aldehyde dehydrogenase-2 suppresses cocaine seeking by generating THP, a cocaine use–dependent inhibitor of dopamine synthesis * Lina Yao1, 4lina.yao@gilead.com Search for this author in: * NPG journals * PubMed * Google Scholar * Peidong Fan1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Arolfo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Zhan Jiang1 Search for this author in: * NPG journals * PubMed * Google Scholar * M Foster Olive2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeff Zablocki1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hai-Ling Sun1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nancy Chu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeongrim Lee3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hee-Yong Kim3 Search for this author in: * NPG journals * PubMed * Google Scholar * Kwan Leung1 Search for this author in: * NPG journals * PubMed * Google Scholar * John Shryock1 Search for this author in: * NPG journals * PubMed * Google Scholar * Brent Blackburn1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ivan Diamond1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1024–1028Year published:(2010)DOI:doi:10.1038/nm.2200Received10 March 2010Accepted21 July 2010Published online22 August 2010Corrigendum26 August 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg There is no effective treatment for cocaine addiction despite extensive knowledge of the neurobiology of drug addiction1, 2, 3, 4. Here we show that a selective aldehyde dehydrogenase-2 (ALDH-2) inhibitor, ALDH2i, suppresses cocaine self-administration in rats and prevents cocaine- or cue-induced reinstatement in a rat model of cocaine relapse-like behavior. We also identify a molecular mechanism by which ALDH-2 inhibition reduces cocaine-seeking behavior: increases in tetrahydropapaveroline (THP) formation due to inhibition of ALDH-2 decrease cocaine-stimulated dopamine production and release in vitro and in vivo. Cocaine increases extracellular dopamine concentration, which activates dopamine D2 autoreceptors to stimulate cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC) in primary ventral tegmental area (VTA) neurons. PKA and PKC phosphorylate and activate tyrosine hydroxylase, further increasing dopamine synthesis in a positive-feedback loop. Monoamine oxi! dase converts dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a substrate for ALDH-2. Inhibition of ALDH-2 enables DOPAL to condense with dopamine to form THP in VTA neurons. THP selectively inhibits phosphorylated (activated) tyrosine hydroxylase to reduce dopamine production via negative-feedback signaling. Reducing cocaine- and craving-associated increases in dopamine release seems to account for the effectiveness of ALDH2i in suppressing cocaine-seeking behavior. Selective inhibition of ALDH-2 may have therapeutic potential for treating human cocaine addiction and preventing relapse. View full text Figures at a glance * Figure 1: ALDH2i reduces intravenous cocaine self-administration, cocaine-primed or cue-induced reinstatement and methamphetamine-induced reinstatement in Sprague Dawley rats. () The number of cocaine infusions recorded during the 2-h cocaine self-administration session (n = 7–12, *P < 0.05, **P < 0.01 compared with vehicle (Veh)). (,) The number of lever presses recorded during the 2-h cocaine-primed () or cue-induced () reinstatement session (n = 6–9 and n = 6–11 for and , respectively; #P < 0.01 compared with extinction (Ext); **P < 0.01 compared with Veh). () The number of lever presses recorded during the 2-h methamphetamine-induced reinstatement session (n = 7, #P < 0.01 compared with Ext; *P < 0.05 compared with Veh). * Figure 2: ALDH2i decreases cocaine-induced dopamine (DA) production and increases THP abundance in PC12 cells. (–) Cells were incubated with or without cocaine (Coca, 1 μM) in the presence or absence of ALDH2i, the D1 antagonist SCH 23390 (SCH, 10 μM), the D2 antagonist spiperone (SPIP, 10 μM) or THP for 24 h. Intracellular and extracellular dopamine (,,), total dopamine () or THP amounts () were determined. *P < 0.05, **P < 0.01 compared with cocaine control. () Dose-response analysis of THP and α-methyl-L-tyrosine (αMLT) on the inhibition of total tyrosine hydroxylase (TH) or phosphorylated tyrosine hydroxylase (p-TH). * Figure 3: Cocaine activates PKA and PKC to phosphorylate tyrosine hydroxylase and increase dopamine production in VTA neurons. (–) Cells were incubated for 24 h with or without cocaine (1 μM) or nomifensine (NMFS, 10 μM) in the presence or absence of the PKA activator Sp-cAMPS (Sp, 1 mM, 10 min), the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nM, 10 min), the D1 antagonist SCH23390 (10 μM), the D2 antagonist spiperone (10 μM), the PKA inhibitor Rp-cAMPS (Rp, 20 μM) or the PKC inhibitor GF 109203X (GF, 1 μM). TH phosphorylation at Ser40 p-40; (–), at Ser19 (p-19) and Ser31 (p-31; ) or the translocation of PKA and εPKC () was detected by western blotting (,–) or by immunostaining (). Green indicates phosphorylated tyrosine hydroxylase at Ser40 (p-TH), red, total tyrosine hydroxylase and yellow, the merged images. C/P, the ratio of PKA or ePKC in the cytosolic fraction (C) versus the particulate fraction (P). (,) Intracellular and extracellular dopamine () or total dopamine and THP (). **P < 0.01 compared with sham or cocaine control. * Figure 4: ALDH2i increases THP production to inhibit tyrosine hydroxylase activity and decrease dopamine production in VTA in cocaine-addicted rats. (,) Dopamine and THP amounts measured in the VTA () and nucleus accumbens () pooled from the cocaine-seeking rats treated with ALDH2i (I, 15 mg per kg body weight, n = 8) or vehicle (V, n = 8) in Figure 1c or from naive rats treated with ALDH2i (15 mg per kg body weight, n = 8) or vehicle (n = 8). The experiment was repeated with similar results. () Tyrosine hydroxylase phosphorylation at Ser40 in the VTA, as detected by western blotting. **P < 0.01 compared with naive vehicle control. () The number of lever presses during the 2-h cue-induced cocaine reinstatement session (n = 8, #P < 0.01 compared with extinction; **P < 0.01 compared with Veh). Change history * Change history * Author information * Supplementary informationCorrigendum 26 August 2010 In the version of this article initially published online, Zhan Jiang's name was incorrectly spelled as Zhang Jiang. The error has been corrected for the print, PDF and HTML versions of this article. Author information * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Lina Yao & * Peidong Fan Affiliations * Gilead Sciences, Palo Alto, California, USA. * Lina Yao, * Peidong Fan, * Maria Arolfo, * Zhan Jiang, * Jeff Zablocki, * Hai-Ling Sun, * Nancy Chu, * Kwan Leung, * John Shryock, * Brent Blackburn & * Ivan Diamond * Center for Drug and Alcohol Programs, Departments of Psychiatry and Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA. * M Foster Olive * Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, USA. * Jeongrim Lee & * Hee-Yong Kim Contributions L.Y. and I.D. designed and supervised the project, analyzed the data and wrote the manuscript. P.F. designed, carried out and analyzed molecular and cell biology studies. M.A. designed, performed and analyzed behavioral studies. Z.J. performed the cell biology experiments. M.F.O. carried out cocaine dose-response experiments. J.Z. and team synthesized CVT-10216. K.L. supervised and H.-L.S. and N.C. performed mass spectrometric analysis of in vitro dopamine and THP. J.L. and H.-Y.K. developed a mass spectrometric analysis method for dopamine and THP and determined their in vivo abundance. J.S. contributed to design and review of PC12 data. B.B. contributed to design and review of in vivo data. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lina Yao (lina.yao@gilead.com) Supplementary information * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (256K) Supplementary Figures 1–4, Supplementary Tables 1 and 2 and Supplementary Methods Additional data - Transgenic mice with a diverse human T cell antigen receptor repertoire
Li LP Lampert JC Chen X Leitao C Popović J Müller W Blankenstein T - Nat Med 16(9):1029-1034 (2010)
Nature Medicine | Technical Report Transgenic mice with a diverse human T cell antigen receptor repertoire * Liang-Ping Li1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * J Christoph Lampert1, 2, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaojing Chen1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Catarina Leitao1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jelena Popović1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Werner Müller3 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Blankenstein1, 2tblanke@mdc-berlin.de Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1029–1034Year published:(2010)DOI:doi:10.1038/nm.2197Received18 February 2010Accepted13 July 2010Published online08 August 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Because of tolerance mechanisms, it has been hard to identify the T cell receptors (TCRs) of high-avidity T cells against self (for example, tumor) antigens. TCRs that are specific for foreign human antigens from the nontolerant T cell repertoire can be identified in mice. Moreover, if mice are constructed to express the human TCR repertoire, they can be used to analyze the unskewed repertoire against human self antigens. Here we generated transgenic mice with the entire human TCRαβ gene loci (1.1 and 0.7 Mb), whose T cells express a diverse human TCR repertoire that compensates for mouse TCR deficiency. A human major histocompatibility class I transgene increases the generation of CD8+ T cells with human compared to mouse TCRs. Functional CD8+ T cells against several human tumor antigens were induced, and those against the Melan-A melanoma antigen used similar TCRs to those that have been detected in T cell clones from individuals with autoimmune vitiligo or melanoma. The! se mice will allow researchers to identify pathogenic and therapeutic human TCRs. View full text Figures at a glance * Figure 1: Generation of mice transgenic for the human TCRα and TCRβ gene loci. (,) Schematic diagram of construction of YACs containing the genomic region of the TCRα (yHuTRA) () and TCRβ (yHuTRB) () loci by homologous recombination of YACs containing partial gene loci on overlapping fragments in yeast cells. The TCR regions contained in the YACs and estimated size of inserts (not drawn to scale) are indicated. Note the further modification after recombination of YACs with pBII-LysUraCen (pLUC) and pBCL-PGKneo-A/B vectors. A detailed depiction of YAC construction is in Supplementary Figures 1–4. (,) PCR analysis of yHuTRA- () and yHuTRB- () transgenic mice with a set of TCR-specific primers. Uracil (URA), lysine (LYS), adenosine (ADE) and tryptophan (TRP) genes are yeast-selectable markers. Centro, yeast centromere; V, variable gene; D, diversity gene; J, joining gene; C, constant gene; CE, constant exon; Enh, TCR enhancer; TRD, TCRδ locus; YACL, YAC left arm sequence; Neo, neomycin gene; W1 and Dad, sequences 3′ of the TRA enhancer; M, DNA size! marker. The red boxes in yHuTRB indicate deletion of Vβ5.1 and Vβ6.1 during homologous recombination of YACs. Details of gene segments in the transgenic mice can be found in Supplementary Table 1. * Figure 2: T cell development in human TCRαβ gene loci transgenic mice with a diverse TCR repertoire. (,) Thymocytes () and blood cells () from the indicated mice at 1–2 months of age were stained with antibodies specific for CD3, CD4 and CD8 and analyzed by flow cytometry. () Top, CD3+ cells in the living thymocyte population; middle, CD4 and CD8 single-positive and double-positive cells gated on the thymocyte population; bottom, CD4 and CD8 single-positive and double-positive cells gated on the CD3+ cell population. Percentage of positive cells is indicated. Data are representative of six analyzed mice per group. () Top, CD3+ cells in the living lymphocyte population; bottom, CD4+ and CD8+ cells gated on the living CD3+ cell population. () Spleen cells of an ABabDII mouse and human peripheral blood lymphocytes were analyzed for Vβ expression on CD4+ and CD8+ T cells. Each staining contained three different antibodies specific for individual Vβ chains, as indicated above the diagrams. Shown are the Vβ+ cells within the CD4+ and CD8+ cell population. Percentages of Vβ+! cells are indicated on the diagrams. PE, phycoerythrin. () RT-PCR analysis of spleen cells of an ABabDII mouse with 5′ Vα primers (specific for TRAV genes as indicated) and a 3′ C-region primer. Data in and are representative of two experiments. * Figure 3: CD8+ T cells in ABabDII mice are functional and use similar TCRs as human CD8+ T cells against an immunogenic antigen. () Flow cytometric analysis of peripheral blood cells of mice immunized with ELA or YMD peptides 7–9 d earlier and stained with ELA-A2 or YMD-A2 tetramer molecules together with CD3- and CD8-specific antibodies. Percentage of peptide-tetramer+ cells of CD8+ cells is indicated. Data are representative of three (YMD) and ten (ELA) experiments. () Analysis of CD8 and intracellular IFN-γ expression in spleen cells of ELA-immunized mice stimulated in vitro with various concentrations of human ELA (hELA), mouse ELA (mELA) and YMD peptides. Left, ELA-specific IFN-γ expression in response to 10−9 M peptide; right, percentages of IFN-γ+ cells of the CD8+ cells. One out of three experiments with similar results is shown. () The rearranged TCRα (ELA-A2 negative, n = 14; ELA-A2 positive, n = 14) and TCRβ (ELA-A2 negative, n = 27; ELA-A2 positive n = 30) sequences in spleen cells of ELA-immunized mice after sorting into the ELA-A2 tetramer–positive (95%) and ELA-A2 tetramer–! negative fractions. Shown is the number of isolates of the indicated genes. Sequences from the ELA-A2 tetramer+ fraction are shown in Supplementary Table 2. () FACS analysis of binding of ELA-A2 tetramers or control tetramers (YMD-A2) on CD8+ TCRαβ-deficient Jurkat cells retrovirally transduced with TCRα 10 and TCRβ 2 or 29 cDNA clones. Shown are cells gated on CD3. Percentage of positive cells of the CD8+ cells is indicated. * Figure 4: Specific CD8+ T cell responses against a panel of human TAAs in ABabDII mice. Mice were immunized with the indicated human TAAs. Seven to eleven days later, pooled spleen and lymph node cells were stimulated in vitro with specific (top; the position of the first amino acid in the peptide is indicated by the subscript) or nonspecific (bottom) peptides and analyzed for expression of CD3, CD8 and intracellular IFN-γ. Shown are CD8+ and IFN-γ+ cells within the CD3+ cell population (percentages indicated by numbers). In parentheses, the percentage of CD8+ and IFN-γ+ T cells within the CD8+ T cell population is given. Shown is one representative out of at least three experiments per experimental group. Unspecific peptides were NY-BR-1 960-968, STEAP 262-270. YMD 369-378 and Melan-A 26-35 (ELA). These peptides used for in vitro stimulation were always different from the peptide used for immunization. 9V in the gp100 peptide indicates an amino acid exchange. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Liang-Ping Li & * J Christoph Lampert Affiliations * Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. * Liang-Ping Li, * J Christoph Lampert, * Xiaojing Chen, * Catarina Leitao, * Jelena Popović & * Thomas Blankenstein * Institute of Immunology, Charité Campus Benjamin Franklin, Berlin, Germany. * Liang-Ping Li, * J Christoph Lampert, * Xiaojing Chen, * Catarina Leitao, * Jelena Popović & * Thomas Blankenstein * Bill Ford Chair in Cellular Immunology, University of Manchester, Faculty of Life Sciences, Oxford Road, Manchester, UK. * Werner Müller Contributions L.-P.L. designed experimental strategies and contributed to writing of the manuscript. He contributed Figures 1c, 2a–c and 3, Supplementary Figures 3, 5–7 and 9, Table 1 and Supplementary Tables 1–3. J.C.L. contributed to experimental design and to Figures 1d and 2c, Supplementary Figure 4 and Supplementary Tables 1 and 3. X.C. contributed to Figures 2d, 3d and 4, Supplementary Figure 10 and Supplementary Table 1. C.L. contributed to Figure 4 and Supplementary Figure 8. J.P. contributed to Figure 4. W.M. was responsible for microinjection of ES cells to obtain chimeric mice. T.B. proposed and supervised the project, interpreted data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas Blankenstein (tblanke@mdc-berlin.de) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (904K) Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary Methods Additional data - Therapeutic cell engineering with surface-conjugated synthetic nanoparticles
Stephan MT Moon JJ Um SH Bershteyn A Irvine DJ - Nat Med 16(9):1035-1041 (2010)
Nature Medicine | Technical Report Therapeutic cell engineering with surface-conjugated synthetic nanoparticles * Matthias T Stephan1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * James J Moon1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Soong Ho Um1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Bershteyn1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Darrell J Irvine1, 2, 3, 4, 5djirvine@mit.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1035–1041Year published:(2010)DOI:doi:10.1038/nm.2198Received07 January 2010Accepted05 May 2010Published online15 August 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg A major limitation of cell therapies is the rapid decline in viability and function of the transplanted cells. Here we describe a strategy to enhance cell therapy via the conjugation of adjuvant drug–loaded nanoparticles to the surfaces of therapeutic cells. With this method of providing sustained pseudoautocrine stimulation to donor cells, we elicited marked enhancements in tumor elimination in a model of adoptive T cell therapy for cancer. We also increased the in vivo repopulation rate of hematopoietic stem cell grafts with very low doses of adjuvant drugs that were ineffective when given systemically. This approach is a simple and generalizable strategy to augment cytoreagents while minimizing the systemic side effects of adjuvant drugs. In addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery. View full text Figures at a glance * Figure 1: Stable conjugation of nanoparticles (NPs) to the surfaces of T cells and HSCs via cell-surface thiols. () Flow cytometry analysis of cell surface thiols on mouse splenocytes detected by fluorophore-conjugated malemide co-staining with lineage-specific surface markers for erythrocytes (Ter-119), T cells (CD3), B cells (B220) and hematopoietic stem cells (c-Kit). () Schematic of maleimide-based conjugation to cell surface thiols. MPB-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]. () Confocal microscopy images of CD8+ effector T cells and lineage−Sca-1+c-Kit+ HSCs immediately after conjugation with fluorescent 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD)-labeled multilamellar lipid nanoparticles (left) and after 4-d in vitro expansion (right). Scale bars, 2 μm. () Flow cytometry analysis of CD8+ T cells after incubation with DiD-labeled multilamellar lipid nanoparticles synthesized with or without maleimide-headgroup lipids. () Quantification of nanoparticle internalization. Immature dendritic cells (DCs), effector CD8+ T! cells or HSCs were conjugated with carboxyfluorescein (CFSE)-tagged maleimide-bearing liposomes. Extracellular trypan blue quenching was used to differentiate surface-bound and internalized liposomes immediately after conjugation or after 4 d in culture. Data represent the mean ± s.e.m. of two independent experiments conducted in triplicate. * Figure 2: Nanoparticle conjugation does not affect key T cell functions. OT-1 ovalbumin-specific CD8+ effector T cells were conjugated with 100 DiD-labeled multilamellar lipid nanoparticles per cell or left unmanipulated as controls. () CFSE dilution of unmodified or nanoparticle-conjugated T cells stimulated in vitro with mature ovalbumin peptide–pulsed dendritic cells. DiD mean fluorescence intensities (MFI)s for distinct CFSE lymphocytes populations are indicated in the bottom right quadrant. () 51Cr release assays of unmanipulated and particle-conjugated OT-1 cells targeting ovalbumin peptide–pulsed or control EL4 tumor cells. (,) Transmigration of OT-1 T cells (with or without surface-bound particles) seeded onto MS1 endothelial cell monolayers in the upper well of a transwell chamber after addition of the chemoattractant monocyte chemoattractant protein-1 to the lower chamber. The fraction of transmigrating T cells () and the profile of cell-bound nanoparticle fluorescence before (UW) and after (LW) transmigration () were quantified by ! flow cytometry (DiD MFI ± s.e.m. from triplicate samples shown in blue). * Figure 3: Nanoparticle-decorated T cells efficiently carry surface-tethered nanoparticles into antigen-expressing tumors. (,) Comparative whole-mouse in vivo bioluminescence (tumors and T cells) and fluorescence imaging (nanoparticles) of mice bearing established subcutaneous extG-luc–expressing EG7-OVA and EL4 tumors on opposite flanks, 2 d after i.v. infusion of firefly luciferase–transgenic Thy1.1+ effector OT-1 T cells (with or without attached DiD-labeled nanoparticles) or an equivalent number of free nanoparticles. Thy1.1+ OT-1 T cells recovered from the EG7-OVA tumors were analyzed for surface-bound DiD nanoparticles by flow cytometry (), and the mean bioluminescent T cell and fluorescent nanoparticle signals from groups of 6 mice are shown in . Respective differences in the bioluminescent or fluorescent photon counts of EL4 compared to EG7-OVA tumors were analyzed by the Student's t test. NS, not significant. ↑ refers to higher fluorescent signal strength of the data points on the right in comparison to respective values on the left. () Confocal images of nanoparticle-decorated OT! -1 T cells infiltrating EG7-OVA tumor 2 d after adoptive transfer. Scale bar, 10 μm. Higher magnification images of nanoparticle-carrying tumor-infiltrating T cells are shown at right. Scale bars, 1.5 μm. Yellow arrowheads highlight evidence for surface localization of nanoparticles. Shown is one of two independent experiments. () Biodistribution of an equivalent number of DiD-labeled nanoparticles injected systemically (open bars) or conjugated to adoptively transferred OT-1 T cells (filled bars) after 48 h. The mean fluorescent signal intensities (± s.d.) of nine mice from three independent experiments are graphed. ID, injected dose. Data shown are pooled from three independent experiments. * Figure 4: Pmel-1 T cells conjugated with IL-15Sa– and IL-21–releasing nanoparticles robustly proliferate in vivo and eradicate established B16 melanomas. Lung and bone marrow tumors were established by tail vein injection of 1 × 106 extG-luc–expressing B16F10 cells into C57BL/6 mice. Tumor-bearing mice were treated after 1 week by sublethal irradiation followed by i.v. infusion of 1 × 107 CBR-luc–expressing Vβ13+CD8+ Pmel-1 T cells. One group of mice received Pmel-1 T cells conjugated with 100 nanoparticles per cell carrying a total dose of 5 μg IL-15Sa and IL-21 (4.03 μg IL-15Sa + 0.93 μg IL-21); control groups received unmodified Pmel-1 T cells and a single systemic injection of the same doses of IL-15Sa and IL-21 or Pmel-1 T cells alone. () Dual longitudinal in vivo bioluminescence imaging of extG-luc–expressing B16F10 tumors and CBR-luc–expressing Pmel-1 T cells. () Frequencies of Vβ13+CD8+ Pmel-1 T cells recovered from pooled lymph nodes of representative mice 16 d after T cell transfer. () CBR-luc T cell signal intensities from sequential bioluminescence imaging every 2 d after T cell transfer. Every line! represents one mouse, with each dot showing the whole-mouse photon count. () Survival of mice after T cell therapy illustrated by Kaplan-Meier curves. Shown are six mice per treatment group pooled from three independent experiments. * Figure 5: HSCs carrying GSK-3β inhibitor–loaded nanoparticles reconstitute recipient animals with rapid kinetics after bone marrow transplants without affecting multilineage differentiation potential. (,) Engraftment kinetics of luciferase-transgenic HSC grafts in lethally irradiated nontransgenic syngeneic recipients. Mice were treated with a single bolus injection of the GSK-3β inhibitor TWS119 (1.6 ng) on the day of transplantation, an equivalent TWS119 dose encapsulated in HSC-attached nanoparticles or no exogenous adjuvant compounds. Transplanted mice were imaged for whole-body bioluminescence every 7 d for 3 weeks. Shown are representative in vivo imaging system images () and whole-mouse photon counts () for nine mice total per treatment condition. () Percentage of donor-derived cells two weeks after transplantation of GFP+ HSCs into lethally irradiated recipients with or without TWS119 adjuvant drug. *P < 0.001. () Average frequency of donor-derived GFP+ B-cells, T cells and myeloid cells in five recipient mice per group (± s.d.) three months after transplantation are shown. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Material Science and Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. * Matthias T Stephan, * James J Moon, * Soong Ho Um, * Anna Bershteyn & * Darrell J Irvine * Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, USA. * Matthias T Stephan, * James J Moon, * Soong Ho Um, * Anna Bershteyn & * Darrell J Irvine * Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. * Soong Ho Um & * Darrell J Irvine * Ragon Institute of Massachusetts General Hospital, MIT and Harvard University, Boston, Massachusetts, USA. * Darrell J Irvine * Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Darrell J Irvine Contributions M.T.S. designed and conducted all experiments and wrote the manuscript. J.J.M. assisted in T cell transmigration assays, optimization of multilamellar lipid nanoparticle synthesis and in vivo nanoparticle biodistribution assays. S.H.U. assisted optimization of multilamellar lipid nanoparticle synthesis. A.B. assisted in initial in vitro T cell assays, collected electron microscopy images and contributed experimental suggestions. D.J.I. supervised all experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Darrell J Irvine (djirvine@mit.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (644K) Maleimide-bearing NPs are not internalized after cell surface conjugation. CD8+ T cells were surface-conjugated with fluorescent DiD-tagged NPs (Fig. 1c). Sequences of single confocal z-sections acquired at 0.4 1m intervals throughout the T cells are shown here and in Supplementary Movie 2. * Supplementary Movie 2 (716K) Maleimide-bearing NPs are not internalized after cell surface conjugation. CD8+ T cells were surface-conjugated with fluorescent DiD-tagged NPs (Fig. 1c). Sequences of single confocal z-sections acquired at 0.4 1m intervals throughout the T cells are here and in Supplementary Movie 1. * Supplementary Movie 3 (672K) Membrane-tethered NPs are retained on the cell surface of tumor homing T lymphocytes. Explanted cross-sectioned EG7-OVA tumors 2 d after T cell injection (Fig 3c). CellTracker green–labeled OT-1 T cells and rhodamine lipid–coated NPs (magenta) were visualized by confocal microscopy. Sequences of single confocal z-sections acquired at 0.4-1m intervals throughout the T cells are shown here and in Supplementary Movie 4. Scale bar, 1.5 1m. * Supplementary Movie 4 (1M) Membrane-tethered NPs are retained on the cell surface of tumor homing T lymphocytes. Explanted cross-sectioned EG7-OVA tumors 2 d after T cell injection (Fig 3c). CellTracker green–labeled OT-1 T cells and rhodamine lipid–coated NPs (magenta) were visualized by confocal microscopy. Sequences of single confocal z-sections acquired at 0.4-1m intervals throughout the T cells are shown here and in Supplementary Movie 3. Scale bar, 1.5 1m. PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–17 and Supplementary Methods Additional data - Clinical microfluidics for neutrophil genomics and proteomics
Kotz KT Xiao W Miller-Graziano C Qian WJ Russom A Warner EA Moldawer LL De A Bankey PE Petritis BO Camp DG Rosenbach AE Goverman J Fagan SP Brownstein BH Irimia D Xu W Wilhelmy J Mindrinos MN Smith RD Davis RW Tompkins RG Toner M the Inflammation and the Host Response to Injury Collaborative Research Program Baker HV Balis UG Billiar TR Calvano SE Cobb JP Cuschieri J Finnerty CC Gamelli RL Gibran NS Harbrecht BG Hayden DL Hennessy L Herndon DN Jeschke MG Johnson JL Klein MB Lowry SF Maier RV Mason PH McDonald-Smith GP Minei JP Moore EE Nathens AB O Keefe GE Rahme LG Remick DG Schoenfeld DA Shapiro MB Sperry J Storey JD Tibshirani R Warren HS West MA Wispelwey B Wong WH - Nat Med 16(9):1042-1047 (2010)
Nature Medicine | Technical Report Clinical microfluidics for neutrophil genomics and proteomics * Kenneth T Kotz1kkotz@partners.org Search for this author in: * NPG journals * PubMed * Google Scholar * Wenzong Xiao1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Carol Miller-Graziano3 Search for this author in: * NPG journals * PubMed * Google Scholar * Wei-Jun Qian4 Search for this author in: * NPG journals * PubMed * Google Scholar * Aman Russom1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth A Warner5 Search for this author in: * NPG journals * PubMed * Google Scholar * Lyle L Moldawer5 Search for this author in: * NPG journals * PubMed * Google Scholar * Asit De3 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul E Bankey3 Search for this author in: * NPG journals * PubMed * Google Scholar * Brianne O Petritis4 Search for this author in: * NPG journals * PubMed * Google Scholar * David G Camp II4 Search for this author in: * NPG journals * PubMed * Google Scholar * Alan E Rosenbach1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeremy Goverman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Shawn P Fagan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernard H Brownstein6 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel Irimia1 Search for this author in: * NPG journals * PubMed * Google Scholar * Weihong Xu2 Search for this author in: * NPG journals * PubMed * Google Scholar * Julie Wilhelmy2 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael N Mindrinos2 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard D Smith4 Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald W Davis2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald G Tompkins1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mehmet Toner1mtoner@hms.harvard.edu Search for this author in: * NPG journals * PubMed * Google Scholar * the Inflammation and the Host Response to Injury Collaborative Research Program * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1042–1047Year published:(2010)DOI:doi:10.1038/nm.2205Received24 August 2009Accepted30 May 2010Published online29 August 2010 Abstract * Abstract * Accession codes * 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 Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting. View full text Figures at a glance * Figure 1: Summary of microfluidic device characterization. (,) Microfluidic chip design () and schematic of the surface functionalization of antibodies to the device (). Green biotinylated CD66b-specific monoclonal antibodies bind red Neutravidin molecules that are covalently linked to the surface. Whole blood flows through each parallel capture channel and cells expressing CD66b antigen are specifically bound to the surface. (,) Chip loading for cells captured () and RNA () with a linear fit (solid line), 95% confidence limits (dashed line) and 95% prediction bands (dotted line). The R value for the fits for and are 0.95 and 0.98, respectively. Error bars for both graphs represent means ± s.d. determined from three or more separate, independent experiments. () Wright-Giemsa stain of isolated cells captured from a burn subject 10 d after injury, showing a mixture of fully segmented neutrophils and band forms. Scale bar, 20 μm. () Immunofluorescence of neutrophils isolated from a healthy volunteer stained with DAPI (blue), antibody! to CD14 conjugated to FITC (green) and antibody to CD16b conjugated to phycoerythrin (red). Scale bar, 25 μm. * Figure 2: Genomic and protemic characterization of neutrophil lysates. (,) Unsupervised cluster analysis for neutrophil validation studies for microarray data () and LC-MS proteomics data (). Red bars indicate upregulated genes, blue bars downregulated genes, orange bars upregulated proteins and green bars downregulated proteins. GM + I, GM-CSF plus IFN-γ. (,) Venn diagrams of significant (FDR < 0.01), gene expression changes () and protein abundance changes () after ex vivo stimulation. () Flow cytometry validation of ex vivo stimulation results, showing the mean fluorescence signal measured in CD66b+ granulocytes for unstimulated blood, LPS-stimulated blood and GM-CSF plus IFN-γ–stimulated blood. () Unsupervised hierarchical clustering of genes (1,690 probe sets with s.d. > 1) from five healthy subjects isolated by microfluidics (M) or bulk Ficoll-dextran (B) methods. There are no significant genes differentially expressed between the microfluidics and bulk isolation at FDR < 5%. * Figure 3: Summary of RNA extractions from cell lysates collected at six different clinical sites. () Histogram of the total RNA isolated from the trauma samples and burn samples. () Histogram of the RIN quality score from both groups in ; RNA is scored on a scale of one to ten (higher is better), and any sample that scored four or higher was processed for microarray expression analysis. () Correlation of the total extracted RNA with clinical polymorphonuclear leukocyte counts taken from a complete blood count with five-part differential; the solid line is a linear fit (R = 0.23) through the origin with 95% confidence limits (dashed line) and 95% prediction bands (dotted line). () Syringe pump unit used at the clinical sites for sample processing. * Figure 4: Summary of the microarray results for a subset of the clinical samples from Figure 3. For the preliminary analysis shown here, we chose transcripts with a statistical significance of ≤ 0.001 (Q value), corresponding to 8,719 genes. () Unsupervised K-means clustering of these 8,719 genes identified from the 187 microarrays in the time-course clinical data, leading to five distinct clusters (from top to bottom): 1, early upregulation with resolution; 2, late upregulation with a peak signal at 7–21 d; 3, early downregulation with resolution at 14–21 d; 4, early downregulation without recovery; and 5, late downregulation without recovery. () Bar graph of the ten most statistically significant (all P < 0.05) upregulated pathways (red) and downregulated pathways from the genes in . Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE22103 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Shriners Hospital for Children, Boston, Massachusetts, USA. * Kenneth T Kotz, * Wenzong Xiao, * Aman Russom, * Alan E Rosenbach, * Jeremy Goverman, * Shawn P Fagan, * Daniel Irimia, * Ronald G Tompkins & * Mehmet Toner * Stanford Genome Technology Center, Palo Alto, California, USA. * Wenzong Xiao, * Weihong Xu, * Julie Wilhelmy, * Michael N Mindrinos & * Ronald W Davis * Department of Surgery, University of Rochester School of Medicine, Rochester, New York, USA. * Carol Miller-Graziano, * Asit De & * Paul E Bankey * Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory (PNNL), Richland, Washington, USA. * Wei-Jun Qian, * Brianne O Petritis, * David G Camp II & * Richard D Smith * Department of Surgery, University of Florida College of Medicine, Gainesville, Florida, USA. * Elizabeth A Warner & * Lyle L Moldawer * Department of Radiation Oncology, Washington University, St. Louis, Missouri, USA. * Bernard H Brownstein * University of Florida, Gainesville, Florida, USA. * Henry V Baker * University of Michigan School of Medicine, Ann Arbor, Michigan, USA. * Ulysses G J Balis & * Stephen F Lowry * University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. * Timothy R Billiar & * Jason Sperry * University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA. * Steven E Calvano * Washington University School of Medicine, St. Louis, Missouri, USA. * J Perren Cobb * University of Washington, Seattle, Washington, USA. * Joseph Cuschieri, * Nicole S Gibran, * Laura Hennessy, * Matthew B Klein, * Ronald V Maier & * Grant E O′Keefe * University of Texas Medical Branch, Galveston, Texas, USA. * Celeste C Finnerty, * David N Herndon & * Marc G Jeschke * Loyola University School of Medicine, Maywood, Illinois, USA. * Richard L Gamelli * University of Louisville, Louisville, Kentucky, USA. * Brian G Harbrecht * Massachusetts General Hospital, Boston, Massachusetts, USA. * Douglas L Hayden, * Philip H Mason, * Grace P McDonald-Smith, * Laurence G Rahme, * David A Schoenfeld, * H Shaw Warren & * Bram Wispelwey * University of Colorado Health Sciences Center, Denver, Colorado, USA. * Jeffrey L Johnson & * Ernest E Moore * University of Texas Southwestern Medical School, Dallas, Texas, USA. * Joseph P Minei * St. Michael's Hospital, Toronto, Ontario, Canada. * Avery B Nathens * Boston University School of Medicine, Boston, Massachusetts, USA. * Daniel G Remick * Northwestern University, Chicago, Illinois, USA. * Michael B Shapiro * Princeton University, Princeton, New Jersey, USA. * John D Storey * Stanford University, Palo Alto, California, USA. * Robert Tibshirani & * Wing H Wong * San Francisco General Hospital, San Francisco, California, USA. * Michael A West * Stanford University, Palo Alto, California, USA. * San Francisco General Hospital, San Francisco, California, USA. Consortia * the Inflammation and the Host Response to Injury Collaborative Research Program * Henry V Baker, * Ulysses G J Balis, * Timothy R Billiar, * Steven E Calvano, * J Perren Cobb, * Joseph Cuschieri, * Celeste C Finnerty, * Richard L Gamelli, * Nicole S Gibran, * Brian G Harbrecht, * Douglas L Hayden, * Laura Hennessy, * David N Herndon, * Marc G Jeschke, * Jeffrey L Johnson, * Matthew B Klein, * Stephen F Lowry, * Ronald V Maier, * Philip H Mason, * Grace P McDonald-Smith, * Joseph P Minei, * Ernest E Moore, * Avery B Nathens, * Grant E O′Keefe, * Laurence G Rahme, * Daniel G Remick, * David A Schoenfeld, * Michael B Shapiro, * Jason Sperry, * John D Storey, * Robert Tibshirani, * H Shaw Warren, * Michael A West, * Bram Wispelwey & * Wing H Wong Contributions K.T.K. performed and analyzed experiments. W. Xiao and W.-J.Q. preformed microarray and proteomics analyses. K.T.K., W. Xu, J.W., M.N.M., W. Xu, A.R., E.A.W., L.L.M., D.I., B.H.B., R.W.D. & M.T. designed genomic experiments. K.T.K., W.-J.Q., D.G.C. II and R.D.S. designed proteomic experiments. J.G., S.P.F., A.E.R. and R.G.T. aided in clinical sample studies at Massachusetts General Hospital. K.T.K., C.M.-G., A.D., L.L.M., W. Xiao, M.N.M., J.W., W.-J.Q., B.O.P., D.G.C. II, A.E.R., P.E.B. and M.T. designed, conducted and analyzed the ex vivo stimulation experiment. K.T.K., C.M.-G., W. Xiao, M.N.M. and L.L.M. wrote the manuscript. All authors contributed to the final editing of the manuscript. Henry V Baker8, Ulysses G J Balis9, Timothy R Billiar10, Steven E Calvano11, J Perren Cobb12, Joseph Cuschieri13, Celeste C Finnerty14, Richard L Gamelli15, Nicole S Gibran13, Brian G Harbrecht16, Douglas L Hayden17, Laura Hennessy13, David N Herndon14, Marc G Jeschke14, Jeffrey L Johnson18, Matthew B Klein13, Stephen F Lowry9, Ronald V Maier13, Philip H Mason17, Grace P McDonald-Smith17, Joseph P Minei19, Ernest E Moore18, Avery B Nathens20, Grant E O′Keefe13, Laurence G Rahme17, Daniel G Remick21, David A Schoenfeld17, Michael B Shapiro22, Jason Sperry10, John D Storey23, Robert Tibshirani24, H Shaw Warren17, Michael A West25, Bram Wispelwey17 & Wing H Wong24 Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Kenneth T Kotz (kkotz@partners.org) or * Mehmet Toner (mtoner@hms.harvard.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (860K) Supplementary Figures 1–3, Supplementary Tables 1–2,4,6-7,9-12 and Supplementary Methods * Supplementary Table 3 (2M) Significantly perturbed genes and proteins following ex vivo stimulation of whole blood by LPS or GM+I. * Supplementary Table 5 (608K) Gene expression across all subjects for the genes in Figure 2f comparing microfluidic isolation with Ficoll-dextran isolation * Supplementary Table 8 (6M) Significantly perturbed genes after severe trauma injury Additional data
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