Hot off the presses! Nov 01 Nat Med

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Latest Articles Include:

• Front-page news
  - Nat Med 16(11):1167 (2010)
  Nature Medicine | Editorial Front-page news Journal name:Nature MedicineVolume: 16 ,Page:1167Year published:(2010)DOI:doi:10.1038/nm1110-1167Published online04 November 2010 Nature Medicine's relationship with the press is probably not what you think it is. View full text Additional data
• Breakup of genetics advisory panel seen as premature
  - Nat Med 16(11):1169 (2010)
  In September, Steven Teutsch received word that the expert panel he chaired, which advised the US Department of Health and Human Services (HHS) on how genetic technologies could be best integrated into health care, was to be abruptly disbanded in two weeks' time."We didn't anticipate the committee would end," says Teutsch, chief science officer of the Los Angeles County Health Department.
• Clinical sabbatical aims to beef up trial-management skills
  - Nat Med 16(11):1170 (2010)
  Clinical psychiatrist Barbara Gracious had a month to spare over the summer while transitioning to her new job at Ohio State University in Columbus. But, instead of jetting off to a vacation spot, she packed her bags for Bethesda, Maryland, home of the US National Institutes of Health (NIH) Clinical Center, to go on sabbatical.
• The knee bone's connected to the... titanium foam?
  - Nat Med 16(11):1170 (2010)
  By some estimates, more than two million bone grafts take place worldwide each year. The best possible graft material is autologous bone taken from the patient—usually shaved from the side of the pelvis.
• Decisions hint that not all biologics are created with equal ease
  - Nat Med 16(11):1171 (2010)
  The Biologics Price Competition and Innovation Act, signed into US law last March, gave the country's drug regulators the authority to establish a pathway to approving 'generic versions' of biologic drugs. Now the US Food and Drug Administration (FDA) must take the next step and set guidelines for evaluating these large, complex organic molecules that are almost—but not quite—copies of some of modern medicine's most important drugs.
• Pathologists scan for options beyond autopsies
  - Nat Med 16(11):1172 (2010)
  LONDON—Despite its value to clinical practice and medical research, use of the autopsy is in decline worldwide, in part as a result of changing attitudes and cost cutting. Researchers met in London last month to discuss how body scans can enhance and possibly one day replace some forms of one of the oldest medical practices.
• Basic animal research on the rise while pharma looks to new options
  - Nat Med 16(11):1172 (2010)
  Drug companies in the EU are increasingly turning to nonanimal strategies to test medicines, but the number of animals used for basic research is on the rise, according to statistics published 30 September by the European Commission.Although the total number of animals used for scientific purposes in the EU's 27 member states has held steady at around 12 million per year, this overall figure masks shifting trends in animal experimentation.
• UK science dealt lighter blow than other sectors in budget cuts
  - Nat Med 16(11):1173 (2010)
  As many European countries adopt austerity measures, lawmakers have chipped away at science budgets. Despite concerns that the UK science budget would be substantially cut as part of a comprehensive spending review, the government announced on 20 October that the budget will be frozen in cash terms.
• Affirmative inaction at the FDA
  - Nat Med 16(11):1173 (2010)
  After an advisory committee to the US Food and Drug Administration (FDA) votes on whether to approve a new medicine, the standard mantra is that the agency doesn't have to follow its panel's advice, but, by and large, it does. However, according to a new analysis from Prevision Policy, a Washington, DC–based healthcare policy group, over the past four years the FDA has followed its committees' advice only 76% of the time.
• Stem cell support cuts across party lines
  - Nat Med 16(11):1173 (2010)
  Over the past decade, federal funding for embryonic stem cell research in the US has been held up continuously by rogue players—first by former President George W. Bush who established an executive order in 2001 limiting funding and twice vetoed legislation to expand the scope of such research, and now by a US district court judge's decision that threatens to halt taxpayer supported embryonic stem cell science altogether.
• News in brief
  - Nat Med 16(11):1174-1175 (2010)
  Sept 21Google filed a civil complaint in US federal court to block so-called "rogue pharmacies," which dispense drugs without requiring proper prescriptions, from advertising on its search results.
• Mountains to Climb
  - Nat Med 16(11):1176-1179 (2010)
  Nature Medicine | News Mountains to Climb * Brendan Borrell1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1176–1179Year published:(2010)DOI:doi:10.1038/nm1110-1176Published online04 November 2010 New medications to fight the altitude sickness suffered by mountain climbers promise to aid peak performance. But the same drugs could also yield new treatments for people with breathing disorders. meets one man at DARPA, the US Defense Department's research agency, who's trying to move mountains for a new therapy. View full text Additional data Affiliations * Brendan Borrell is a journalist based in New York.
• Straight talk with...Leonor Beleza
  - Nat Med 16(11):1180 (2010)
  Nature Medicine | News Straight talk with...Leonor Beleza * Lucas Laursen Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:1180Year published:(2010)DOI:doi:10.1038/nm1110-1180Published online04 November 2010 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Portuguese businessman António Champalimaud surprised his family when his will, opened after his 2004 death, revealed that he was bequeathing 500 million ($690 million), about a quarter of his estate, to establish a foundation for applied biomedical research. He also surprised law professor and one-time Portuguese Health Minister Leonor Beleza, whom he named to lead the foundation. Beleza, who met Champalimaud just once, agreed in principle to run his proposed foundation during a phone call in 2000 but did not hear any further until his death. She has now returned from a global tour of medical research institutions and foundations lasting over a year to determine how best to spend Champalimaud's millions. On 5 October, the Champalimaud Foundation opened its seaside Center for the Unknown in Lisbon, Portugal. The center will host about 600 researchers and physicians and 300 patients when it reaches full staffing levels. recently called Beleza to ask how she laid the groundwork and what lies ahead. View full text Additional data
• The new age of global health governance holds promise
  - Nat Med 16(11):1181 (2010)
  Nature Medicine | News The new age of global health governance holds promise * Tikki Pang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nils Daulaire2 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerald Keusch3 Search for this author in: * NPG journals * PubMed * Google Scholar * Rose Leke4 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter Piot5 Search for this author in: * NPG journals * PubMed * Google Scholar * Srinath Reddy6 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrzej Rys7 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Szlezak8, 9 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:1181Year published:(2010)DOI:doi:10.1038/nm1110-1181Published online04 November 2010 The recognition that many diseases present worldwide challenges has spurred nations and institutions to participate in the development of what is known as 'global health governance'. But this new form of governance will only succeed with strengthened country commitment, collaborations across disparate sectors and improved accountability. View full text Additional data Affiliations * Research Policy & Cooperation, World Health Organization, Geneva, Switzerland. * Tikki Pang * Office of Global Health Affairs, US Department of Health and Human Services, Washington, DC, USA. * Nils Daulaire * Boston University, Boston, Massachusetts, USA. * Gerald Keusch * University of Yaoundé 1, Yaoundé, Cameroon. * Rose Leke * London School of Hygiene & Tropical Medicine, London, UK. * Peter Piot * Public Health Foundation of India, New Delhi, India. * Srinath Reddy * European Commission, Luxembourg. * Andrzej Rys * McKinsey & Company, Berlin, Germany * Nicole Szlezak * Sustainability Science Program, John F. Kennedy School of Government, Harvard University, Cambridge, Massachusetts, USA. * Nicole Szlezak
• Mental illness mania
  - Nat Med 16(11):1183 (2010)
  Robert Whitaker has done it again! He has written a controversial book that asks if the number of disabled mentally ill in the US has tripled over the past two decades due to the increasing use of psychotropic prescription drugs such as antidepressants, anxiolytics and antipsychotics.
• The T-ALL paradox in cancer
  - Nat Med 16(11):1185-1186 (2010)
  The TLX1 oncogene (encoding the transcription factor T cell leukemia homeobox protein-1) has a major role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). However, the specific mechanisms of T cell transformation downstream of TLX1 remain to be elucidated. Here we show that transgenic expression of human TLX1 in mice induces T-ALL with frequent deletions and mutations in Bcl11b (encoding B cell leukemia/lymphoma-11B) and identify the presence of recurrent mutations and deletions in BCL11B in 16% of human T-ALLs. Most notably, mouse TLX1 tumors were typically aneuploid and showed a marked defect in the activation of the mitotic checkpoint. Mechanistically, TLX1 directly downregulates the expression of CHEK1 (encoding CHK1 checkpoint homolog) and additional mitotic control genes and induces loss of the mitotic checkpoint in nontransformed preleukemic thymocytes. These results identify a previously unrecognized mechanism contributing to chromosomal miss! egregation and aneuploidy active at the earliest stages of tumor development in the pathogenesis of cancer.
• Keep the 'phospho' on MAPK, be happy
  - Nat Med 16(11):1187-1188 (2010)
  The lifetime prevalence (~16%)1 and the economic burden ($100 billion annually)2, 3 associated with major depressive disorder (MDD) make it one of the most common and debilitating neurobiological illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology of MDD have not been identified. Here we use whole-genome expression profiling of postmortem tissue and show significantly increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1, encoded by DUSP1, but hereafter called MKP-1) in the hippocampal subfields of subjects with MDD compared to matched controls. MKP-1, also known as dual-specificity phosphatase-1 (DUSP1), is a member of a family of proteins that dephosphorylate both threonine and tyrosine residues and thereby serves as a key negative regulator of the MAPK cascade4, a major signaling pathway involved in neuronal plasticity, function and survival5, 6. We tested the role of altered MKP-1 expression ! in rat and mouse models of depression and found that increased hippocampal MKP-1 expression, as a result of stress or viral-mediated gene transfer, causes depressive behaviors. Conversely, chronic antidepressant treatment normalizes stress-induced MKP-1 expression and behavior, and mice lacking MKP-1 are resilient to stress. These postmortem and preclinical studies identify MKP-1 as a key factor in MDD pathophysiology and as a new target for therapeutic interventions.
• Autophagy thwarts muscle disease
  - Nat Med 16(11):1188-1190 (2010)
  Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI–knockout (Col6a1−/−) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B–interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype o! f Col6a1−/− mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.
• Bitter treats for better breathing
  - Nat Med 16(11):1190-1191 (2010)
  Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants! decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.
• Omega-3 oil: a fishy protection for the heart
  - Nat Med 16(11):1192-1193 (2010)
  Adding n–3 fatty acids from fish oil—eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)—to our diet is likely to lower our risk of cardiovascular disease, according to some previous studies and basic research but other studies in people with cardiac disease have shown negative, disappointing results. Daan Kromhout et al.1 now show that low doses of a mixture of EPA and DHA (EPA-DHA) or the plant-derived lipid α-linoleic acid (ALA) did not have a beneficial effect in reducing sudden cardiac death in people that had suffered myocardial infarction in the past.
• Inside the microbial and immune labyrinth: Totally gutted
  - Nat Med 16(11):1194-1195 (2010)
  Nature Medicine | Between Bedside and Bench Inside the microbial and immune labyrinth: Totally gutted * Thomas T MacDonald1t.t.macdonald@qmul.ac.uk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1194–1195Year published:(2010)DOI:doi:10.1038/nm1110-1194Published online04 November 2010 In Crohn's disease, immune damage to the gut wall is both induced and modified by the gut microflora, challenging researchers to solve the maze of interactions exploitable for therapeutic benefit. Whether these microbial 'guests' are worsening or helping in this scenario is still open to debate. In 'Bench to Bedside', Warren Strober highlights mice studies showing that certain microbes in the gut have a protective role promoting a shift towards an increased regulatory response that protects from recurrence of the disease. In 'Bedside to Bench', Thomas MacDonald examines how human studies using strategies to block soluble proinflammatory cytokines—despite solid supporting data from animal models—have shown disappointing results compared with therapies that neutralize soluble cytokines but also deplete proinflammatory cells, calling into question whether targeting a single soluble cytokine will ever be useful to treat people with Crohn's disease. View full text Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Thomas T. MacDonald is at the Centre for Immunology and Infectious Disease, Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Thomas T MacDonald (t.t.macdonald@qmul.ac.uk) Additional data
• Inside the microbial and immune labyrinth: Gut microbes: friends or fiends?
  - Nat Med 16(11):1195-1197 (2010)
  There is now a general consensus that the two major inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, arise from an abnormal immune response to one or more bacterial components of the very rich community of commensal flora in the large bowel and the more distal parts of the small bowel1. Theoretically, this abnormal response can have two origins: an increased proinflammatory response to a bacterial component or a decreased regulatory response, which may lead to an excessive effector immune response.
• Research Highlights
  - Nat Med 16(11):1198 (2010)
  
• The Herrenhausen Symposium on Neurodegeneration
  - Nat Med 16(11):1200 (2010)
  Nature Medicine | Commentary The Herrenhausen Symposium on Neurodegeneration Journal name:Nature MedicineVolume: 16 ,Page:1200Year published:(2010)DOI:doi:10.1038/nm1110-1200Published online04 November 2010 Although the pathological hallmarks of neurodegenerative diseases have been described for over 100 years, our understanding of the molecular events leading to neuronal death has emerged over the past two decades. Despite all of this progress in basic research, why do we still lack disease-modifying therapeutics? View full text Additional data
• Initiation and propagation of neurodegeneration
  - Nat Med 16(11):1201-1204 (2010)
  Nature Medicine | Commentary Initiation and propagation of neurodegeneration * Christian Haass1, 2chaass@med.uni-muenchen.de Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1201–1204Year published:(2010)DOI:doi:10.1038/nm.2223Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Although substantial progress has been made in understanding the molecular and pathological bases of neurodegeneration, there have been few successes in the clinic and a number of fundamental questions remain unanswered. Is this skepticism misplaced, or do the words of Sir Isaac Newton hold true, that "what we know is a drop, what we don't know is an ocean"? View full text Author information * Abstract * Author information Affiliations * DZNE—German Center for Neurodegenerative Diseases, Munich, Germany. * Christian Haass * Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany. * Christian Haass Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Christian Haass (chaass@med.uni-muenchen.de) Additional data
• Degeneration and repair in central nervous system disease
  - Nat Med 16(11):1205-1209 (2010)
  Nature Medicine | Commentary Degeneration and repair in central nervous system disease * Eng H Lo1lo@helix.mgh.harvard.edu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1205–1209Year published:(2010)DOI:doi:10.1038/nm.2226Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Divergent disease triggers in neurodegeneration may induce convergent endogenous pathways in neuronal, glial and vascular elements as the central nervous system (CNS) attempts to compensate, remodel and recover. Dissecting these multicellular mechanisms and the integrative responses in cerebral blood flow and metabolism may allow us to understand the balance between injury and repair, validate new targets and define therapeutic time windows for neurodegeneration. View full text Author information * Abstract * Author information Affiliations * Eng H. Lo is at the Neuroprotection Research Laboratory, Departments of Neurology and Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Eng H Lo (lo@helix.mgh.harvard.edu) Additional data
• The benefits and limitations of animal models for translational research in neurodegenerative diseases
  - Nat Med 16(11):1210-1214 (2010)
  Nature Medicine | Commentary The benefits and limitations of animal models for translational research in neurodegenerative diseases * Mathias Jucker1mathias.jucker@uni-tuebingen.de Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1210–1214Year published:(2010)DOI:doi:10.1038/nm.2224Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Age-related neurodegenerative diseases are largely limited to humans and rarely occur spontaneously in animals. Genetically engineered mouse models recapitulate aspects of the corresponding human diseases and are instrumental in studying disease mechanisms and testing therapeutic strategies. If considered within the range of their validity, mouse models have been predictive of clinical outcome. Translational failure is less the result of the incomplete nature of the models than of inadequate preclinical studies and misinterpretation of the models. This commentary summarizes current models and highlights key questions we should be asking about animal models, as well as questions that cannot be answered with the current models. View full text Author information * Abstract * Author information Affiliations * Mathias Jucker is at the Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, and the DZNE-German Center for Neurodegenerative Diseases, Tübingen, Germany. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Mathias Jucker (mathias.jucker@uni-tuebingen.de) Additional data
• The future of genetic research on neurodegeneration
  - Nat Med 16(11):1215-1217 (2010)
  Nature Medicine | Commentary The future of genetic research on neurodegeneration * Christine Van Broeckhoven1christine.vanbroeckhoven@molgen.vib-ua.be Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1215–1217Year published:(2010)DOI:doi:10.1038/nm.2225Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Why, with all the progress in the field of neurodegeneration, do we still lack disease-modifying drugs that tackle the primary defect of severe cell loss? How much progress has been made toward this goal? Have we spent our time and resources wisely? And, most important, is there room for improvement? This commentary highlights several problems faced by researchers in studying the genetic etiology of neurodegenerative diseases and seeks to provide direction in overcoming some of these obstacles. View full text Author information * Abstract * Author information Affiliations * Christine Van Broeckhoven is at the Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Antwerpen, Belgium, and the Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerpen, Belgium. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Christine Van Broeckhoven (christine.vanbroeckhoven@molgen.vib-ua.be) Additional data
• Biomarkers in Alzheimer's disease drug development
  - Nat Med 16(11):1218-1222 (2010)
  Nature Medicine | Commentary Biomarkers in Alzheimer's disease drug development * Kaj Blennow1kaj.blennow@neuro.gu.se Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1218–1222Year published:(2010)DOI:doi:10.1038/nm.2221Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Biomarkers may be of great value in Alzheimer's disease drug development to select the most optimal drug candidates for large and expensive phase 3 clinical trials. Biomarkers will also be important to provide evidence that a drug affects the underlying pathophysiology of the disease, which, together with a beneficial effect on the clinical course, will be essential for labeling the drug as having a disease-modifying effect. View full text Author information * Abstract * Author information Affiliations * Kaj Blennow is at the Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy at University of Gothenburg, Mölndal, Sweden. Competing financial interests The author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Corresponding author Correspondence to: * Kaj Blennow (kaj.blennow@neuro.gu.se) Additional data
• Clinical trials of disease-modifying therapies for neurodegenerative diseases: the challenges and the future
  - Nat Med 16(11):1223-1226 (2010)
  Nature Medicine | Commentary Clinical trials of disease-modifying therapies for neurodegenerative diseases: the challenges and the future * Anthony E Lang1lang@uhnresearch.ca Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1223–1226Year published:(2010)DOI:doi:10.1038/nm.2220Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease represent a crucial and exponentially increasing challenge to health care systems throughout the world. There is an urgent need for effective treatments that will both delay their onset and slow their inexorable progression. Many obstacles stand in the way of realizing these goals. It is expected that future advances will have a major impact on how and when the diagnosis will be made. It is hoped that these will eventually make it possible to initiate effective disease-modifying therapies long before the neurodegenerative process becomes established and symptomatic. View full text Author information * Abstract * Author information Affiliations * Anthony E. Lang is in the Department of Medicine (Neurology) at the University of Toronto, Toronto, Canada. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Anthony E Lang (lang@uhnresearch.ca) Additional data
• Bridging the Valley of Death of therapeutics for neurodegeneration
  - Nat Med 16(11):1227-1232 (2010)
  Nature Medicine | Commentary Bridging the Valley of Death of therapeutics for neurodegeneration * Steven Finkbeiner1sfinkbeiner@gladstone.ucsf.edu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1227–1232Year published:(2010)DOI:doi:10.1038/nm.2222Published online21 September 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Neurodegenerative diseases are the sixth leading cause of death in the US. The market for disease-modifying drugs is enormous, but no drug exists. Academic scientists are increasingly pursuing the discovery and development of therapeutics. Their progress could potentially reduce the risk of failure sufficiently to warrant greater industry investment and movement of leads into clinical trials. Here we consider the many obstacles to the development of therapeutics for neurodegenerative disease within academia, with a special focus on organizational issues. View full text Figures at a glance * Figure 1: The Valley of Death. Drug discovery and development is an orderly but fraught step-wise process. Traditionally, it has begun with NIH-supported basic research and the identification of a therapeutic target. Ultimately, it leads to the development of a small molecule whose efficacy is tested in people by industry. The steps in between are known as the Valley of Death, partly because of the high failure rate and partly because the expertise and resources available to execute them are critically lacking. Consequently, the vast majority of leads that start the journey fail somewhere along the way. Recently, individuals and institutions have begun to create new mechanisms to bridge the valley. ADME, absorption, distribution, metabolism and excretion. * Figure 2: A model nonprofit biotechnology incubator to facilitate academic drug discovery and development. The Taube-Koret Center acts as a bidirectional bridge between academia and industry to reduce the risk in the development of therapeutics. Discoveries with therapeutic potential are made in academic laboratories and are transferred to the Taube-Koret Center within the same institution. The Center maintains the necessary expertise in drug discovery and medicinal chemistry to validate targets and discover and optimize small molecules to modulate these targets. Contract research organizations are used by the Center as needed to carry out specific steps in drug development. Leads that fail to meet performance targets are returned to the academic labs that discovered them for further optimization. Leads that meet milestones are advanced until partners can be found to co-develop them further. The potential paths to therapeutics via partners (black arrows) can involve foundations, the NIH, existing biotechnology companies, new companies created by venture capitalists, or major phar! maceutical (pharma) companies. Since the Center's overarching goal is to find effective therapies for neurodegenerative diseases, rather than to make a profit from proprietary programs, it can make its new technologies, assays and disease models available to external entities that want to evaluate the efficacy of their lead programs (gray arrows). Author information * Abstract * Author information Affiliations * Steven Finkbeiner is at the Gladstone Institute of Neurological Disease, Taube-Koret Center for Huntington's Disease Research, Consortium for Fronto-temporal Dementia Research, and Departments of Neurology and Physiology, University of California, San Francisco, California, USA. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Steven Finkbeiner (sfinkbeiner@gladstone.ucsf.edu) Additional data
• Pain, from bench to bedside
  - Nat Med 16(11):1236 (2010)
  Nature Medicine | Introduction Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Pain, from bench to bedside Journal name:Nature MedicineVolume: 16 ,Page:1236Year published:(2010)DOI:doi:10.1038/nm1110-1236Published online04 November 2010 There has been substantial progress in understanding the neurobiological basis of pain, but these advances have yet to translate into new and improved analgesics. View full text Additional data
• Animalgesic effects
  - Nat Med 16(11):1237-1240 (2010)
  Nature Medicine | News Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Animalgesic effects * Elie Dolgin1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1237–1240Year published:(2010)DOI:doi:10.1038/nm1110-1237Published online04 November 2010 Animal experiments have produced an explosion of information about pain, but this knowledge has failed to yield new painkillers for use in humans. This abysmal track record has led to calls to overhaul the design of preclinical studies. goes to great pains to learn how monitoring rodents' facial expressions and brain activity might offer a more effective and humane way to test drug candidates. View full text Additional data Affiliations * Elie Dolgin is a news editor with Nature Medicine in New York.
• Overcoming obstacles to developing new analgesics
  - Nat Med 16(11):1241-1247 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Overcoming obstacles to developing new analgesics * Clifford J Woolf1clifford.woolf@childrens.harvard.edu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1241–1247Year published:(2010)DOI:doi:10.1038/nm.2230Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Despite substantial investment by the pharmaceutical industry over several decades, there has been little progress in developing new, efficacious and safe analgesics. As a result, many large pharmaceutical companies are leaving the area of pain medication. Nevertheless, the chances of success could increase if analgesic drug development strategy changed. To achieve such a paradigm shift we must understand why development of drugs for pain relief is so challenging. View full text Figures at a glance * Figure 1: The standard analgesic drug development pathway. New small-molecule analgesics are currently developed by a series of linear steps, starting with selecting a target, and ending with marketing the drug with a label for pain relief in particular patients. In this model, preclinical research is distinct from clinical development. Failure late in the path, in phase 2b or phase 3, is expensive and time consuming. If the preclinical pathway, driven by biased hypotheses typically obtained from rodent surrogate pain models, selects a target irrelevant to human pain conditions or does not suggest the most relevant patient cohort and outcome, clinical development will probably fail. The notable lack of success in producing new analgesics over the past two decades has been driven by this model, as well as by the difficulty in validating lead candidates on human targets in human cells, and by the differences between rodent and human pharmacokinetic profiles. ADME, absorption, distribution, metabolism and excretion; PK, pharmacokinetic! s; POP, proof of principle; RCT, randomized control trials. * Figure 2: A proposed new analgesic drug development pathway. Several changes to the analgesic drug development model may increase the chances of success: the choice of target must be driven by data from patients using unbiased screens; screening and validation must focus on the native human target expressed in human cells relevant to the target's action in pain; preclinical toxicity studies must be done on human cells; phase 1 must include pharmacokinetic data showing engagement or occupancy of the target; phase 2b must maximize the chance of a genuine efficacy signal by detailed phenotyping, objective outcome measures, use of biomarkers and reduction of placebo; phase 3 studies must identify responders and this should drive drug labeling; drug labeling must change as data accumulate in the postmarketing phase; treatment must be personalized, identifying which patient requires which treatment. The feedback from the different elements in the scheme should iteratively improve the pathway and the distinction between preclinical and clini! cal studies should be abolished. iPS, induced pluripotent stem cell; PET, positron emission tomography; POP, proof of principle. Author information * Abstract * Author information Affiliations * F.M. Kirby Neurobiology Center and Program in Neurobiology, Department of Neurology, Children's Hospital Boston and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. * Clifford J Woolf Competing financial interests Clifford J. Woolf declares grant support from GlaxoSmithKline and Endo Pharmaceuticals and is a consultant to Endo Pharmaceuticals, Taisho Pharmaceutical and Solace Pharmaceuticals. Corresponding author Correspondence to: * Clifford J Woolf (clifford.woolf@childrens.harvard.edu) Additional data
• Nociceptor sensitization in pain pathogenesis
  - Nat Med 16(11):1248-1257 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Nociceptor sensitization in pain pathogenesis * Michael S Gold1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerald F Gebhart1gebhartgf@upmc.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1248–1257Year published:(2010)DOI:doi:10.1038/nm.2235Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The incidence of chronic pain is estimated to be 20–25% worldwide. Few patients with chronic pain obtain complete relief from the drugs that are currently available, and more than half report inadequate relief. Underlying the challenge of developing better drugs to manage chronic pain is incomplete understanding of the heterogeneity of mechanisms that contribute to the transition from acute tissue insult to chronic pain and to pain conditions for which the underlying pathology is not apparent. An intact central nervous system (CNS) is required for the conscious perception of pain, and changes in the CNS are clearly evident in chronic pain states. However, the blockage of nociceptive input into the CNS can effectively relieve or markedly attenuate discomfort and pain, revealing the importance of ongoing peripheral input to the maintenance of chronic pain. Accordingly, we focus here on nociceptors: their excitability, their heterogeneity and their role in initiating and main! taining pain. View full text Figures at a glance * Figure 1: Heterogeneity of nociceptors. (–) Nociceptors can be subclassified by an array of anatomical, physiological and biochemical criteria. One common criterion is the response profile of the afferent: afferents that respond to mechanical, thermal and chemical stimuli are referred to as polymodal nociceptors (), those that respond to mechanical and cold stimuli are referred to as C-MC (mechano-cold) fibers (), and those that do not respond to mechanical stimuli are referred to as MIAs (). Even within these classifications there is tremendous heterogeneity, with differences in the particular molecules that underlie transduction, action potential initiation and propagation, as well as in the channels and receptors that can modulate each of these processes. There are also differences in the transmitters that are released, with more recent data pointing to differences in the proteins that are responsible for vesicle filling. With increasing evidence that MIAs are particularly important in various pain states, th! e identification of unique patterns of chemosensitivity and the mechanisms that underlie the emergence of mechanosensitivity in these afferents might yield new approaches for the treatment of pain. RTK, receptor tyrosine kinase; SK, small-conductance Ca2+-dependent potassium channel; VGCC, voltage-gated calcium channel. * Figure 2: Activation and sensitization of nociceptors. () Transduction can involve both direct and indirect pathways. The ion channel TRPV1, for example, can be directly opened by increases in temperature or by chemicals released from resident (mast) and recruited (polymorphonuclear leukocyte; PMNL) immune cells, epithelial cells, Schwann cells, fibroblasts and sympathetic post-ganglionic neurons (SPGN). () There are multiple points of interaction between second messenger pathways that are engaged after nociceptor activation, including at the levels of signaling molecules such as Ca2+, effector molecules such as PKCε, and common targets, such as TRPV1 and NaV1.8 (not shown) for the pathways activated. For clarity, we have omitted positive modulation of TRPV1 by ceramide, p38, PI3K, PKCε and PKA. Also not shown is the translocation of TRPV1 to the cell surface, which may contribute to injury-induced increases in channel activity. () Sensitization of nociceptors also involves positive feedback. Activation of ion channels such as! TRPV1 results in membrane depolarization and Ca2+ influx through TRPV1 and VGCC. Ca2+ influx can drive the release of neuropeptides (stored in dense core vesicles; pink) and glutamate (stored in clear vesicles; yellow), both of which can drive further activation of receptors on nociceptors and release mediators from the sources described in . PGs, prostaglandins; OLAMs, oxidized linoleic acid meabolites; NE, norepinephrine; ER/GPR30, estrogen receptor/G protein receptor-30; 5-HT, serotonin; CaM, calmodulin; PLC, phospholipase C. DAG, diacylglycerol; IP3, inositol triphosphate; AC, adenylate cyclase; EPAC, cAMP-activated guanine exchange factor; PI3K, phosphoinositide 3-kinase; ERK1/2, extracellular signal–regulated kinases 1 and 2; TNFR, tumor necrosis factor receptor. Author information * Abstract * Author information Affiliations * Center for Pain Research, Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. * Michael S Gold & * Gerald F Gebhart Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Gerald F Gebhart (gebhartgf@upmc.edu) Additional data
• Central mechanisms of pathological pain
  - Nat Med 16(11):1258-1266 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Central mechanisms of pathological pain * Rohini Kuner1rohini.kuner@pharma.uni-heidelberg.de Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1258–1266Year published:(2010)DOI:doi:10.1038/nm.2231Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Chronic pain is a major challenge to clinical practice and basic science. The peripheral and central neural networks that mediate nociception show extensive plasticity in pathological disease states. Disease-induced plasticity can occur at both structural and functional levels and is manifest as changes in individual molecules, synapses, cellular function and network activity. Recent work has yielded a better understanding of communication within the neural matrix of physiological pain and has also brought important advances in concepts of injury-induced hyperalgesia and tactile allodynia and how these might contribute to the complex, multidimensional state of chronic pain. This review focuses on the molecular determinants of network plasticity in the central nervous system (CNS) and discusses their relevance to the development of new therapeutic approaches. View full text Figures at a glance * Figure 1: Pain circuits. (,) A schematic overview of the main circuits mediating physiological pain () and some manifestations of chronic pain (). * Figure 2: Disease-induced functional and structural plasticity in neural substrates of pain. () Different levels of activity-dependent functional plasticity. Molecules may become functionally sensitized (top), synaptic transmission may become potentiated by presynaptic mechanisms (second row, arrow to the left) or by postsynaptic plasticity (arrow to the right), cells may respond to noxious stimuli with increased activity and expanded receptive fields after injury (third row) and network function may change so that more cell ensembles respond to noxious stimuli, collectively leading to a higher net spinal output after injury or inflammation (bottom). () Examples of nociceptive activity-induced structural plasticity. From the top, synaptic spines may increase in size and density; axons may sprout or degenerate; and cells may atrophy (for example, loss of inhibitory interneurons) or proliferate (for example, microglia and astrocytes). * Figure 3: Spinal mechanisms of physiological pain and disease-induced pain hypersensitivity. (,) The diagrams show a few prominent of many possible mediators and cell-cell interactions in the spinal cord dorsal horn in physiological states () and disease states (). Putative changes in pathological states include mechanisms involving suppression of inhibition, potentiation of presynaptic release and postsynaptic excitability, increases in synapse-to-nucleus communication and gene transcription, release of neuromodulators from activated microglia and astrocytes and a net increase in nociceptive input onto higher brain structures. Glu, glutamate; sP, substance P. * Figure 4: Overview of typical signaling pathways used by pronociceptive molecules that mediate disease-induced pain hypersensitivity. Shown are typical ligands and the types of receptors and signaling mediators that they use to induce changes in the expression or function of target proteins, thereby leading to characteristic functional changes over diverse timescales. 5HT, serotonin; CGRP: calcitonin gene-related peptide; CRF, corticotrophin releasing factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IP3R, inositol 1,4,5-triphosphate receptor; DAG, diacylglycerol; P2X3, ATP-gated ion channel; 5-HT3, serotonin-gated ion channel; NK1: neurokinin receptor-1; PAR1-4, protease-activated receptors 1-4; ETA, endothelin receptor A; EP1, prostaglandin receptor-1; CCK, cholecystokinin; TrkA, neurotrophin receptor A; TrkB, neurotrophin receptor B; G-/GM-CSFR, G-CSF receptor and GM-CSF receptor pJAK, phosphorylated Janus-activated kinase; pSTAT, phosphorylated signal transducer and activator of transcription; PI3-K, phosphoinositol 3-kinase; pAKT, phosphopr! otein kinase B; sGC, soluble guanylyl cyclase; PIP2, phosphoinositol diphosphate. Author information * Abstract * Author information Affiliations * Pharmacology Institute, University of Heidelberg, Im Neuenheimer Feld, 366 Heidelberg, Germany. * Rohini Kuner Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Rohini Kuner (rohini.kuner@pharma.uni-heidelberg.de) Additional data
• Interactions between the immune and nervous systems in pain
  - Nat Med 16(11):1267-1276 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Interactions between the immune and nervous systems in pain * Ke Ren1kren@umaryland.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald Dubner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1267–1276Year published:(2010)DOI:doi:10.1038/nm.2234Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Immune cells and glia interact with neurons to alter pain sensitivity and to mediate the transition from acute to chronic pain. In response to injury, resident immune cells are activated and blood-borne immune cells are recruited to the site of injury. Immune cells not only contribute to immune protection but also initiate the sensitization of peripheral nociceptors. Through the synthesis and release of inflammatory mediators and interactions with neurotransmitters and their receptors, the immune cells, glia and neurons form an integrated network that coordinates immune responses and modulates the excitability of pain pathways. The immune system also reduces sensitization by producing immune-derived analgesic and anti-inflammatory or proresolution agents. A greater understanding of the role of the immune system in pain processing and modulation reveals potential targets for analgesic drug development and new therapeutic opportunities for managing chronic pain. View full text Figures at a glance * Figure 1: Immune activation and nociceptor sensitization after injury. Injury initiates the release of mediators that activate TLRs on keratinocytes (top) and mast cells (MC) close to the nerve terminal. Vasodilators are also released, promoting adhesion and transmigration of immune cells including T cells (T), neutrophils (N) and monocytes (MN), and recruitment of macrophages (Mφ). These cells, once activated, release a battery of inflammatory mediators that act on receptors expressed on adjacent nociceptor nerve terminals, leading to peripheral nociceptor sensitization. Targets include cytokine receptors (CytR), G protein–coupled receptors (GPCR), ligand-gated channels (LGC) and tyrosine kinase receptor type 1 (TrkA). Three examples of interactions between immune cells and nerve terminals are depicted. (1) Mast cell degranulation requires direct contact between mast cells and nerve terminals, mediated by N-cadherin (N-cad). The metalloproteinase MMP-24 prevents mast cell degranulation by digesting N-cad. (2) Release of TNF-α and IL-15 by ! peripheral nerves and Schwann cells activates MMP-9 and facilitates recruitment of macrophages. (3) Nociceptive nerve terminals can secrete substance P (SP) and CGRP through antidromic activation of neighboring nerve terminal branches (see text). Substance P and CGRP promote vasodilation and extravasation of immune cells. Neutral endopeptidase (NEP) restrains neuroinflammation by degrading substance P and CGRP. * Figure 2: Modulation of sensory nerve activity in dorsal root ganglia by SGCs. () Nerve injury reduces Kir4.1 expression in SGCs, resulting in reduced K+ buffering and increased neuronal excitability. () A reciprocal paracrine signaling loop involving NO, COX, PGE2, CGRP and IL-1β. Macrophages infiltrate into the space between SGCs and neurons and secrete inflammatory mediators. () Chemokine-mediated regulation of neuronal TRP channels through paracrine (Schwann cell–derived CCL3 and neuronal CCR1) and autocrine (neuron-derived CCL2 and neuronal CCR2) signaling. () P2X7R in SGC tonically inhibits P2X3R in neurons by activating neuronal P2Y1. * Figure 3: Activation of glia and neurons in the dorsal horn of the spinal cord after peripheral injury. () Microglia-neuron interactions. Upon activation, afferent nerve terminals release neurotransmitters, substance P, CGRP, glutamate (Glu), ATP and BDNF, as well as inflammatory mediators including IL-6 and CCL2 and the growth and differentiation factor neuregulin-1 (NRG-1), into the spinal cord. Three examples are shown. (1) Neuronal NRG-1 acts on microglial erbB2, leading to IL-1β release. (2) Microglial cathepsin S (catS) cleaves neuronal CX3CL1, which binds CX3CR1 and stimulates phosphorylation of p38 MAPK in microglia. This pathway may be inhibited by protein-coupled receptor kinase 2 (GRK2). (3) ATP binds P2X4 and induces BDNF release from microglia, which upon binding TrkB receptor induces a shift in the chloride anion gradient and GABAA receptor-mediated depolarization in dorsal horn neurons. () Astrocyte-neuron interactions. (1) Astrocytes release glutamate and D-serine, which bind extrasynaptic and synaptic NMDA receptors on neurons, respectively. (2) Injury-induce! d downregulation of astrocytic GLT-1 alters glutamate homeostasis in the synaptic cleft. (3) TNF-α activates the JNK1 pathway, which leads to release of CCL2 and alterations in NMDAR and AMPAR activity. () Cross-talk between nerve terminals, astrocytes and glia. (1) TLR priming and purinergic signaling increase IL-1β release by glia, which modulates NMDA receptor activity on postsynaptic neurons. TIMPs in astrocytes inhibit MMP-mediated cleavage of pro–IL-1β. (2) Microglial IL-18 binds IL18R on astrocytes and induces NF-κB activity and upregulation of inflammatory cytokines. Dashed lines represent multiple intermediate signaling events. Author information * Abstract * Author information Affiliations * Department of Neural and Pain Sciences, Dental School & Program in Neuroscience, University of Maryland, Baltimore, Maryland, USA. * Ke Ren & * Ronald Dubner Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ke Ren (kren@umaryland.edu) Additional data
• Getting the pain you expect: mechanisms of placebo, nocebo and reappraisal effects in humans
  - Nat Med 16(11):1277-1283 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Getting the pain you expect: mechanisms of placebo, nocebo and reappraisal effects in humans * Irene Tracey1irene@fmrib.ox.ac.uk Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1277–1283Year published:(2010)DOI:doi:10.1038/nm.2229Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The perception of pain is subject to powerful influences. Understanding how these are mediated at a neuroanatomical and neurobiological level provides us with valuable information that has a direct impact on our ability to harness positive and minimize negative effects therapeutically, as well as optimize clinical trial designs when developing new analgesics. This is particularly relevant for placebo and nocebo effects. New research findings have directly contributed to an increased understanding of how placebo and nocebo effects are produced and what biological and psychological factors influence variances in the magnitude of the effect. The findings have relevance for chronic pain states and other disorders, where abnormal functioning of crucial brain regions might affect analgesic outcome even in the normal therapeutic setting. View full text Figures at a glance * Figure 1: Factors influencing pain perception and the neural basis for endogenous pain modulation, placebo and nocebo effects. (,) Schematic illustration of key brain regions involved in generating a pain experience (green, blue and purple) with core brain regions that comprise the cognitive and descending pain modulatory networks (blue) () and a description of the various factors that influence the pain experience listed in the text boxes (). () The regions highlighted in blue indicate the core descending endogenous pain and cognitive modulatory networks that many of these factors, including placebo and nocebo effects, use to elicit their influence on nociceptive processing and resultant pain perception. The hippocampal region (purple) is important for amplifying pain experiences during nocebo or increased anxiety. () Schematic illustration indicating where endogenous opioid and dopamine neurotransmission occurs in the human brain during placebo analgesia. Note the overlap with many of the brain regions involved in cognitive modulation of pain, and for some brain regions (NAc) there is a bidirectio! nal response of both opioid and dopamine release that produces either placebo (increased release) or nocebo (decreased release) effects. vmPFC, ventromedial prefrontal cortex; Amy, amygdala; Hypo, hypothalamus; Hipp, hippocampus; S2, secondary somatosensory cortex; S1, primary somatosensory cortex; dlPFC, dorsolateral prefrontal cortex; rACC, rostral anterior cingulate cortex; mACC, midanterior cingulate cortex; CCK, cholecystokinin. * Figure 2: The patient environment. (–) Schematic of a treatment environment where both drug and therapeutic context interact to produce resultant pain report (), where without drug only the therapeutic context influences pain report () and where, due to conditions that affect the key brain regions listed in Figure 1, there is only the drug and its pharmacodynamics able to influence the pain report (). Author information * Abstract * Author information Affiliations * Nuffield Department of Anaesthetics and Oxford Centre for Functional Magnetic Resonance Imaging of the Brain, Department of Clinical Neurology, University of Oxford, Oxford, UK. * Irene Tracey Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Irene Tracey (irene@fmrib.ox.ac.uk) Additional data
• Advances in clinical research methodology for pain clinical trials
  - Nat Med 16(11):1284-1293 (2010)
  Nature Medicine | Review Focus on Pain Focus issue: November 2010 Volume 16, No 11 * * Reviews * Sponsor * * Introduction * News Advances in clinical research methodology for pain clinical trials * John T Farrar1jfarrar@upenn.edu Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Pages:1284–1293Year published:(2010)DOI:doi:10.1038/nm.2249Published online14 October 2010 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Pain is a ubiquitous phenomenon, but the experience of pain varies considerably from person to person. Advances in understanding of the growing number of pathophysiologic mechanisms that underlie the generation of pain and the influence of the brain on the experience of pain led to the investigation of numerous compounds for treating pain. Improved knowledge of the subjective nature of pain, the variations in the measurement of pain, the mind-body placebo effect and the impact of differences in the conduct of a clinical trial on the outcome have changed approaches to design and implement studies. Careful consideration of how these concepts affect the choice of study population, the randomization and blinding process, the measurement and collection of data, and the analysis and interpretation of results should improve the quality of clinical trials for potential pain therapies. View full text Figures at a glance * Figure 1: Schematic diagram of the crucial components of a prospective randomized, double blind, placebo controlled clinical trial. * Figure 2: Normal density curves of simulated data for a single normal distribution control group and a combined (bimodal) treatment group. The combined treatment group is created by adding the values of the two normally distributed treatment subgroups each with 50% of the subjects. One subgroup includes subjects with the potential to respond to the treatment, resulting in a mean value that is larger than the control group. The other subgroup includes subjects without the potential to respond, which mimics the control group in mean change and distribution. The values on the horizontal axes represent the change in pain intensity (in percentage) from baseline to study endpoint. The vertical axis is the subject frequency, which indicates the number of individuals who achieved each level of change. The cumulative distribution function curves shown in Figure 3 were constructed from the normal distribution curves shown here. * Figure 3: Cumulative distribution function curve for a simulated analysis of two groups, placebo and combined treatment created from data used for the graph in Figure 2. The x axis represents cut-off points in percentage of change in pain intensity, and the y axis shows the cumulative proportion of subjects that have achieved that level of response or higher. For example, for the combined treatment group line at the 50% response mark, approximately 19% of subjects achieved that level or more. The distance between the two lines (placebo and combined treatment) is the absolute risk difference (ARD) between the two groups, and 1 / ARD provides an estimate of the number needed to treat (NNT). Author information * Abstract * Author information Affiliations * Associate Professor of Epidemiology and Anesthesia, Clinical Associate in Neurology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. * John T Farrar Competing financial interests The author declares competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Corresponding author Correspondence to: * John T Farrar (jfarrar@upenn.edu) Additional data
• Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1
  - Nat Med 16(11):1295-1298 (2010)
  Nature Medicine | Brief Communication Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1 * Eva Herker1, 2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles Harris2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Céline Hernandez5 Search for this author in: * NPG journals * PubMed * Google Scholar * Arnaud Carpentier5 Search for this author in: * NPG journals * PubMed * Google Scholar * Katrin Kaehlcke1 Search for this author in: * NPG journals * PubMed * Google Scholar * Arielle R Rosenberg5 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert V Farese Jr2, 3, 4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Melanie Ott1, 2, 3mott@gladstone.ucsf.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1295–1298Year published:(2010)DOI:doi:10.1038/nm.2238Received15 April 2010Accepted10 September 2010Published online10 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Hepatitis C virus (HCV) infection is closely tied to the lipid metabolism of liver cells. Here we identify the triglyceride-synthesizing enzyme diacylglycerol acyltransferase-1 (DGAT1) as a key host factor for HCV infection. DGAT1 interacts with the viral nucleocapsid core and is required for the trafficking of core to lipid droplets. Inhibition of DGAT1 activity or RNAi-mediated knockdown of DGAT1 severely impairs infectious virion production, implicating DGAT1 as a new target for antiviral therapy. View full text Figures at a glance * Figure 1: DGAT1 activity is necessary for HCV particle assembly at lipid droplets. () Infection of shRNA-expressing Huh7.5 cells with low concentrations of EGFP-Jc1 viral stock. Spreading viral infection was measured by flow cytometry of EGFP (mean ± s.e.m.; n = 4; *P < 0.05, **P < 0.01). The shRNA numbers correspond to the shRNA targeting sequence in the genes. () Real-time RT-PCR analysis of DGAT1 or DGAT2 mRNAs in knockdown cells (mean ± s.e.m.; n = 4) and western blot analysis of DGAT1 protein expression. The available antibodies against DGAT2 do not reliably detect endogenous human DGAT2 in our hands. () Dose-dependent decrease of infectious titers in Huh7.5 cells transfected with Luc-Jc1 or Luc-JFH1 viral RNA and incubated with increasing concentrations of the DGAT1 inhibitor or DMSO for 48 h. Naive Huh7.5 cells were infected with cell supernatants of treated cells and lysed 48 h after infection to analyze luciferase activity (relative light units (RLU) expressed as percentage relative to DMSO control; mean ± s.e.m.: n = 3). () Dose-dependent decr! ease of infectious virus titers released from freshly isolated primary human hepatocytes infected with HCV-Jc1 viral stock and treated with increasing amounts of the DGAT1 inhibitor or DMSO for 3 d. Shown are infectivity titers (in focus-forming units (FFU)) expressed as percentage of DMSO control. A single experiment is shown. () Real-time RT-PCR analysis of HCV RNA isolated from cells or from supernatants of Huh7.5 cells electroporated with EGFP-Jc1 RNA and treated with DGAT1 inhibitor (20 μM) or DMSO. Results are expressed as HCV RNA copy numbers per 1 μg total cellular RNA normalized to 18S rRNA (intracellular) or per 1 ml culture supernatant (secreted) at day 4 after transfection (mean ± s.d.; n = 6; **P < 0.01). () Western blot analysis of cell extracts described in lysed at day 4 after transfection. () Infection of naive Huh7.5 cells with either intracellular or secreted viral particles isolated from Huh7.5 transfected with Luc-Jc1 RNA and treated with the DGAT1 i! nhibitor (20 μM) or DMSO. Shown are luciferase values express! ed as percentage of DMSO control (mean ± s.d.; n = 3; **P < 0.01). () Representative images and quantification of epifluorescence of oil red O (ORO)-stained Huh7 Lunet cells electroporated with EGFP-Jc1 RNA and treated with DMSO or DGAT1 inhibitor (20 μM). Lipid droplet area: mean of 1,000 cells ± s.e.m.; lipid droplet diameter: mean of >1,600 lipid droplets (LDs) ± s.e.m.; lipid droplet number: mean of >50 cells ± s.e.m. LDs, lipid droplets. () Western blot analysis of cell extracts or isolated lipid droplet fractions from cells described in . ADRP, adipose differentiation-related protein; CRT, calreticulin; TG, extracted triglycerides analyzed by thin-layer chromatography. () Indirect immunofluorescence of double-stranded RNA (anti-dsRNA) at lipid droplets (ORO) in the cells described in (scale bar, 10 μm) and quantification of overlap of the signals for double-stranded RNAs and ORO per cell (mean of 30 cells ± s.e.m.). * Figure 2: Specific interaction of the HCV core protein with DGAT1. () Coimmunoprecipitation assays in 293T cells transfected with expression vectors for the HCV core protein and Flag-Dgat1 or Flag-Dgat2 proteins and treated with DGAT1 inhibitor (20 μM) or DMSO. After immunoprecipitation with agarose conjugated to antibodies specific for Flag, the core protein was detected by western blotting with antibodies to the core protein. () Coimmunoprecipitation of the HCV core protein with endogenous DGAT1 in Huh7 hepatoma cells transduced with a core-expressing lentiviral vector. () Coimmunoprecipitation of the HCV core protein with endogenous DGAT1 in Huh7.5 and Huh7 Lunet cells electroporated with EGFP-Jc1 RNA. () Indirect immunofluorescence of core and endogenous DGAT1 in Huh7 cells transfected with wild-type or mutant (SPMT) core expression vectors (scale bar, 10 μm). () Coimmunoprecipitation assays in 293T cells transfected with expression vectors for wild-type (WT), mutant (SPMT) or truncated (1–173) core protein and the Flag-Dgat1–expr! essing plasmid. Arrows mark unprocessed (top) and processed (bottom) core protein. () Model of HCV core recruitment to DGAT1-generated lipid droplets. The HCV core protein interacts with DGAT1 at the ER and thereby gains access to DGAT1-generated lipid droplets. This step is crucial to recruit HCV RNA replication complexes to ER membranes in the vicinity of lipid droplets and to initiate assembly of progeny virions. When the formation of DGAT1-generated lipid droplets is prevented by the treatment with the DGAT1 inhibitor, the translocation of the HCV core protein to lipid droplets is disrupted, and infectious HCV particles cannot form, despite the presence of DGAT2-generated droplets. Author information * Author information * Supplementary information Affiliations * Gladstone Institute of Virology and Immunology, University of California, San Francisco, USA. * Eva Herker, * Katrin Kaehlcke & * Melanie Ott * Liver Center, University of California, San Francisco, USA. * Eva Herker, * Charles Harris, * Robert V Farese Jr & * Melanie Ott * Department of Medicine, University of California, San Francisco, USA. * Eva Herker, * Charles Harris, * Robert V Farese Jr & * Melanie Ott * Gladstone Institute of Cardiovascular Disease, San Francisco, USA. * Charles Harris & * Robert V Farese Jr * Université Paris Descartes, EA 4474 Virologie de l'Hépatite C, Paris, France. * Céline Hernandez, * Arnaud Carpentier & * Arielle R Rosenberg * Department of Biochemistry and Biophysics, University of California, San Francisco, USA. * Robert V Farese Jr Contributions E.H. designed, performed and analyzed most of the experiments and wrote the manuscript. C. Harris and R.V.F. Jr. provided the DGAT1 inhibitor. C. Harris also performed DGAT activity assays, analyzed data and edited the manuscript. C. Hernandez and A.C. performed the experiments in human primary hepatocytes. K.K. constructed the DGAT1 mutants and performed some of the immunoprecipitation experiments. A.R.R., R.V.F. Jr. and M.O. supervised this work, analyzed data and edited (A.R.R. and R.V.F.) or wrote (M.O.) the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Melanie Ott (mott@gladstone.ucsf.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–7 and Supplementary Methods Additional data
• Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction
  - Nat Med 16(11):1299-1304 (2010)
  Nature Medicine | Article Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction * Deepak A Deshpande1 Search for this author in: * NPG journals * PubMed * Google Scholar * Wayne C H Wang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth L McIlmoyle1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kathryn S Robinett1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rachel M Schillinger1 Search for this author in: * NPG journals * PubMed * Google Scholar * Steven S An2 Search for this author in: * NPG journals * PubMed * Google Scholar * James S K Sham3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephen B Liggett1, 4sligg001@umaryland.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1299–1304Year published:(2010)DOI:doi:10.1038/nm.2237Received04 February 2010Accepted09 September 2010Published online24 October 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 Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2+]i) in ASM in a Gβγ–, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP3) receptor–dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2+]i response at the cell membrane, which opens large-conductance Ca2+-activated K+ (BKCa) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decre! ased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants. View full text Figures at a glance * Figure 1: Bitter tastants of diverse structures evoke increases in [Ca2+]i in human airway smooth muscle cells. Studies were performed with cultured primary ASM cells loaded with Fluo-4 AM. (,) [Ca2+]i transients and dose response curves to saccharin () and chloroquine (). The mean ± s.e.m. dose-response curves were from five or six experiments, and the transients shown are representative of single experiments. () Maximal [Ca2+]i responses to 1.0 mM of the bitter tastants aristocholic acid (aristo), chloroquine, colchicine, denatonium, quinine, saccharin, salicin, strychnine and yohimbine and the bronchoconstrictive Gq-coupled agonists bradykinin (0.01 mM) and histamine (0.1 mM). Results are means ± s.e.m. from four to six experiments. *P < 0.01 versus basal; #P < 0.05 versus denatonium. () The [Ca2+]i response to bitter tastants is ablated by the PLC inhibitor U73122 and the βγ antagonist gallein, and attenuated by the IP3 receptor antagonist 2APB. These studies were performed in the absence of extracellular calcium. Results shown are from a single representative experiment of at! least three performed. * Figure 2: Bitter tastants induce relaxation of intact mouse tracheas in a non–cAMP-dependent manner. () Dose-response curves of relaxation for the β-adrenergic agonist isoproterenol (iso) and the bitter taste receptor agonists chloroquine (chloro), denatonium (denat) and quinine, derived from intact mouse tracheas contracted with 1.0 mM acetylcholine (n = 7 experiments). () Relaxation by chloroquine and quinine of intact mouse tracheas contracted by 1.0 mM serotonin (n = 4 experiments). () cAMP production in cultured human ASM cells incubated with 1.0 mM chloroquine for the indicated times, or for 15 min with 30 μM isoproterenol, as determined by radioimmunoassay. There was no evidence for chloroquine-induced cAMP accumulation (n = 3 experiments). Inset, immunoblot of VASP and phosphorylated VASP (P-VASP) in cultured human ASM cells exposed to 1.0 mM chloroquine or saccharin (sacc), or 10 μM forskolin (forsk). Forskolin, which stimulates cAMP production, resulted in phosphorylation of VASP as indicated by the upper band. () Bitter tastant reversibility and additivity stu! dies with intact mouse tracheas. Intact mouse tracheas were contracted with 1.0 mM acetylcholine (ach) which was maintained in the bath when chloroquine (200 μM) or isoproterenol (30 μM), or both drugs, were added. After exposure to chloroquine alone, the rings were washed and then rechallenged with the same dose of acetylcholine. *P < 0.05 versus acetylcholine alone; #P < 0.01 versus acetylcholine + isoproterenol, or chloroquine alone. Results are from four experiments. Data are presented as means ± s.e.m. * Figure 3: Isolated airway smooth muscle responses to bitter tastants as assessed by single cell mechanics and membrane potentials. () Cell stiffness of isolated ASM cells in response to 10 μM isoproterenol (iso), 1.0 mM chloroquine (chloro), 1.0 mM saccharin (sacc) or 1.0 μM histamine (hist). () Relaxation of isolated ASM cells in response to 1.0 mM saccharin in the presence of 1 μM of the PLCβ inhibitor U73122, 10 nM of the BKCa antagonists iberiotoxin (IbTx) and charybdotoxin (ChTx) or 100 nM of the PKA inhibitor H89. () The relaxation response to 1.0 mM chloroquine in isolated mouse airway contracted by 10 μM methacholine (Mch) in the absence or presence of 100 nM of the BKCa antagonist IbTx. Results are representative of five to eight experiments. () Membrane potential effects of saccharin and chloroquine. ASM cells loaded with a fluorescence-based membrane potential-sensitive dye were exposed to 1.0 mM chloroquine or saccharin, 1.0 μM histamine or 60 mM KCl (representative of four experiments). A decrease in relative fluorescence units (RFU) indicates hyperpolarization. () Effects of the BKCa! antagonist IbTx (100 nM) on chloroquine and saccharin-promoted ASM hyperpolarization in intact ASM cells. Results represent the peak responses from four experiments. *P < 0.01 vs. vehicle control. Data are presented as means ± s.e.m. * Figure 4: Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells. (,) Sequential confocal images of Fluo-3–loaded cells showing localized [Ca2+]i increases in the cell upon exposure of ASM cells to 0.3 mM saccharin (). The images represent Fluo-3 fluorescence after background subtraction and baseline normalization (F / F0) with intensity encoded by pseudocolor. The arrows highlight local [Ca2+]i 'hot-spots'. The numbers over areas of the cells represent regions of interest (ROI) which correspond to the numbered intensity tracings (). (,) [Ca2+]i images () and intensity tracings of ROIs (), in ASM cells (loaded as in and ) in response to exposure to 1.0 μM histamine. () Confocal line-scan imaging showing spatially and temporally resolved local [Ca2+]i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 μm parallel to the cell membrane at one end of an elongated ASM cell, as shown at left. Arrows indicate several local [Ca2+]i events that occurred before the more defined increase with! in the isolated region. At the bottom is the spatially averaged normalized fluorescence signal (F / F0) generated from the line scan. Results are from single experiments representative of five performed. * Figure 5: Bitter taste receptor agonists attenuate bronchoconstriction in a mouse model of asthma. (,) Photomicrographs from sections of control () and ovalbumin-challenged () mouse lungs showing eosinophilic inflammation of the airway, epithelial hyperplasia and basement membrane thickening in ovalbumin challenged airways (H&E stain). Br, bronchus; Bm, basement membrane; Eo, eosinophil; Ep, epithelium; Bl, blood vessel. (,) Airway resistance in control () and ovalbumin-challenged () mice measured at baseline, in response to aerosolized methacholine (mch) and in response to single doses of quinine 150 μg or the β-agonist albuterol (3 μg) given during the bronchoconstrictive phase (n = 5 experiments). The studies were carried out with a dose of methacholine that resulted in a four- to five-fold increase in airway resistance over baseline (≥16 mg ml−1 in control mice and 8 mg ml−1 in ovalbumin-challenged mice). *P < 0.01 versus methacholine; #P < 0.05 versus methacholine. Data are presented as means ± s.e.m. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Deepak A Deshpande, * Wayne C H Wang, * Elizabeth L McIlmoyle, * Kathryn S Robinett, * Rachel M Schillinger & * Stephen B Liggett * Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA. * Steven S An * Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA. * James S K Sham * Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA. * Stephen B Liggett Contributions D.A.D., single-cell mechanics and imaging, data analysis and manuscript preparation; W.C.H.W., expression studies, gene knockdown, airway physiology, data analysis and manuscript preparation; E.L.M., calcium signaling and data analysis; K.S.R., intact airway studies, expression studies, data analysis and manuscript preparation; R.M.S., airway physiology; S.S.A., single cell mechanics, data analysis, manuscript preparation; J.S.K.S., confocal calcium imaging, data analysis, manuscript preparation; S.B.L. directed all studies, data analysis and interpretation and is the primary author of the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Stephen B Liggett (sligg001@umaryland.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–8 Additional data
• CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung
  - Nat Med 16(11):1305-1312 (2010)
  Nature Medicine | Article CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung * Cyrille Mionnet1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Vanessa Buatois1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Akira Kanda2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie Milcent1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sebastien Fleury2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * David Lair2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Marie Langelot2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Yannick Lacoeuille2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Edith Hessel6 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Coffman6 Search for this author in: * NPG journals * PubMed * Google Scholar * Antoine Magnan2, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * David Dombrowicz2, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Glaichenhaus1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Valerie Julia1, 2vjulia@unice.fr Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1305–1312Year published:(2010)DOI:doi:10.1038/nm.2253Received30 July 2010Accepted04 October 2010Published online31 October 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 Allergic asthma is a T helper type 2 (TH2)-dominated disease of the lung. In people with asthma, a fraction of CD4+ T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4+ T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT TH2 cells. We found that CX3CR1 signaling promoted TH2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient TH2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for TH1 cells upon airway inflammation but not under ho! meostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma. View full text Figures at a glance * Figure 1: Airway disease in CX3CR1-deficient mice. (–) Cx3cr1GFP/GFP (GFP/GFP) and Cx3cr1+/+ (+/+) littermates were sensitized with LACK, challenged with five consecutive aerosols, and analyzed 1 d () or 2 d (–) later. () Dynamic lung resistance and compliance, as monitored by plethysmography in Cx3cr1GFP/GFP and Cx3cr1+/+ mice after LACK or PBS challenge. Data show means ± s.e.m. in one representative experiment out of three. n = 6–8 mice per group. P values were obtained by comparing Cx3cr1GFP/GFP and Cx3cr1+/+ mice upon LACK sensitization and challenge. () Quantification of a FACS analysis of BALF cells from Cx3cr1GFP/GFP and Cx3cr1+/+ mice. E, eosinophils; N, neutrophils; L, lymphocytes. Data show means ± s.e.m. of three pooled experiments. n = 18–24 mice per group. () Representative microscopic images of lung sections at a tenfold magnification after MSB staining. () Quantification of mucus after periodic acid–Schiff staining. Data show means ± s.e.m. of two experiments. n = 6 mice per group. () IL-4, IL-5,! IL-13 and IFN-γ contents in lung extracts of LACK-sensitized mice challenged with LACK or PBS. Data show one representative experiment out of three, with bars indicating the means. n = 6–8 mice per group. () IL-4 and IL-5 intracellular staining of LACK-stimulated, pooled lung cells from Cx3cr1GFP/GFP and Cx3cr1+/+ mice. Data show means ± s.e.m. of two experiments. *P < 0.05, **P < 0.01. * Figure 2: AHR, airway inflammation and cytokine response in mice treated with CX3CR1 blocking reagents. () Experimental protocol. BALB/c mice were sensitized on days 0 and 7 with two intraperitoneal injections of 10 μg of LACK in alum and challenged on day 17 with 40 μg of LACK intranasally (i.n.). For prophylactic treatments, mice were treated on days 16, 18 and 21 with either 50 μg of polyclonal antibodies to CX3CR1 intravenously, or 50 μg of FKN-AT or PBS i.n. For curative treatments, mice were treated on days 18, 20 and 21 with 50 μg of FKN-AT i.n. () AHR, as measured by whole-body plethysmography. Data show means ± s.e.m. of one representative experiment out of five for the prophylactic treatments and out of three for the curative treatments. n = 6 mice per group. () Dynamic lung resistance and compliance, as monitored by plethysmography. Data show means ± s.e.m. in one representative experiment out of two. n = 6–8 mice per group. () Quantification of a FACS analysis of BALF cells. Data show means ± s.e.m. of three pooled experiments. n = 8–12 mice per group. ! () IL-4, IL-5 and IL-13 concentrations in the supernatants of lung cells from individual mice incubated with or without LACK for 72 h. Data show means ± s.e.m. in one representative experiment out of two. n = 4–6 mice per group. NS, non significant; *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 3: Airway disease in T cell–injected mice. (,) CD4+ T cells (5 × 106) from Cx3cr1GFP/GFP or Cx3cr1+/+ mice were injected into the indicated recipients. LACK-sensitized mice were challenged with LACK or PBS. Data show means ± s.e.m. in one representative experiment out of two. n = 6 mice per group. () AHR, as measured by whole-body plethysmography. () Quantification of a FACS analysis of BALF cells. (–) LACK-specific Thy1.1+ polyclonal TH2 cells (2 × 106) were injected into Cx3cr1GFP/GFP recipients. Recipients were treated with FKN-AT or PBS, challenged with LACK or PBS, and analyzed 5 d () or 6 d (–) later. () AHR, as monitored by whole-body plethysmography in mice treated with FKN-AT or PBS. LACK-sensitized, PBS-challenged mice were used as controls. Data show means ± s.e.m. in one experiment out of two. n = 6 mice per group. The black circles are obscured by the gray ones. () Quantification of a FACS analysis of BALF cells from FKN-AT–treated or PBS-treated mice. Data show means ± s.e.m. of two experimen! ts. n = 12 mice per group. () Quantification of a FACS analysis of MLN, lung, spleen and BALF cells from FKN-AT–treated and PBS-treated mice. Data show means ± s.e.m. n = 6 mice per group. () IL-4, IL-5 and IL-13 concentrations in the supernatants of lung cells incubated with or without LACK. Data show means ± s.d. of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 4: Number and phenotype of CX3CR1-expressing CD4+ T cells. (–) Cx3cr1+/GFP (,,) and WT () mice were sensitized and challenged with LACK and killed 1 d after the last aerosol. () GFP expression, after gating on live CD3+CD4+ cells, in the indicated organs in which blood cells were removed by cardiac perfusion with PBS. Data show representative FACS profiles. Numbers indicate the mean frequency ± s.e.m. of GFP+ cells among CD4+ T cells. n = 12 mice per group pooled from three experiments. () Cx3cr1 mRNA levels, as measured by quantitative PCR, in CD4+ T cells purified from spleen, MLN, lung and BAL. Data are expressed as Cx3cr1 / 18s rRNA ratio. () FACS analysis of T1/ST2 expression. Data show mean frequencies after gating on live CD3+CD4+ cells. n = 6 mice per group. () T cell cytokine secretion assessed by ELISA and intracellular staining. Top, 2 × 104 sorted GFP+ or GFP− cells were incubated with LACK and 5 × 105 T cell–depleted spleen cells for 72 h. Supernatants were analyzed for IL-4, IL-5, IL-13 and IFN-γ contents. Da! ta show means ± s.d. of three experiments. Bottom, quantification of intracellular staining of sorted GFP+ or GFP− cells. Data show means ± s.e.m. of three experiments. *P < 0.05; **P < 0.01. * Figure 5: Frequency and phenotype of injected TH2 cells. (–) Thy1.1+Thy1.2−Cx3cr1GFP/GFP and Thy1.1+Thy1.2+Cx3cr1+/GFP TCR-transgenic TH2 cells were co-injected into naive (,) or OVA-sensitized (,) BALB/c mice (1.5 × 106 cells of each per mouse). Recipients were exposed to LACK aerosols, and cells were analyzed by FACS. () The frequency of Cx3cr1GFP/GFP and Cx3cr1+/GFP donor cells in individual mice in two experiments. n = 8 mice per group, except for MLN before aerosol and BALF after the third aerosol, for which each dot represents pools of four and two mice, respectively. () Representative profiles after gating on CX3CR1-proficient (black histogram) and CX3CR1-deficient (empty histogram) donor or endogenous CD4+ T cells (gray histogram). Numbers indicate mean fluorescence intensities (MFI) ± s.e.m. of eight mice from two experiments. (,) Mice were exposed to five aerosols of OVA only or LACK and OVA, and lung and BALF cells were analyzed by FACS 1 d later. () Representative FACS profiles after gating on live CD4+ T cells. ! Numbers indicate mean frequencies ± s.e.m. of donor cells in one representative experiment out of two. n = 4 mice per group. () GFP expression by donor and endogenous CD4+ T cells in OVA-sensitized, challenged mice. Data show representative FACS profiles after gating on CX3CR1-proficient (black histogram), CX3CR1-deficient (empty histogram) and endogenous CD4+ T cells (gray histogram). Numbers indicate mean fluorescence intensities ± s.e.m. of eight mice from two experiments. * Figure 6: T cell proliferation and apoptosis. (–) Equal numbers of LACK-specific Cx3cr1GFP/GFP and Cx3cr1+/GFP TCR-transgenic TH2 (–) or TH1 () cells were injected into naive mice. () Apoptosis of transferred cells, as assessed by TUNEL. Recipients were challenged with LACK aerosols and analyzed 24 h after the 3rd aerosol. Data show representative FACS profiles after gating on CX3CR1-proficient or CX3CR1-deficient cells. Data show mean frequencies ± s.e.m. of TUNEL+ cells among CX3CR1-proficient (black histograms) and CX3CR1-deficient (gray histograms) cells. One representative experiment out of two is shown. n = 6 mice per group. () Frequencies of infected T cells following aerosol challenges. CX3CR1-proficient and CX3CR1-deficient (TH2 cells) were infected with a BCL-2–encoding or a control retroviral vector. Equal numbers of transduced cells were injected into naive recipients. Data show donor cell frequency in individual mice, with bars indicating the mean in one representative experiment out of two. () Quant! ification of a FACS analysis of BALF cells 1 d after Cx3cr1GFP/GFP recipients were injected with transduced TH2 cells and exposed to LACK aerosols. Data show means ± s.e.m. of two pooled experiments. n = 6 mice per group. () Quantification of a FACS analysis of donor cells 1 or 3 weeks after injection. Data show mean frequency ± s.e.m. of CX3CR1-proficient and CX3CR1-deficient donor cells in a representative experiment out of two. n = 6 mice per group. () Quantification of a FACS analysis of lymph node cells 4 d after the mice were immunized with LACK in CFA or CFA only 1 d after cell injection. Data show mean frequency ± s.e.m. of CX3CR1-proficient and CX3CR1-deficient donor cells in a representative experiment out of two. n = 3 mice per group. () Quantification of a FACS analysis of CD4+ donor TH1 cells from mice exposed to LACK aerosols. Data show the frequency of CX3CR1-proficient or CX3CR1-deficient donor cells in individual mice in one representative experiment out! of two. n = 5, except for BALF before and after the first aer! osol, for which each dot represents a pool of three mice. *P < 0.05; **P < 0.01; ***P < 0.001. Author information * Abstract * Author information * Supplementary information Affiliations * University of Nice-Sophia Antipolis, U924, Valbonne, France. * Cyrille Mionnet, * Vanessa Buatois, * Valerie Milcent, * Nicolas Glaichenhaus & * Valerie Julia * Institut National de la Santé et de la Recherche Médicale, Paris, France. * Cyrille Mionnet, * Vanessa Buatois, * Akira Kanda, * Valerie Milcent, * Sebastien Fleury, * David Lair, * Marie Langelot, * Yannick Lacoeuille, * Antoine Magnan, * David Dombrowicz, * Nicolas Glaichenhaus & * Valerie Julia * University of Lille 2, U1011, Lille, France. * Akira Kanda, * Sebastien Fleury & * David Dombrowicz * Institut Pasteur de Lille, Lille, France. * Akira Kanda, * Sebastien Fleury & * David Dombrowicz * University of Nantes, UMR915, Institut du Thorax, Nantes, France. * David Lair, * Marie Langelot, * Yannick Lacoeuille & * Antoine Magnan * Dynavax Technologies, Berkeley, California, USA. * Edith Hessel & * Robert Coffman Contributions C.M. and V.B. conducted most of the experiments and contributed to data analysis. A.K., S.F. and D.D. measured lung resistance and compliance and performed lung histology. V.M. provided technical help. D.L., M.L., Y.L. and A.M. performed experiments with HDM. E.H. and R.C. contributed to early experiments aimed at monitoring chemokine receptor expression in lung T cells. N.G. and V.J. wrote the manuscript. V.J. conceived of and directed the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Valerie Julia (vjulia@unice.fr) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–14 Additional data
• Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration
  - Nat Med 16(11):1313-1320 (2010)
  Nature Medicine | Article Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration * Paolo Grumati1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Luisa Coletto2, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Patrizia Sabatelli3 Search for this author in: * NPG journals * PubMed * Google Scholar * Matilde Cescon1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alessia Angelin4 Search for this author in: * NPG journals * PubMed * Google Scholar * Enrico Bertaggia2 Search for this author in: * NPG journals * PubMed * Google Scholar * Bert Blaauw5 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Urciuolo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Tania Tiepolo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Luciano Merlini6 Search for this author in: * NPG journals * PubMed * Google Scholar * Nadir M Maraldi3, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Bernardi4 Search for this author in: * NPG journals * PubMed * Google Scholar * Marco Sandri2, 4marco.sandri@unipd.it Search for this author in: * NPG journals * PubMed * Google Scholar * Paolo Bonaldo1bonaldo@bio.unipd.it Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume: 16 ,Pages:1313–1320Year published:(2010)DOI:doi:10.1038/nm.2247Received02 June 2010Accepted24 September 2010Published online31 October 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 Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI–knockout (Col6a1−/−) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B–interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6! a1−/− mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies. View full text Figures at a glance * Figure 1: Autophagy is impaired in Col6a1−/− mice. () Western blot for LC3 lipidation in diaphragm of fed wild-type and Col6a1−/− mice. MW, molecular weight; WT, wild-type; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. () Electron-microscopic quantification of myofibers containing autophagic vesicles in diaphragms of fed and 24-h–fasted mice (***P < 0.001; n = 5, each group). Error bars indicate s.d. () Electron micrograph of a double-membrane autophagosome (arrowheads) containing mitochondria (mit) and sarcoplasmic reticulum (sr) in diaphragms of 24-h–fasted wild-type mice. Scale bar, 400 nm. () Western blot for LC3 lipidation in diaphragm (left) and tibialis anterior (right) of fed and 24-h–fasted mice. () Quantification of TUNEL-positive nuclei in tibialis anterior transfected with vector expressing either scrambled or Map1lc3b-targeting shRNA (*P < 0.05; NS, not significant; n = 5, each group). Error bars indicate s.e.m. () Western blot for LC3 lipidation in diaphragm (left) and tibialis anterior (right) of ! untreated mice (−) or mice treated with chloroquine diphosphate at 50 mg per kg body weight per day for 10 d (+). () Western blot for LC3 and p62 in diaphragm (left) and tibialis anterior (right) of fed, 6-h–fasted and 12-h–fasted mice. () Western blot for beclin-1 and Bnip3 in diaphragm (left) and tibialis anterior (right) of fed and 24-h–fasted mice (top) and densitometric quantification of beclin-1 (middle) and Bnip3 (bottom) (***P < 0.001; *P < 0.05; n = 3). Error bars indicate s.e.m. () Quantitative RT-PCR (qRT-PCR) analysis of Bnip3 mRNA in tibialis anterior of fed and 24-h–fasted mice (*P < 0.05; n = 7). Error bars indicate s.e.m. () Western blot for Akt and 4E-BP1 phosphorylation (p-Akt and p–4E-BP1, respectively) in diaphragm (left) and tibialis anterior (right) of fed or 24-h–fasted mice. * Figure 2: Prolonged starvation induces autophagy in Col6a1−/− mice. () Western blot for LC3, beclin-1 and Bnip3 in diaphragm (left) and tibialis anterior (right) of fed and 30-h–fasted mice. () Densitometric quantification of Bnip3 (top) and beclin-1 (bottom) after western blotting of diaphragm (left) and tibialis anterior (right) (***P < 0.001; *P < 0.05, n = 3). Error bars indicate s.e.m. () qRT-PCR analysis of Bnip3 mRNA in tibialis anterior of fed and 30-h–fasted mice (*P < 0.05; n = 7). Error bars indicate s.e.m. () Immunoblot analysis for Akt and 4E-BP1 phosphorylation in diaphragm (left) and tibialis anterior (right) of fed and 30-h–fasted mice. The right graphs show the densitometric quantification of the western blots for p-Akt. (***P < 0.001; *P < 0.05; n = 3). Error bars indicate s.e.m. () Quantification of myofibers containing autophagic vesicles in diaphragms of 30-h–fasted mice (***P < 0.001; n = 5, each group). Error bars indicate s.d. () Immunogold labeling of LC3 in 30-h–fasted Col6a1−/− diaphragm. LC3 labeling! (black dots marked by arrowheads) is associated with the membrane of an autophagosome containing a mitochondrion (mit). Scale bar, 300 nm. () Fluorescence microscopy of tibialis anterior cryosections from wild-type and Col6a1−/− mice transfected with YFP-LC3 and starved for 30 h. LC3 puncta (arrowheads) are indicated. Scale bar, 50 μm. * Figure 3: Induction of autophagy ameliorates the dystrophic phenotype. () Electron micrographs of diaphragm from Col6a1−/− mice in fed conditions (left) and after 30 h starvation (right). Abnormal mitochondria (arrowheads) and dilated sarcoplasmic reticulum cisternae (arrows) in myofibers of the fed Col6a1−/− mice are indicated. Scale bar, 1 μm. () Percentage of myofibers with morphologically altered mitochondria in diaphragm of fed, 24-h–fasted and 30-h–fasted mice (***P < 0.001; n = 5, each group). Error bars indicate s.d. () Mitochondrial response to oligomycin in myofibers isolated from FDB muscles of fed and fasted mice. Where indicated, 6 μM oligomycin (arrow) or 4 μM of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) (arrowhead) were added. Each trace represents the tetramethylrhodamine methyl ester (TMRM) fluorescence of a single fiber. The fraction of myofibers with depolarizing mitochondria is indicated for each condition, where fibers are considered as depolarizing when they lose more than 10! % of initial value of TMRM fluorescence after oligomycin addition (n = 5, each group). () Quantification of TUNEL-positive nuclei in diaphragm (left) and tibialis anterior (right) of fed, 24-h–fasted and 30-h–fasted mice (***P < 0.001, **P < 0.01, *P < 0.05; n = 5, each group). Error bars indicate s.e.m. * Figure 4: Beclin-1 protein abundance is decreased in muscle biopsies of subjects with UCMD or Bethlem myopathy, and its expression counteracts muscle apoptosis in Col6a1−/− mice. (,) Quantification of TUNEL-positive nuclei in wild-type and Col6a1−/− tibialis anterior transfected with a vector expressing scramble or Becn1 shRNA () or Bnip3 shRNA (). Western blot confirmation of RNAi-mediated knockdown of Becn1 and Bnip3 in tibialis anterior of wild-type mice is included above each set of graphs (***P < 0.001; **P < 0.01; *P < 0.05; n = 10, each group). Error bars indicate s.e.m. () Fluorescence microscopy of tibialis anterior cryosections from wild-type and Col6a1−/− mice transfected with beclin-1 and YFP-LC3 expression vectors and maintained in fed condition. LC3 puncta (arrowheads) are indicated. Scale bar, 50 μm. () Right, quantification of TUNEL-positive nuclei in transfected and nontransfected myofibers of wild-type and Col6a1−/− tibialis anterior muscle after in vivo transfection with beclin-1–EGFP expression vector. Transfected fibers were revealed by fluorescence microscopy (left). Scale bar, 50 μm (***P < 0.001; n = 10, each g! roup). Error bars indicate s.e.m. () Western blot for beclin-1 and Bnip3 in protein lysates of human muscle biopsies from two healthy (normal) controls (C1, C2), five individuals with UCMD (UCMD1–5) and four individuals with Bethlem myopathy (BM1–4). Data are representative of three independent experiments. * Figure 5: LPD rescues the dystrophic phenotype of Col6a1−/− mice. () Electron micrographs of diaphragm from standard diet (SD)-fed and LPD-fed Col6a1−/− mice. Abnormal mitochondria (arrowheads) and dilated sarcoplasmic reticulum cisternae (arrows) in Col6a1−/− myofibers are indicated. nu, nucleus. Scale bar, 500 nm. () Percentage of myofibers with morphologically altered mitochondria in diaphragms of mice fed with SD or LPD. (***P < 0.001; n = 5, each group). Error bars indicate s.d. () Western blot for LC3, beclin-1 and Bnip3 in diaphragm (left) and tibialis anterior (right) of mice fed with SD or LPD. () Percentage of myofibers containing autophagic vesicles in diaphragm of wild-type and Col6a1−/− mice fed with SD or LPD (*P < 0.05; n = 5 each group). Error bars indicate s.d. () Mitochondrial response to oligomycin in FDB myofibers of Col6a1−/− fed with SD or LPD. The fraction of depolarizing myofibers is provided as in Figure 3c (n = 5, each group). () Quantification of TUNEL-positive nuclei in diaphragm (left) and tibia! lis anterior (right) of mice fed with SD or LPD (***P < 0.001; **P < 0.01; *P < 0.05; n = 5, each group). Error bars indicate s.e.m. () In vivo tetanic force measurements in the gastrocnemius muscle of mice fed with SD or LPD (***P < 0.001; *P < 0.05; n = 12, each group). Error bars indicate s.e.m. * Figure 6: Pharmacological treatments induce autophagy and ameliorate the myopathic phenotype of Col6a1−/− mice. () Quantification of TUNEL-positive nuclei in diaphragm (left) and tibialis anterior (right) of wild-type and Col6a1−/− mice treated with vehicle (ethanol) or with rapamycin (Rap) for 15 d (***P < 0.001; *P < 0.05; n = 5, each group). Error bars indicate s.e.m. () Representative micrographs of a transverse section of diaphragm from untreated (left) and rapamycin-treated (right) Col6a1−/− mice. Abnormal mitochondria (white arrowheads) and dilated sarcoplasmic reticulum (white arrows) in the muscle of untreated mice are indicated, as is autophagosome formation in the treated mice (inset). Scale bar, 500 nm. () Percentage of myofibers with morphologically altered mitochondria in the diaphragm of Col6a1−/− mice treated with rapamycin (+) or left untreated (−) for 15 d (***P < 0.001; n = 5 each group). Error bars indicate s.d. () Western blot analysis for LC3, beclin-1 and Bnip3 in diaphragm (left) and tibialis anterior (right) of wild-type and Col6a1−/− mice tr! eated with vehicle (olive oil) (−) or with cyclosporin A (CsA) (+) for 4 d and maintained in fed or 24-h–fasting conditions. () Quantification of TUNEL-positive nuclei in diaphragm (left) and tibialis anterior (right) of wild-type and Col6a1−/− mice treated with vehicle or with cyclosporin A for 4 d (***P < 0.001; n = 4, each group). Error bars indicate s.e.m. () In vivo tetanic force measurements in the gastrocnemius muscle of mice treated with vehicle or with cyclosporin A for 10 d (***P < 0.001; **P < 0.01; *P < 0.05; n = 8, each group). Error bars indicate s.e.m. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Paolo Grumati & * Luisa Coletto Affiliations * Department of Histology, Microbiology & Medical Biotechnology, University of Padova, Padova, Italy. * Paolo Grumati, * Matilde Cescon, * Anna Urciuolo, * Tania Tiepolo & * Paolo Bonaldo * Dulbecco Telethon Institute, Venetian Institute of Molecular Medicine, Padova, Italy. * Luisa Coletto, * Enrico Bertaggia & * Marco Sandri * Institute of Medical Genetics—National Research Council, Bologna, Italy. * Patrizia Sabatelli & * Nadir M Maraldi * Department of Biomedical Sciences, University of Padova, Padova, Italy. * Alessia Angelin, * Paolo Bernardi & * Marco Sandri * Department of Human Anatomy & Physiology, University of Padova, Padova, Italy. * Bert Blaauw * Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy. * Luciano Merlini * Department of Anatomical Sciences, University of Bologna, Bologna, Italy. * Nadir M Maraldi Contributions P.G. performed biochemical analyses, autophagy assays, mouse treatments, analysis and interpretation of data, and contributed to manuscript preparation. L.C. carried out RNA analysis, muscle transfections, molecular biology, analysis and interpretation of data, and contributed to manuscript preparation. P.S. performed electron microscopy. M.C. performed TUNEL and histology. A.A. performed tetramethylrhodamine methyl ester (TMRM) analysis. E.B. carried out muscle transfections and mitochondria isolation. B.B. analyzed muscle mechanics. A.U. performed TUNEL analysis. T.T. genotyped and maintained mice. L.M., P. Bernardi and N.M.M. were involved in data analysis. P. Bonaldo and M.S. designed the study, analyzed data and wrote the paper. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Paolo Bonaldo (bonaldo@bio.unipd.it) or * Marco Sandri (marco.sandri@unipd.it) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–8 and Supplementary Tables 1–3 Additional data
• The TLX1 oncogene drives aneuploidy in T cell transformation
  - Nat Med 16(11):1321-1327 (2010)
  Nature Medicine | Article The TLX1 oncogene drives aneuploidy in T cell transformation * Kim De Keersmaecker1, 2, 3, 28 Search for this author in: * NPG journals * PubMed * Google Scholar * Pedro J Real1, 27, 28 Search for this author in: * NPG journals * PubMed * Google Scholar * Giusy Della Gatta1, 28 Search for this author in: * NPG journals * PubMed * Google Scholar * Teresa Palomero1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Luisa Sulis1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Valeria Tosello1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pieter Van Vlierberghe1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kelly Barnes1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mireia Castillo4 Search for this author in: * NPG journals * PubMed * Google Scholar * Xavier Sole6, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Hadler1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jack Lenz8 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter D Aplan9 Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle Kelliher10 Search for this author in: * NPG journals * PubMed * Google Scholar * Barbara L Kee11 Search for this author in: * NPG journals * PubMed * Google Scholar * Pier Paolo Pandolfi12 Search for this author in: * NPG journals * PubMed * Google Scholar * Dietmar Kappes13 Search for this author in: * NPG journals * PubMed * Google Scholar * Fotini Gounari14 Search for this author in: * NPG journals * PubMed * Google Scholar * Howard Petrie15 Search for this author in: * NPG journals * PubMed * Google Scholar * Joni Van der Meulen16 Search for this author in: * NPG journals * PubMed * Google Scholar * Frank Speleman16 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth Paietta17, 18 Search for this author in: * NPG journals * PubMed * Google Scholar * Janis Racevskis17, 18 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter H Wiernik17, 18 Search for this author in: * NPG journals * PubMed * Google Scholar * Jacob M Rowe19 Search for this author in: * NPG journals * PubMed * Google Scholar * Jean Soulier20, 21 Search for this author in: * NPG journals * PubMed * Google Scholar * David Avran20, 21 Search for this author in: * NPG journals * PubMed * Google Scholar * Hélène Cavé22 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicole Dastugue23 Search for this author in: * NPG journals * PubMed * Google Scholar * Susana Raimondi24 Search for this author in: * NPG journals * PubMed * Google Scholar * Jules P P Meijerink25 Search for this author in: * NPG journals * PubMed * Google Scholar * Carlos Cordon-Cardo4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrea Califano1, 26 Search for this author in: * NPG journals * PubMed * Google Scholar * Adolfo A Ferrando1, 4, 5af2196@columbia.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1321–1327Year published:(2010)DOI:doi:10.1038/nm.2246Received31 March 2010Accepted21 September 2010Published online24 October 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 The TLX1 oncogene (encoding the transcription factor T cell leukemia homeobox protein-1) has a major role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). However, the specific mechanisms of T cell transformation downstream of TLX1 remain to be elucidated. Here we show that transgenic expression of human TLX1 in mice induces T-ALL with frequent deletions and mutations in Bcl11b (encoding B cell leukemia/lymphoma-11B) and identify the presence of recurrent mutations and deletions in BCL11B in 16% of human T-ALLs. Most notably, mouse TLX1 tumors were typically aneuploid and showed a marked defect in the activation of the mitotic checkpoint. Mechanistically, TLX1 directly downregulates the expression of CHEK1 (encoding CHK1 checkpoint homolog) and additional mitotic control genes and induces loss of the mitotic checkpoint in nontransformed preleukemic thymocytes. These results identify a previously unrecognized mechanism contributing to chromosomal missegrega! tion and aneuploidy active at the earliest stages of tumor development in the pathogenesis of cancer. View full text Figures at a glance * Figure 1: TLX1-induced T cell leukemias in mice. () Kaplan-Meier survival curves of p56Lck-TLX1–transgenic mice and littermate controls (WT) from three independent founder lines. () H&E staining showing infiltration of thymus, spleen, bone marrow and liver by immature lymphoblasts. Scale bars, 100 μm. () Western blot analysis of TLX1 expression in mouse T cell tumors. (,) Immunohistochemical analysis of TLX1 () and CD3 () expression in mouse TLX1-induced T-ALL cells. Scale bar, 100 μm. (,) Immunophenotype distribution () and representative flow cytometry plots () showing heterogeneous expression of CD4 and CD8 in TLX1-induced leukemias. DN, double negative; DP, double positive; SP, single positive. () Clonality analysis by expression of Tcrb chains in TLX1-induced tumors. Polyclonal expression of Tcrb in normal thymocytes is shown as control (top). * Figure 2: Molecular signatures associated with TLX1-induced transformation. () Heat-map diagram of the 50 top ranking differentially expressed genes by t test in mouse TLX1-induced leukemias (Supplementary Table 2). () GSEA analysis of differentially expressed genes associated with TLX1-induced transformation in mice, showing enrichment of this signature in human TLX1- and TLX3- expressing T-ALLs. Gene set: human orthologs of mouse TLX1 signature genes. Data set: TLX1- and TLX3-positive versus TLX1- and TLX3-negative human leukemias. Enrichment plots (left) and heat map representations of the 50 top ranking genes in the leading edge (right) are shown. Genes in heat maps are shown in rows; each individual sample is shown in one column. The scale bar shows color-coded differential expression from the mean in s.d. (σ) units, with red indicating higher expression and blue lower expression. * Figure 3: Developmental defects in thymocyte development in TLX1-transgenic mice. () Thymus size (inset image; WT on left and TLX1 on right), weight and cellularity in preleukemic TLX1-transgenic mice at 6 weeks of age compared with littermate controls. Error bars represent s.d. Scale bar, 10 mm. () Cell cycle analysis of control and preleukemic TLX1-transgenic thymocytes via propidium iodide staining of DNA content, as analyzed by flow cytometry. () Flow cytometry analysis of T cell development in preleukemic TLX1-transgenic mice. Accumulation of CD44+CD25+ cells shows a differentiation block at the DN2 stage of thymocyte development. () Apoptosis analysis of control and TLX1-transgenic preleukemic thymocytes via annexin V and propidium iodine staining. Error bars represent s.d. () TUNEL staining on thymus tissue sections. Scale bars, 100 μm. (–) Effects of transgenic expression of BCL2 on apoptosis () thymic weight () and cellularity () in preleukemic TLX1 and BCL2 double-transgenic mice. Error bars represent s.d. * Figure 4: Numerical and structural chromosomal alterations in TLX1-induced mouse T-ALLs. () A mouse chromosomal ideogram showing the areas of genetic gain and loss identified by aCGH in TLX1-induced thymic tumors. Red bars on the right of the ideograms represent areas of gain. Green bars on the left of the ideograms represent areas of copy number loss. Socs1, suppressor of cytokine signaling-1. () Schematic representation of the chromosome 12q commonly deleted region encompassing the Bcl11b locus in mouse TLX1-induced tumors. Setd3, SET domain–containing-3. () aCGH plot showing a focal deletion of the Bcl11b gene in a mouse TLX1-induced T-ALL. () Schematic representation of Bcl11b mutations identified in mouse TLX1-induced T-ALLs. () DNA sequence chromatograms corresponding to Bcl11b mutations identified in mouse TLX1-induced T-ALLs. Mutations (nucleotide position and sequence change) are indicated on the left. Inserted and deleted nucleotides are in brackets. * Figure 5: BCL11B is a TLX1 target gene mutated in human T-ALL. () aCGH plots showing focal deletions in chromosomal band 14q32.2 encompassing the BCL11B locus in human T-ALL. CCDC85C, coiled-coil domain–containing 85C; HHIPL1, hedgehog-interacting protein–like-1. () Schematic representation of BCL11B mutations identified in human T-ALL. () DNA sequence analysis of BCL11B in diagnostic and remission T-ALL samples. () ChIP analysis of TLX1 binding of BCL11B regulatory sequences in ALL-SIL cells. Representative results of two independent ChIPs are shown. () Western blot analysis of TLX1 and RT-PCR analysis of BCL11B expression in ALL-SIL cells electroporated with control or TLX1-targeting siRNAs. Error bars represent s.d. * Figure 6: Numerical chromosomal alterations and defects in the mitotic checkpoint in TLX-transgenic mice. () Spectral karyotyping analysis of a mouse TLX1-induced tumor with trisomy 15. Tumor karyotype is indicated at the bottom. () Distribution of numerical chromosomal aberrations found by spectral karyotyping and aCGH analysis in mouse TLX1-induced leukemias. () Cell cycle analysis of vehicle-only–, paclitaxel- and nocodazole-treated mouse T-ALLs, showing defective activation of the mitotic checkpoint in CUMTLL1 mouse TLX1-positive leukemia cells but not in the TLX1-negative St4113 cell line. () GSEA analysis of mitotic regulators identified as TLX1 direct target genes by ChIP-on-chip in sorted thymocytes (DN2 cells) from wild-type and preleukemic TLX1 transgenic mice. Gene set: TLX1 direct targets in mitotic cell cycle (Gene Ontology: 0000278). Data set: TLX1-transgenic preleukemic cells versus wild type cells. The enrichment plot (left) and heat map representation of the top 25 mitotic genes in the rank of transcripts differentially expressed in TLX1-preleukemic cells (rig! ht) are shown. The scale bar at the bottom shows color-coded differential expression from the mean in s.d. units, with red indicating higher expression and blue lower expression. () RT-PCR analysis of Chek1 expression in thymocytes isolated from wild-type or TLX1-transgenic mice. () ChIP analysis of TLX1 binding to CHEK1 regulatory sequences in ALL-SIL cells. () RT-PCR analysis of TLX1 and CHEK1 expression in ALL-SIL cells electroporated with control or TLX1-targeting siRNAs. Error bars represent s.d. () Cell cycle analysis of vehicle-only− and paclitaxel-treated mouse thymocytes from BCL2-transgenic and TLX1- and BCL2-transgenic mice showing defective activation of the mitotic checkpoint in mouse TLX1- and BCL2-expressing preleukemic cells. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE19499 * GSE10609 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Kim De Keersmaecker, * Pedro J Real & * Giusy Della Gatta Affiliations * Institute for Cancer Genetics, Columbia University, New York, New York, USA. * Kim De Keersmaecker, * Pedro J Real, * Giusy Della Gatta, * Teresa Palomero, * Maria Luisa Sulis, * Valeria Tosello, * Pieter Van Vlierberghe, * Kelly Barnes, * Michael Hadler, * Andrea Califano & * Adolfo A Ferrando * Department of Molecular and Developmental Genetics, VIB, Leuven, Belgium. * Kim De Keersmaecker * Center for Human Genetics, K.U. Leuven, Leuven, Belgium. * Kim De Keersmaecker * Department of Pathology, Columbia University Medical Center, New York, New York, USA. * Teresa Palomero, * Mireia Castillo, * Carlos Cordon-Cardo & * Adolfo A Ferrando * Department of Pediatrics, Columbia University Medical Center, New York, New York, USA. * Maria Luisa Sulis & * Adolfo A Ferrando * Biomarkers and Susceptibility Unit, Catalan Institute of Oncology, Institut d′Investigació Biomèdica de Bellvitge, L'Hospitalet, Barcelona, Spain. * Xavier Sole * Biomedical Research Centre Network for Epidemiology and Public Health, Catalan Institute of Oncology, IDIBELL, L'Hospitalet, Barcelona, Spain. * Xavier Sole * Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, USA. * Jack Lenz * The Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. * Peter D Aplan * Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Michelle Kelliher * Department of Pathology, University of Chicago, Chicago, Illinois, USA. * Barbara L Kee * Departments of Medicine and Pathology, Beth Israel Deaconess Cancer Center, Harvard Medical School, Boston, MA, USA. * Pier Paolo Pandolfi * Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA. * Dietmar Kappes * Department of Medicine, University of Chicago, Chicago, Illinois, USA. * Fotini Gounari * Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida, USA. * Howard Petrie * Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium. * Joni Van der Meulen & * Frank Speleman * Montefiore Medical Center–North Division, New York, New York, USA. * Elisabeth Paietta, * Janis Racevskis & * Peter H Wiernik * New York Medical College, New York, New York, USA. * Elisabeth Paietta, * Janis Racevskis & * Peter H Wiernik * Rambam Medical Center and Technion, Israel Institute of Technology, Haifa, Israel. * Jacob M Rowe * Assistance publique—Hôpitaux de Paris Hematology Laboratory and Institut National de la Santé et de la Recherche Médicale U944, Hôpital Saint-Louis, Paris, France. * Jean Soulier & * David Avran * Université Paris 7-Denis Diderot, Institut Universitaire d'Hematology, Hôpital Saint-Louis, Paris, France. * Jean Soulier & * David Avran * Assistance publique—Hôpitaux de Paris, Hôpital Robert Debré, Département de Génétique, Université Paris 7-Denis Diderot, Paris, France. * Hélène Cavé * Laboratoire d′Hématologie, Hôpital Purpan, Toulouse, France. * Nicole Dastugue * Department of Pathology, St Jude Children′s Research Hospital, Memphis, Tennessee, USA. * Susana Raimondi * Department of Pediatric Oncology/Hematology, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands. * Jules P P Meijerink * Joint Centers for Systems Biology, Columbia University, New York, New York, USA. * Andrea Califano * Current address: Andalusian Stem Cell Bank, Centro de Investigacion Biomedica, Granada, Spain. * Pedro J Real Contributions K.D.K. performed cellular, genetic and molecular characterization of TLX1-induced tumors and preleukemic thymocytes, identified BCL11B mutations in mouse and human tumors and wrote the manuscript. P.J.R. generated the TLX1-transgenic mice and characterized the tumor phenotype. G.D.G. analyzed ChIP-on-chip data and gene expression signatures in human and mouse tumors. T.P. performed ChIP-on-chip. V.T. characterized mouse thymocytes. P.V.V. and K.D.K. analyzed aCGH data. M.L.S. performed mouse tumor microarray analysis. K.B. and M.H. analyzed TLX1-transgenic lines. M.C. performed histological and immunohistochemical studies. J.L., P.D.A., M.K., B.L.K., P.P.P., D.K. and F.G. provided mouse tumor samples. H.P. provided gene expression data on normal mouse thymocytes. X.S. analyzed ChIP-on-chip data. J.V.d.M. and F.S. analyzed BCL11B mutations in human T-ALL samples. S.R., H.C., N.D., J.S. and D.A. provided cytogenetic data on human T-ALLs. E.P., J.R., P.H.W. and J.M.R. provided ! human T-ALL specimens from Eastern Cooperative Oncology Group clinical trials. J.P.P.M. generated human expression profiling data and characterized human T-ALL samples. C.C.-C. supervised histological and immunohistochemical studies. A.C. supervised the bioinformatic data analysis. A.A.F. designed the study, supervised research and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Adolfo A Ferrando (af2196@columbia.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Results, Supplementary Methods, Supplementary Figures 1–7 and Supplementary Tables 1–15 Additional data
• A negative regulator of MAP kinase causes depressive behavior
  - Nat Med 16(11):1328-1332 (2010)
  Nature Medicine | Letter A negative regulator of MAP kinase causes depressive behavior * Vanja Duric1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mounira Banasr1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pawel Licznerski1 Search for this author in: * NPG journals * PubMed * Google Scholar * Heath D Schmidt1 Search for this author in: * NPG journals * PubMed * Google Scholar * Craig A Stockmeier2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Arthur A Simen1 Search for this author in: * NPG journals * PubMed * Google Scholar * Samuel S Newton1 Search for this author in: * NPG journals * PubMed * Google Scholar * Ronald S Duman1ronald.duman@yale.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1328–1332Year published:(2010)DOI:doi:10.1038/nm.2219Received09 March 2010Accepted24 August 2010Published online17 October 2010 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The lifetime prevalence (~16%)1 and the economic burden ($100 billion annually)2, 3 associated with major depressive disorder (MDD) make it one of the most common and debilitating neurobiological illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology of MDD have not been identified. Here we use whole-genome expression profiling of postmortem tissue and show significantly increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1, encoded by DUSP1, but hereafter called MKP-1) in the hippocampal subfields of subjects with MDD compared to matched controls. MKP-1, also known as dual-specificity phosphatase-1 (DUSP1), is a member of a family of proteins that dephosphorylate both threonine and tyrosine residues and thereby serves as a key negative regulator of the MAPK cascade4, a major signaling pathway involved in neuronal plasticity, function and survival5, 6. We tested the role of altered MKP-1 expression in rat! and mouse models of depression and found that increased hippocampal MKP-1 expression, as a result of stress or viral-mediated gene transfer, causes depressive behaviors. Conversely, chronic antidepressant treatment normalizes stress-induced MKP-1 expression and behavior, and mice lacking MKP-1 are resilient to stress. These postmortem and preclinical studies identify MKP-1 as a key factor in MDD pathophysiology and as a new target for therapeutic interventions. View full text Figures at a glance * Figure 1: MKP-1 is dysregulated in major depressive disorder (MDD). () Microarray analysis of MDD postmortem brain samples showing alterations in the expression of DUSP genes in hippocampal subfields. () qPCR analysis of MKP-1 gene expression in samples from the same cohort as in . Data are expressed as mean fold change ± s.e.m. (n = 6); *P ≤ 0.05 compared to the healthy controls (Student's t test). () Representative autoradiographs and quantitative analysis of hippocampal MKP-1 mRNA levels by in situ hybridization in a separate cohort of subjects with MDD and matched controls (scale bar, 5 mm). Results are shown as percentage increase for each control and MDD-affected subject. *P < 0.02 compared to the healthy controls (Student's t test). DG, dentate gyrus. (,) Microarray-based expression levels of MAPKs () and downstream transcription factors and target genes (). CREBBP, CREB binding protein (CBP); NPY, neuropeptide Y; RAF1, v-raf-1 murine leukemia viral oncogene homolog-1;RPS6KA2, ribosomal S6 kinase (RSK). () Model for neurotrophic an! d growth factor receptor activation of MAPK, downstream transcription factors, and target genes. Microarray results (, and ) are shown as an average fold change (dentate gyrus, n = 14; CA1, n = 15); *P < 0.05, †P < 0.06 compared to the healthy controls (permutation tests, P value adjusted to false discovery rate at 0.05). Fold change for specific splice variants is reported for DUSP19.2, DUSP24.2, RPS6KA5.2 and VEGFa.2. FLK-1, fetal liver kinase-1; TRKA–C, tyrosine kinase receptors A,B and C; IGF-1R, insulin-like growth factor-1 receptor; FGFR1-4, fibroblast growth factor receptors 1, 2, 3 and 4. * Figure 2: Influence of CUS and antidepressant treatment on behavior and MKP-1 expression. () Schematic of the rat CUS paradigm. Rats were exposed to CUS or control conditions and were then given either saline or fluoxetine (FLX) for 21 d. Locomotor activity (LA), active avoidance test (AAT) and sucrose preference test (SPT) behaviors were determined. () Behavioral results for AAT and SPT, expressed as mean ± s.e.m. (n = 8). () Representative autoradiographs (top) and quantitative analysis (bottom) of Mkp-1 mRNA levels by in situ hybridization on coronal sections of rat hippocampus (scale bar, 1.0 mm). Results are expressed as mean ± s.e.m. (n = 4 or 5). () Western blot analysis showing the effects of CUS and FLX treatments on hippocampal Mkp-1 protein abundance. Tissue amounts of β-actin were used as loading controls. Data are expressed as mean ± s.e.m. percentage change over nonstressed control group (n = 5); *P < 0.05 compared to the nonstressed control group, *P < 0.05 compared to CUS group (two-way ANOVA and Fisher's protected least significant difference! post hoc analysis). * Figure 3: Influence of MKP-1 overexpression on behavior in rodent models of depression. () Depiction of the rAAV engineered to locally overexpress MKP-1 (rAAV–Mkp-1), and compared to a control vector that expresses green fluorescent protein (rAAV-GFP). ITR, inverted terminal repeats; CMV, cytomegalovirus promoter. (,) Rats received bilateral intrahippocampal infusions of rAAV-Mkp-1 or rAAV-GFP. Expression levels of GFP protein () and Mkp-1 mRNA () are shown (representative images; scale bars in , 100 μm; scale bar in , 500 μm). (–) Effects of rAAV- Mkp-1 infusions on rat behavior, compared to rAAV-GFP controls, for the percentage sucrose consumed compared to total fluid consumption (water and sucrose) in the SPT (), the number of escape failures in the AAT () and latency to feed in the novelty suppressed feeding test (). Behavioral data are expressed as mean ± s.e.m. (n = 6–9); *P < 0.02, †P < 0.10 compared to the rAAV-GFP control group (Student's t test). * Figure 4: Influence of MKP-1 deletion on behavioral models of depression. () Experimental paradigm for behavioral testing and CUS exposure of Mkp-1–knockout mice (Mkp-1−/−; n = 9 or 10) and WT littermates (Mkp-1+/+; n = 8). SCT, sucrose consumption test; WCT, water consumption test; OFT, open field test; EPM, elevated plus maze. () Baseline sucrose consumption on day 0, followed by post-stress measurements conducted on days 17 and 30. Water consumption was also measured throughout the stress paradigm (data shown for water test on day 20). () The number of entries into the open or closed arms, and the total time spent in the open arms during the elevated plus maze test. Results are expressed as mean ± s.e.m.; *P < 0.05 compared to the WT control group and *P < 0.05 compared to the WT stress group (sucrose and water consumption: repeated-measures ANOVA and Dunnett's test; elevated plus maze: Student's t test). () Representative blots (top) and quantitative results (bottom) of western blot analysis showing the effects of CUS on hippocampal pho! spho-Erk (pErk) abundance in WT and Mkp-1−/− mice compared to nonstressed WT control mice. Tissue amounts of total Erk and β-actin were used as loading controls. Optical density values are expressed as a ratio of phospho-Erk to total Erk. Data are expressed as mean ± s.e.m. percentage change over nonstressed WT control (n = 3); *P < 0.05 compared to WT control group and *P < 0.05 compared to the WT stress group (ANOVA and Student-Newman-Keuls' post hoc analysis). Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE24095 Author information * Accession codes * Author information * Supplementary information Affiliations * Department of Psychiatry, Yale University, New Haven, Connecticut, USA. * Vanja Duric, * Mounira Banasr, * Pawel Licznerski, * Heath D Schmidt, * Arthur A Simen, * Samuel S Newton & * Ronald S Duman * Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, Mississippi, USA. * Craig A Stockmeier * Department of Psychiatry, Case Western Reserve University, Cleveland, Ohio, USA. * Craig A Stockmeier Contributions V.D. prepared the original draft of the manuscript and was involved in all aspects of the experimental design and research, including execution of all microarray and molecular experiments, as well as behavioral tests in rats and mice. M.B. conducted the behavioral aspects of the rat CUS study, assisted with rat surgeries and was involved in analysis and interpretation of behavioral tests. P.L. was responsible for optimization, construction and preparation of recombinant AAVs. H.D.S. conducted baseline behavior tests in Mkp-1−/− mice. C.A.S. was responsible for human tissue generation and preparation of relevant human subjects' information tables and methodology. A.A.S. conducted statistical analysis of microarray experiments. S.S.N. assisted in the development and optimization of microarray experiments. R.S.D. was involved in all aspects of study design, data analysis, interpretation of results and preparation of the manuscript and figures. All authors discussed the resu! lts presented in the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Ronald S Duman (ronald.duman@yale.edu) Supplementary information * Accession codes * Author information * Supplementary information Excel files * Supplementary Data 1 (1.6M) Dysregulated genes in dentate gyrus * Supplementary Data 2 (508K) Dysregulated genes in CA1 PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–3, Supplementary Tables 1–6 and Supplementary Methods Additional data
• A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes
  - Nat Med 16(11):1333-1340 (2010)
  Nature Medicine | Technical Report A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes * Isamu Z Hartman1, 7, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * AeRyon Kim1, 8 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert J Cotter2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kimberly Walter3 Search for this author in: * NPG journals * PubMed * Google Scholar * Sarat K Dalai3 Search for this author in: * NPG journals * PubMed * Google Scholar * Tatiana Boronina4 Search for this author in: * NPG journals * PubMed * Google Scholar * Wendell Griffith2 Search for this author in: * NPG journals * PubMed * Google Scholar * David E Lanar5 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert Schwenk5 Search for this author in: * NPG journals * PubMed * Google Scholar * Urszula Krzych5 Search for this author in: * NPG journals * PubMed * Google Scholar * Robert N Cole4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Scheherazade Sadegh-Nasseri1, 3ssadegh@jhmi.edu Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume: 16 ,Pages:1333–1340Year published:(2010)DOI:doi:10.1038/nm.2248Received16 October 2009Accepted23 July 2010Published online31 October 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 Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4+ T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4+ T cells from H5N1-immunized HLA-DR1–transgenic mice and LSA-NRC–vacc! inated HLA-DR1–positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens. View full text Figures at a glance * Figure 1: Sensitivity to cathepsins and identification of rHA1-derived peptides by mass spectrometry. () Sensitivity to cathepsin B (CatB) and cathepsin H (CatH). HA306–318–DR1 (top) and rHA1 (bottom) were treated with CatB and CatH at various concentrations of each enzyme. Samples were resolved by gentle SDS-PAGE where samples were not boiled to preserve peptide-DR1αβ complex37. () Sensitivity of DM and DR1 to cathepsins. Left, conventional SDS-PAGE of empty DR1 (lanes 1 and 2), pre-formed HA306–318–DR1 (lanes 3 and 4) and DM (lanes 5 and 6) incubated in the presence or absence of 200 nM CatB and CatH. rHA1 (lanes 8 and 9) served as a positive control for digestion by CatB and CatH. Right, gentle SDS-PAGE (12% acrylamide, silver stained) of empty DR1 (lane 3), HA306–318–DR1 (lane 5) and DM (lane 7) treated with 100 nM CatS. (–) Mass spectra of rHA1-derived peptides eluted from DR1 for m/z 1,950–2,550 Da. is background for , and is background for . Mass species in bold represent rHA1 fragments eluted from DR1 containing HA306–318 epitope (Supplementary F! igs. 3 and ), underlining represents other rHA1-derived peptides, and in gray are the background peaks. Experiments were repeated more than three times. Pointed arrows indicate peaks that disappeared in . (,) In vitro proliferation of rHA1-immunized cells, as measured by [3H]thymidine incorporation (two individual mice out of five tested). * Figure 2: Identification of type II collagen-derived peptides eluted from DR1. (,) Mass spectra of CII-derived peptides eluted from DR1. () DR1 was incubated with the following components: MMP-9–fragmented bovine CII20, DM, CatB and CatH. () The negative control reactions carried out without including MMP-9–fragmented CII. (CII 273–305)4OH represents the sequence of collagen peptide containing four hydroxylations of proline and lysine (QTGEPOHGIAGFKGEQGPKOHGEPOHGPAGVQGAPOHGPAG). The experiment was repeated more than three times. () Expanded spectrum of between m/z 2,800 and 3,500 Da. Peptide modifications: hexose, 162 Da; hydroxylation, 16 Da; Na+ adduction, 22Da. () Proliferation of cells immunized with native CII protein in CFA incubated with increasing doses of CII280–294 (AGFKGEQGPKGEPGP), CLIP89–105 and heat-denatured CII protein in vitro, as measured by [3H]thymidine incorporation (one out of three representative individual mice tested). * Figure 3: Identification of DR1 restricted epitope of H5N1 rHA1 by the cell-free antigen processing system. (–) Mass spectra of H5N1 rHA1 (A/Vietnam/1203/2004 H5N1 strain, Genbank AY651334)-derived peptides eluted from DR1. DR1 and DM were incubated with native H5N1 rHA1 (), heat-denatured H5N1 rHA1 () or no protein antigens (), followed by addition of CatB, CatH and CatS, and then immunoprecipitation and peptide elution. Mass species that are underlined represent H5N1-derived HA259–274 and HA259–278 peptides eluted from DR1. Background mass species are labeled in gray. () Tandem mass spectra of HA259–274 at m/z 1,814.82 Da. These spectra represent one of four repeated experiments. * Figure 4: Biological validation of the immunodominant epitope of H5N1 rHA1 identified by the reductionist antigen processing system. () Proliferation of T cells isolated from DR1-transgenic mice immunized with native H5N1 rHA1 protein in CFA in response to stimulation with HA259–274 (SNGNFIAPEYAYKIVK), CLIP89–105 or H5N1 rHA1 protein in vitro. Cellular proliferation was measured by [3H]thymidine incorporation (representative of one mouse out of three individual mice tested). (,) IL-2 () and IFN-γ () ELISA performed from supernatant collected from in vitro culture of another three individual mice immunized as in . Cell culture supernatants were removed after 24 h, 48 h and 72 h culture. () Draining lymph node cells (pooled from four mice) freshly isolated from H5N1 rHA1 protein–immunized mice on day 8 and directly stained for T cells specific for HA259–274–DR1 or CLIP–DR1. Cells from three mock-immunized mice were also stained for the presence of T cell receptors specific for HA259–274–DR1 and served as controls. Cells were stained for 2 h at 37 °C with varying concentrations of tetramers! as shown followed by staining with monoclonal antibodies for CD4, CD8, CD44, F4/80 and B220. () Cells from were expanded with 0.07 μM H5N1 rHA1 for an additional 7 d in vitro. Protein-stimulated cells were stained with the tetramers (1.5 μg ml−1) and the antibodies described in . Numbers represent the percentage of tetramer-positive events among parent CD4+ (CD4+CD44+CD8−F4/80−B220−7AAD−) cells. * Figure 5: Identification of the DR1-restricted epitope of LSA-NRC and biological validation. (,) Mass spectra of LSA-NRC–derived peptides eluted from DR1. LSA-NRC (top) or no protein antigens (bottom) were incubated with DR1 and DM followed by addition of CatB, CatH and CatS. LSA-NRC–derived peptides captured by DR1 are underlined. () Expanded spectrum at 1,700–2,430 Da. () Spectrum at m/z 3,435–3,515 Da. The experiments were repeated more than five times. () Tandem mass spectrum of m/z ~2,278 Da. (,) LSA-NRC–immunized cells were incubated with LSA323–337 (VQYDNFQDEENIGIY), LSA429–443 (VDELSEDITKYFMKL), LSA436–449 (ITKYFMKLGGSGSP), CLIP89–105 and LSA-NRC in vitro. () T cell proliferation, as measured by [3H]thymidine incorporation (one out of three individual mice tested). () Cell culture supernatant from another two mice under a similar experimental set up as in were removed 24 h or 48 h later, and the amount of IL-2 produced was determined by ELISA. () Proliferation results of PBMCs from vaccinated individuals and their HLA-DR types. PBMCs from e! ight volunteers were obtained before or after immunization with an LSA-NRC liposome-based vaccine and were stimulated with the intact LSA-NRC protein (0.2 μM) as a positive control or different concentrations of LSA436–449 and CLIP89–105 and were assayed by [3H]thymidine incorporation. Samples of each individual are shown by different colors matching their HLA-DR haplotypes shown on top of the figure. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions GenBank * AY651334 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Isamu Z Hartman & * AeRyon Kim Affiliations * Graduate Program in Immunology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Isamu Z Hartman, * AeRyon Kim & * Scheherazade Sadegh-Nasseri * Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Robert J Cotter & * Wendell Griffith * Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Kimberly Walter, * Sarat K Dalai & * Scheherazade Sadegh-Nasseri * Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Tatiana Boronina & * Robert N Cole * Division of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA. * David E Lanar, * Robert Schwenk & * Urszula Krzych * Mass Spectrometry and Proteomics Facility, Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. * Robert N Cole * Present address: Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas, USA. * Isamu Z Hartman Contributions I.Z.H. and A.K. designed and executed experiments, analyzed data and wrote the paper; R.J.C., W.G., T.B. and R.N.C. provided mass spectrometry data and analyses; K.W. cloned DM; S.K.D. contributed to in vivo testing; R.S., D.E.L. and U.K. did LSA-1 experiments in humans; and S.S.-N. designed experiments, supervised the project, obtained funding and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Scheherazade Sadegh-Nasseri (ssadegh@jhmi.edu) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (776K) Supplementary Figures 1–14 and Supplementary Tables 1–4 Additional data
• Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: Evidence in Crohn disease and experimental colitis in vivo
  - Nat Med 16(11):1341 (2010)
  Nature Medicine | Erratum Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: Evidence in Crohn disease and experimental colitis in vivo * R Atreya Search for this author in: * NPG journals * PubMed * Google Scholar * J Mudter Search for this author in: * NPG journals * PubMed * Google Scholar * S Finotto Search for this author in: * NPG journals * PubMed * Google Scholar * J Müllberg Search for this author in: * NPG journals * PubMed * Google Scholar * T Jostock Search for this author in: * NPG journals * PubMed * Google Scholar * S Wirtz Search for this author in: * NPG journals * PubMed * Google Scholar * M Schütz Search for this author in: * NPG journals * PubMed * Google Scholar * B Bartsch Search for this author in: * NPG journals * PubMed * Google Scholar * M Holtmann Search for this author in: * NPG journals * PubMed * Google Scholar * C Becker Search for this author in: * NPG journals * PubMed * Google Scholar * D Strand Search for this author in: * NPG journals * PubMed * Google Scholar * J Czaja Search for this author in: * NPG journals * PubMed * Google Scholar * J F Schlaak Search for this author in: * NPG journals * PubMed * Google Scholar * H A Lehr Search for this author in: * NPG journals * PubMed * Google Scholar * F Autschbach Search for this author in: * NPG journals * PubMed * Google Scholar * G Schürmann Search for this author in: * NPG journals * PubMed * Google Scholar * N Nishimoto Search for this author in: * NPG journals * PubMed * Google Scholar * K Yoshizaki Search for this author in: * NPG journals * PubMed * Google Scholar * H Ito Search for this author in: * NPG journals * PubMed * Google Scholar * T Kishimoto Search for this author in: * NPG journals * PubMed * Google Scholar * P R Galle Search for this author in: * NPG journals * PubMed * Google Scholar * S Rose-John Search for this author in: * NPG journals * PubMed * Google Scholar * M F Neurath Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume: 16 ,Page:1341Year published:(2010)DOI:doi:10.1038/nm1110-1341Published online04 November 2010 Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.6, 583–588 (2000); corrected after print 4 November 2010 In the version of this article initially published, the fourth image in was a duplication of the third image. The error has been corrected in the HTML and PDF versions of the article. Additional data