Hot off the presses! May 01 Nature medicine

The May 01 issue of the Nature medicine is now up on Pubget (About Nature medicine): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Crossing the gender divide
  - Nature Medicine 16(5):491 (2010)
  Nature Medicine | Editorial Crossing the gender divide Journal name:Nature MedicineVolume:16,Page:491Year published:(2010)DOI:doi:10.1038/nm0510-491 A program at the US National Science Foundation (NSF) has tackled the problem of the under-representation of women in academic bioscience by providing grants to foster institutional change. A recent study shows that more progress is needed—but universities and other research centers can make a big difference in sometimes small ways. View full text Additional data
• Regulators must step up stem cell oversight
  - Nature Medicine 16(5):492 (2010)
  Nature Medicine | Editorial Regulators must step up stem cell oversight Journal name:Nature MedicineVolume:16,Page:492Year published:(2010)DOI:doi:10.1038/nm0510-492 A growing number of clinics are offering cell therapies that remain untested in rigorous clinical trials. Although the scientific community has chided the use of unproven treatments, we need less talk and more action in regulating stem cell therapies. View full text Additional data
• Vaccine contamination prompts safety review
  - Nature Medicine 16(5):493 (2010)
  When Eric Delwart couldn't find the right email addresses online to contact GlaxoSmithKline (GSK) in early February, he posted a good old-fashioned letter to the Belgian headquarters of the pharma giant to inform the company that one of its vaccines was contaminated with a pig virus.Months earlier, Delwart, a viral genomicist at the University of California–San Francisco, and his colleagues began what they thought would be a run-of-the-mill experiment to sequence the viral nucleic acids in a suite of live, attenuated vaccines, including common shots for measles, rubella, yellow fever and four other viral diseases.
• Mayo tries new model in clinical trial business
  - Nature Medicine 16(5):494 (2010)
  "The worlds of academia and market economics don't have to clash," Gary Lubin says. But he quickly adds that, in the field of clinical trials, he'll have to prove that in the face of conflicting evidence.
• Myriad 'after-math'
  - Nature Medicine 16(5):494 (2010)
  When a US federal judge rejected patents by Myriad Genetics on the BRCA1 and BRCA2 genes in late March, it put many in the biotech industry on edge after years of making similar claims of their own. Kyle Jensen, a director with the nonprofit PIPRA, has been tracking the patent push, and in 2005 he coauthored a study on human genome patenting (Science 310, 239–240, 2005).
• Survey details stem cell clinics ahead of regulatory approval
  - Nature Medicine 16(5):495 (2010)
  A growing number of clinics offer unproven and possibly unsafe stem cell treatments to patients who are willing to travel thousands of miles in pursuit of a cure. To help people make more informed decisions, an international society of stem cell clinicians has published a preliminary survey of centers.
• Telemedicine has more than a remote chance in prisons
  - Nature Medicine 16(5):496 (2010)
  Telemedicine is at a tipping point in the US. Correctional systems in more than two dozen states are already relying on the approach, which uses video and other transmittable information rather than direct patient-doctor contact.
• 'Universal' immunizations get a boost in India
  - Nature Medicine 16(5):497 (2010)
  The economic liberalization that began in India two decades ago might have produced an unwanted side effect—the production of vaccines against illnesses such as measles and tetanus seems to be threatened by the disappearance of more than a dozen government-owned vaccine producers. Now, in response partly to calls made by some parliament members, the Indian health ministry might move to reverse its shift toward leaning on the private sector and pay greater attention to these public sector producers (in which the government owns a majority stake) when it comes to vaccine production.
• Science seen as olive branch to the Muslim world
  - Nature Medicine 16(5):497 (2010)
  In an effort to bolster scientific partnerships in the Middle East, two US congressmen have introduced a bill that would fund research and education in Muslim-majority countries. The move follows a speech made last summer by President Barack Obama at Cairo University in Egypt in which he promised to ramp up science diplomacy in the Arab world.
• Medical insights from mummies
  - Nature Medicine 16(5):498 (2010)
  The health status of people living in ancient times has remained under wraps—quite literally so in the case of Egyptian mummies. But thanks in part to developments in medical imaging technologies, various research teams have scanned mummies and gained insight into which diseases may have most troubled these populations.
• Treatments aim to topple papillomavirus before cancer begins
  - Nature Medicine 16(5):499 (2010)
  Between the recently approved prophylactic vaccines against the human papillomavirus (HPV), and the ease with which surgery can treat most cervical cancers, therapeutic treatments for the virus have been low on most clinicians' priority lists. But with increasing incidence of other HPV-associated cancers that are more difficult to treat surgically—for example, oral cancer—a new generation of therapies aimed at HPV-induced precancerous lesions and warts could be on the horizon.
• US health reform burden falls on medical devices
  - Nature Medicine 16(5):500 (2010)
  Like many other industries, makers of medical devices are reeling from the anticipated effects of US healthcare reform. But, unlike other businesses, which might only have a faint idea of what the coming changes mean, medical device companies have a concrete notion of the future effects on their revenue, namely a 2.
• Ariad patent decision points to description dilemma
  - Nature Medicine 16(5):500 (2010)
  Much of the recent press attention relating to gene patents has focused on a US federal judge's decision to overturn Myriad Genetics' patent on two breast cancer genes, BRCA1 and BRCA2. But around the same time another important ruling was issued.
• Animal rights activists try a more creative legal tactic
  - Nature Medicine 16(5):501 (2010)
  At a monthly meeting last year, Eric Sandgren, the director of animal resources at the University of Wisconsin-Madison, mentioned that some sheep being used to study decompression sickness, what divers call 'the bends', had died during an experiment.The comment set off a flurry of action by a local animal rights group, the Alliance for Animals.
• News in brief
  - Nature Medicine 16(5):502-503 (2010)
  Mar 23Around 40 representatives from religious groups pledged a stronger response to HIV, with tenets such as increasing prevention efforts and decreasing stigmatization of those affected. The pledge capped off the first ever High Level Summit of Religious Leaders on HIV in the Netherlands.
• Aiding adherence: five approaches to following prescriptions
  - Nature Medicine 16(5):504 (2010)
  Poor adherence to medications costs the US as much as $290 billion a year in increased healthcare costs, according to the not-for-profit New England Healthcare Institute. That cost arises from roughly half of all patients failing to take medications as prescribed (N. Engl. J. Med
• Straight talk with...Melinda Moree
  - Nature Medicine 16(5):505 (2010)
  Nature Medicine | News Straight talk with...Melinda Moree * Christian Torres Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:505Year published:(2010)DOI:doi:10.1038/nm0510-505 Abstract Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg For many people, the biotech industry and global health community might seem like strange bedfellows. One is driven by commercial success, the other largely by philanthropy. Innovation is key, however, for both fields, and the Washington, DC–based not-for-profit BIO Ventures for Global Health (BVGH) is trying to draw on that commonality. Melinda Moree, who officially became BVGH's chief executive at the start of this year, has experience bringing new technologies to the poorest and unhealthiest of communities. A former director of the Malaria Vaccine Initiative, Moree oversaw a successful proof-of-concept study for a malaria vaccine. spoke with Moree about how BVGH engenders partnerships between biotech and global health and how it might produce the next big achievement for both. View full text Additional data
• On the pill
  - Nature Medicine 16(5):506-508 (2010)
  Nature Medicine | News On the pill * Ellen Friedrichs1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:506–508Year published:(2010)DOI:doi:10.1038/nm0510-506 This month marks the fiftieth anniversary of the approval of oral contraception in the US, and the Pill's age is starting to show. Problems abound with its delivery, dosage and side effects. investigates how researchers have conceived of new ways to deliver birth control. View full text Additional data Affiliations * Ellen Friedrichs is a writer and health educator living in Brooklyn, New York.
• Contraceptive compliance lags behind the science
  - Nature Medicine 16(5):509 (2010)
  Nature Medicine | News Contraceptive compliance lags behind the science * Anna Glasier1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:509Year published:(2010)DOI:doi:10.1038/nm0510-509 The causes underlying unintended pregnancies are many and complex. But there is no doubt that the vast majority could be prevented by contraceptives that are already available and easily accessible. People just need to stick with them. View full text Additional data Affiliations * Anna Glasier is the director of family planning and well woman services at National Health Service Lothian in Edinburgh. She holds honorary professorships at the Universities of Edinburgh and London and advises the Population Council, the World Health Organization and many pharmaceutical companies about contraception, including those offering emergency contraception and IUDs.
• Regenerating physician-scientists
  - Nature Medicine 16(5):511 (2010)
  The academic niche for physician-scientists has been degenerating for over three decades. In 1979, the director of the US National Institutes of Health (NIH), James Wyngaarden, initially highlighted an alarming drop-off in the number of physician-scientists and their success rates in NIH funding.
• β-catenin does not regulate memory T cell phenotype
  - Nature Medicine 16(5):513-514 (2010)
  Nature Medicine | Correspondence β-catenin does not regulate memory T cell phenotype * Gregory Driessens1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Zheng1 Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas F Gajewski1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:513–514Year published:(2010)DOI:doi:10.1038/nm0510-513 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg To the Editor: The Wnt–β-catenin axis constitutes an evolutionarily conserved pathway that is important for hematopoietic stem cell self-renewal capacities and other developmental processes in vivo. In the absence of Wnt-induced signaling, cytoplasmic β-catenin is constitutively phosphorylated by glycogen synthase kinase-3β (GSK-3β) and targeted for degradation by the proteasome. Upon exposure to Wnt ligands, GSK-3β becomes phosphorylated and β-catenin protein accumulates and translocates to the nucleus, where it binds Lef/Tcf family proteins and facilitates transcriptional activation1. Despite evidence that the β-catenin pathway regulates thymocyte development, its function in post-thymic T cells is poorly understood. It was recently suggested by Gattinoni et al.2 that activation of this pathway may arrest effector T cell differentiation and generate CD8+ memory stem cells. These experiments were almost exclusively performed after addition of a GSK-3β inhibitor during T cell pri! ming, and the investigators observed accumulation of an unusual population of activated CD62LhighCD44low cells. Upon adoptive transfer in vivo, those cells showed more potent antitumor capabilities than other subpopulations of activated T cells. A better understanding of the mechanism of this effect could improve immunotherapies for clinical translation. However, in contrast to the results of Gattinoni et al.2, acquired with a pharmacologic inhibitor, we have used a genetic approach and find no evidence that the β-catenin pathway regulates T cell memory phenotype. View full text Author information * Author information * Supplementary information Affiliations * Department of Pathology, The University of Chicago, Chicago, Illinois, USA. * Gregory Driessens, * Yan Zheng & * Thomas F Gajewski * Department of Medicine, The University of Chicago, Chicago, Illinois, USA. * Thomas F Gajewski Contributions G.D. and T.F.G. designed the research; G.D. and Y.Z. performed the research; G.D. and T.F.G. analyzed the data; G.D. and T.F.G. wrote the paper. G.D. is a Fellow of the Leukemia and Lymphoma Society. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Thomas F Gajewski (tgajewsk@medicine.bsd.uchicago.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Figure and Methods (300K) Supplementary Fig 1 and Methods Additional data
• Reply to: "β-catenin does not regulate memory T cell phenotype"
  - Nature Medicine 16(5):514-515 (2010)
  We recently reported that the activation of the Wnt–β-catenin pathway during T cell priming arrests effector T cell differentiation and promotes the generation of CD8+ memory stem cells (TSCM cells), which we found to be more effective than other memory T cell subsets in treating tumors in mice after adoptive cell transfer1. We generated TSCM cells by activating naive CD8+ T cells in the presence of a naturally occurring ligand, Wnt3a, or inhibitors of glycogen synthase kinase-β (GSK-3β) to stabilize β-catenin and mimic downstream events of the Wnt signaling cascade.
• 'Denialism' has no place in scientific debate
  - Nature Medicine 16(5):515-516 (2010)
  I was intrigued to find my name on Megan Scudellari's short list of "today's most vocal denialists spreading ideas that counter the consensus in health fields"1. I am a prominent and published critic of the assertions of everyone else on the list, except for Wolfgang Wodarg—who doesn't belong there either.
• Improved UK clinical trial capacity through the North West Exemplar Programme
  - Nature Medicine 16(5):516 (2010)
  Laursen1 highlights the decline in clinical trials activity within the UK's National Health Service (NHS) over the past ten years and, in particular, the marked reduction in the UK's global market share in commercial clinical trials from 6% to 2% between 2000 and 2006. His article attributes this largely to the introduction of regulation to protect the interests of trial participants and to ensure data integrity.
• The enemy within: dormant retroviruses awaken
  - Nature Medicine 16(5):517-518 (2010)
  Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell–derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberr! antly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.
• Fighting off pain with resolvins
  - Nature Medicine 16(5):518-520 (2010)
  Inflammatory pain, such as arthritis pain, is a growing health problem1. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a unique family of lipid mediators, including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, have shown marked potency in treating disease conditions associated with inflammation2, 3. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 in mice potently reduces inflammatory pain behaviors induced by intraplantar injection of formalin, carrageenan or complete Freund's adjuvant (CFA), without affecting basal pain perception. Intrathecal RvE1 injection also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and tumor necrosis factor-α (TNF-α). RvE1 has anti-inflammatory activity by reducing neutrophil infiltration, paw edema and proinflammator! y cytokine expression. RvE1 also abolishes transient receptor potential vanilloid subtype-1 (TRPV1)- and TNF-α–induced excitatory postsynaptic current increases and TNF-α–evoked N-methyl-D-aspartic acid (NMDA) receptor hyperactivity in spinal dorsal horn neurons via inhibition of the extracellular signal–regulated kinase (ERK) signaling pathway. Thus, we show a previously unknown role for resolvins in normalizing the spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the potency of resolvins and the well-known side effects of opioids and COX inhibitors, resolvins may represent new analgesics for treating inflammatory pain.
• T cell receptors and cancer: gain gives pain
  - Nature Medicine 16(5):520-521 (2010)
  The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy–induced autoimmune pathology.
• Calling all antigens
  - Nature Medicine 16(5):522-523 (2010)
  Giardia lamblia is a human intestinal pathogen. Like many protozoan microorganisms, Giardia undergoes antigenic variation, a mechanism assumed to allow parasites to evade the host's immune response, producing chronic and/or recurrent infections. Recently, we found that the mechanism controlling variant-specific surface protein (VSP) switching in Giardia involves components of the RNA interference machinery and that disruption of this pathway generates trophozoites simultaneously expressing many VSPs. Here we use these altered trophozoites to determine the role of antigenic variation in a gerbil model of giardiasis. Our results show that either primary infection with trophozoites simultaneously expressing many VSPs or immunization with purified VSPs from the transgenic cells protects gerbils from subsequent Giardia infections. These results constitute, to our knowledge, the first experimental evidence that antigenic variation is essential for parasite survival within ho! sts and that artificial disruption of this mechanism might be useful in generating vaccines against major pathogens that show similar behavior.
• Fibrosis under arrest
  - Nature Medicine 16(5):523-525 (2010)
  Fibrosis is responsible for chronic progressive kidney failure, which is present in a large number of adults in the developed world. It is increasingly appreciated that acute kidney injury (AKI), resulting in aberrant incomplete repair, is a major contributor to chronic fibrotic kidney disease. The mechanism that triggers the fibrogenic response after injury is not well understood. In ischemic, toxic and obstructive models of AKI, we demonstrate a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome. G2/M-arrested proximal tubular cells activate c-jun NH2-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production. Treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney. Hence, epithelial cell cycle arrest at G2/M and its subsequent downstream signaling are hitherto ! unrecognized therapeutic targets for the prevention of fibrosis and interruption of the accelerated progression of kidney disease.
• Blame your genes, but not your copy numbers
  - Nature Medicine 16(5):526 (2010)
  Numerous studies have pinned single nucleotide polymorphisms (SNPs) to common diseases such as type 1 diabetes and breast cancer. A new study looks at another major source of genetic variation, copy number variants (CNVs)—extra or missing pieces of the genome.
• Revisiting Reproduction: What a difference a gene makes
  - Nature Medicine 16(5):527-529 (2010)
  Nature Medicine | Between Bedside and Bench Revisiting Reproduction: What a difference a gene makes * Bruce D Murphy1 Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Pages:527–529Year published:(2010)DOI:doi:10.1038/nm0510-527 Much can go wrong during the nine-month journey from single cell to birth—with infertility stopping the process as it begins and premature birth completing it before its time. These two major problems in reproductive biology are examined by Bruce D. Murphy, Yasushi Hirota, Jeeyeon Cha and Sudhansu K. Dey. In 'Bench to Bedside', Murphy analyzes studies showing how a single gene, FOXL2, may mediate many processes required for fertility. In 'Bedside to Bench', Dey and colleagues take a look at conflicting clinical findings testing progesterone as a therapy for premature birth: they conclude that much more work needs to be done at the bench, particularly in developing mouse models of parturition, before clinicians can successfully intervene to prevent birth from occurring prematurely. View full text Author information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Affiliations * Bruce D. Murphy is at the Centre de recherche en reproduction animale, Université de Montréal, St-Hyacinthe, Québec, Canada. Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * Bruce D Murphy (bruce.d.murphy@umontreal.ca) Additional data
• Revisiting Reproduction: Prematurity and the puzzle of progesterone resistance
  - Nature Medicine 16(5):529-531 (2010)
  Parturition is a complex and involved process. Within the protected confines of the mother, the fetus grows rapidly for 37–42 weeks until the right combination of signals, stemming from both endocrine and mechanical stimulation, induces parturition, culminating in birth.
• Research Highlights
  - Nature Medicine 16(5):532-533 (2010)
  
• Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury
  Yang L Besschetnova TY Brooks CR Shah JV Bonventre JV - Nature Medicine 16(5):535-543 (2010)
  Nature Medicine | Article Epithelial cell cycle arrest in G2/M mediates kidney fibrosis after injury * Li Yang1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Tatiana Y Besschetnova1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Craig R Brooks1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jagesh V Shah1, 3, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph V Bonventre1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:535–543Year published:(2010)DOI:doi:10.1038/nm.2144Received21 January 2010Accepted05 April 2010Published online02 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Fibrosis is responsible for chronic progressive kidney failure, which is present in a large number of adults in the developed world. It is increasingly appreciated that acute kidney injury (AKI), resulting in aberrant incomplete repair, is a major contributor to chronic fibrotic kidney disease. The mechanism that triggers the fibrogenic response after injury is not well understood. In ischemic, toxic and obstructive models of AKI, we demonstrate a causal association between epithelial cell cycle G2/M arrest and a fibrotic outcome. G2/M-arrested proximal tubular cells activate c-jun NH2-terminal kinase (JNK) signaling, which acts to upregulate profibrotic cytokine production. Treatment with a JNK inhibitor, or bypassing the G2/M arrest by administration of a p53 inhibitor or the removal of the contralateral kidney, rescues fibrosis in the unilateral ischemic injured kidney. Hence, epithelial cell cycle arrest at G2/M and its subsequent downstream signaling are hitherto unreco! gnized therapeutic targets for the prevention of fibrosis and interruption of the accelerated progression of kidney disease. View full text Figures at a glance * Figure 1: Clinical-pathological features of AKI models. () Changes of serum creatinine over time in AKI mouse models, including moderate and severe IRI and AAN (left) and unilateral IRI (UIRI) and UUO (right) models (n = 8 for each time point). *P < 0.001, #P < 0.01 versus day 0 before procedure. () Histology of the fibrotic outcomes of the AKI models (n = 3 mice in each group, Masson's trichrome staining showing fibrosis with blue color). () Sircol assay of kidney collagen content in the five AKI models (n = 3 mice in each group). *P < 0.001 versus control, #P < 0.001 versus moderate IRI. () Immunostaining of collagen IV (left, green (anti–collagen IV)) and α-SMA (right, red (anti–α-SMA)) in AKI models (n = 3 mice in each group). () The percentage of total tissue area that stained positively for collagen IV, collagen I and α-SMA in the kidneys at 42 d (moderate IRI, severe IRI, UIRI and AAN) or 14 d (UUO). *P < 0.001 versus control, #P < 0.001 versus moderate IRI. Scale bars, 50 μm. Error bars represent s.d. * Figure 2: Repair of renal tubular cells in models of AKI. () Number (per 400× field) of Ki-67–positive (left), BrdU-positive (middle) or p-H3–positive (right) tubular cells in moderate IRI, UIRI and AAN mice (n = 3 mice at each time point in each group). *P < 0.01 versus sham, #P < 0.01 versus control. () Cell cycle distribution (G1, S and G2/M) of tubular cells in moderate IRI (left), UIRI (middle) and AAN (right) models as a function of time after the insult (n = 3 mice of each time point in each group). () Percentage of the proliferating (Ki-67–positive) tubular cells that are in the G2/M phase of the cell cycle in the AKI models in moderate IRI, UIRI and AAN (top) and UIRI and UUO models (bottom), showing both the injured kidney and the contralateral kidney (contr). *P < 0.001, #P < 0.01 versus control or sham (n = 3 mice of each time point in each group). () Coimmunostaining with antibodies to Ki-67 (anti–Ki-67) and p-H3 (anti–p-H3) on day-7 kidneys from the moderate and UIRI groups. () Coimmunostaining with antibod! ies to p-H3 and Kim-1 (anti–Kim-1) in UIRI mice. The tubular basement membrane is outlined. () Western blot analysis of cyclin D1 and cyclin B1 in isolated tubules from AKI kidneys (top) and ratio of cyclin B1 to cyclin D1 densities standardized to β-actin (bottom). *P < 0.001. () Percentage of BrdU-positive tubular cells that are in G2/M at various times after BrdU administration in the moderate IRI, AAN and UUO groups. *P < 0.001. () Staining with antibodies to BrdU and p-H3, as well as of nuclei with DAPI, in moderate IRI, AAN and UUO mice that were injected with BrdU at 12 h before killing. Scale bars, 50 μm. Error bars represent s.d. * Figure 3: Profibrogenic factor production in G2/M-arrested proximal tubular cells in vitro and in AKI models in vivo. () Cell cycle analysis by propidium iodide staining and flow cytometry in HK-2, LLC-PK1 or IRPTC cells at baseline (top three graphs) and after treatment with aristolochic acid at 5 μg ml−1 for 48 h (bottom three graphs). (n = 10, n = 3 and n = 3 experiments for the three cell types, respectively.) () Quantification of mRNA levels of profibrogenic genes in HK-2, LLC-PK1 and IRPTC cells treated with aristolochic acid (AA) at 5 μg ml−1 for 48 h, expressed as fold increases over controls (n = 6, n = 3 and n = 3 experiments). *P < 0.001, **P < 0.01 versus control. () TGF-β1 concentration (left) and fold increase in CTGF protein concentrations (right) in the supernatant of HK-2 cells treated with aristolochic acid for 48 h. *P < 0.001, #P < 0.05. () Changes in mRNA levels of profibrogenic factors in cells in various cell cycle phases with or without previous aristolochic acid treatment (n = 6). *P < 0.01, **P < 0.05 versus control G0/G1; #P < 0.01, ##P < 0.05 versus contro! l G2/M. () Effect of conditioned medium from G2/M-arrested HK-2 cells on cell proliferation of serum-starved fibroblasts. *P < 0.05. (n = 3.) () Collagen secretion (left) and collagen IV production (right) in fibroblasts treated with conditioned medium from control or G2/M-arrested HK-2 cells, (n = 3.) *P < 0.01. () mRNA levels of profibrogenic factors as a function of time in AKI models, including moderate IRI, severe IRI, UIRI, AAN and UUO (n = 3 mice per time point in each group). *P < 0.001, **P < 0.01, #P < 0.05 versus control or sham (set arbitrarily to 1). () Western blot analysis of TGF-β1 and CTGF in isolated tubules from moderate IRI and aristolochic acid–treated mice. () Co-staining of CTGF or TGF-β1 with p-H3. Nuclei (N) are outlined in the top image. Scale bar, 10 μm. Error bars represent s.d. * Figure 4: Reversal of G2/M arrest rescues the fibrogenic effect in aristolochic acid-treated HK-2 cells and in the UIRI mouse model. () Number of proximal tubule cells per 600× field that were positive for p-ATM (Ser1981) for all of the models on day 3 (left) and a temporal curve of p-ATM–positive cells in a 400× field for the moderate IRI and AAN insults (right, n = 3 mice per time point in each group). *P < 0.05, **P < 0.01, #P < 0.001 versus control or sham. () Western blot analysis of p-ATM, p-Chk2 (Ser68) and p-p53 (Ser15) in HK-2 cells treated with aristolochic acid (AA). () Effects of KU 55933 (an ATM inhibitor) on cell cycle changes (top) and profibrogenic gene expression (bottom) in HK-2 cells (left), LLC-PK1 cells (middle) and IRPTC cells (right) treated with AA for 48 h (n = 3). *P < 0.01, **P < 0.05 versus control; #P < 0.05 versus AA. () Cell cycle distribution (top) and profibrogenic factor production (bottom) in HK-2 cells and LLC-PK1 cells with or without previous ATM shRNA treatment. (n = 3.) *P < 0.01, #P < 0.05. Symbols in the cell cycle data panels refer to the comparison of G2/M p! hases. () Percentage of proliferating tubular cells in G2/M in kidneys from UIRI mice and UIRI mice that had undergone contralateral nephrectomy (Nx) or PIF-α treatment 10 d after the UIRI. *P < 0.01. () The mRNA levels of whole-kidney Tgfb1, Ctgf, Col4a1 and Col1a1 in UIRI mice with contralateral Nx or PIF-α treatment. *P < 0.01, versus sham; #P < 0.05 versus UIRI. () The effect of Nx or PIF-α treatment on the degree of interstitial fibrosis in the UIRI kidneys (examined 42 d after UIRI injury), as indicated by staining with Masson's trichrome (left) and collagen content determined with the Sircol assay (right). Scale bar, 50 μm. *P < 0.01. Error bars represent s.d. For the mRNA levels in and , the control values were orbitrarily set to 1. * Figure 5: Prolonged G2/M arrest, induced by alternative strategies, causes a profibrotic phenotype both in vitro and in vivo. () Cell cycle changes in HK-2 and LLC-PK1 cells treated with RO3306 (RO) for 48 h. () Cell cycle changes (top) and profibrotic gene expression (bottom) in HK-2 and LLC-PK1 cells treated with RO3306 for 24 h (RO24 h) or 48 h (RO 48 h). (n = 3.) In one group, the RO3306 drug was washed out after 24 h and the cells examined 24 h later (RO 24 h/WO 24 h). **P < 0.001, *P < 0.01, #P < 0.05. () Cell cycle distribution of normal cycling HK-2 cells (control), cells treated with RO3306 for 24 h (RO 24 h), cells treated with RO3306 for 48 h (RO 48 h) and cells treated with RO3306 for 48 h and then collected 6 h after washout of the RO3306 (RO 48 h WO) (top) and mRNA levels of profibrogenic genes: TGFB1, CTGF, COL4A1 and ACTA2 in cells in G0/G1 or G2/M phase (bottom) (n = 3 experiments). *P < 0.001, **P < 0.05 versus control G0/G1; #P < 0.001, ##P < 0.05 versus control G2/M; &P < 0.01 versus RO 48 h G2/M. () Cell cycle changes in HK-2 cells treated with paclitaxel at various doses for 2! 4 h. (n = 3.) *P < 0.001. () The CTGF protein (top western blot) and mRNA levels of TGFB1, CTGF, COL4A1 and COL1A1 (bottom) in HK-2 cells treated with paclitaxel for 24 h. n = 5. *P < 0.001. For mRNA levels in and , control values were arbitrarily set to 1. () The cell cycle changes in proliferating tubular cells in moderate IRI models without (day 1) or with (>day 1) paclitaxel treatment. *P < 0.01, #P < 0.05. Symbols in the cell cycle data graphs refer to the comparison of G2/M phases. () Masson's trichrome staining of moderate IRI kidneys with or without paclitaxel treatment (21 d after injury, n = 3 mice in each group). Scale bar, 50 μm. () The serum creatinine changes in moderate IRI models with or without paclitaxel treatment. Error bars represent s.d. * Figure 6: JNK signaling activation mediates G2/M arrest–induced profibrogenic cytokines upregulation. () Western blot analysis of MAPK pathway activation (top) and the corresponding cell cycle distribution (bottom) of HK-2 cells treated with aristolochic acid, RO3306 or paclitaxel. (n = 3–5.) () Effects of the JNK inhibitor (SP600125, SP) on cell cycle distribution (top) and profibrogenic gene expression (bottom) in HK-2 cells treated with aristolochic acid, RO3306 (RO) or paclitaxel. (n = 3.) mRNA data are presented as fold induction over the untreated cells. *P < 0.05, **P < 0.001. Symbols in the cell cycle graphs refer to the comparison of G2/M phases. () The protein amounts of CTGF in the supernatant from HK-2 cells treated with aristolochic acid or aristolochic acid together with SP600125 (AA + SP) versus untreated cells (CON). Data are presented as fold induction over the control cells (n = 3). *P < 0.001. () The proliferation of fibroblasts incubated with conditioned medium (CM) from HK-2 cells treated with aristolochic acid or from HK-2 cells treated with aristoloc! hic acid together with SP600125. (n = 3.) *P < 0.01. () The collagen content, expressed as percentage change over control, in the supernatant from fibroblasts incubated with CM from aristolochic acid–treated HK-2 cells or from HK-2 cells treated with aristolochic acid together with SP600125 (SP). (n = 3.) *P < 0.01. () Co-localization of p-JNK or p–c-jun with p–histone H3 in AAN kidney on day 7. Scale bar, 10 μm. () Masson's trichrome staining of UIRI kidneys with or without SP600125 treatment on day 28 after UIRI. Scale bar, 50 μm. () Quantitative kidney interstitial fibrosis score (left) and collagen content in the kidneys on day 28, as determined by the Sircol assay (right). *P < 0.001, #P < 0.05. () Effect of JNK inhibition on profibrogenic gene expression in UIRI-treated kidneys. Data are presented as fold induction over the untreated cells. *P < 0.001, #P < 0.05. Error bars represent s.d. Author information * Abstract * Author information * Supplementary information Affiliations * Renal Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA. * Li Yang, * Tatiana Y Besschetnova, * Craig R Brooks, * Jagesh V Shah & * Joseph V Bonventre * Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China. * Li Yang * Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. * Tatiana Y Besschetnova & * Jagesh V Shah * Harvard–Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, Massachusetts, USA. * Jagesh V Shah & * Joseph V Bonventre Contributions L.Y. and J.V.B. designed the experiments and wrote the manuscript. L.Y. performed experiments and collected and analyzed data. J.V.B. supervised the project. J.V.S. designed the in vitro rescue experiment and advised on cell biology. T.Y.B. helped collect data and edited the manuscript. C.R.B. helped with making lentivirus shRNA specific for ATM. All authors discussed the results and implications and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Joseph V Bonventre (joseph_bonventre@hms.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (3M) Supplementary Figures 1–3 Additional data
• Methylation determines fibroblast activation and fibrogenesis in the kidney
  Bechtel W McGoohan S Zeisberg EM Müller GA Kalbacher H Salant DJ Müller CA Kalluri R Zeisberg M - Nature Medicine 16(5):544-550 (2010)
  Nature Medicine | Article Methylation determines fibroblast activation and fibrogenesis in the kidney * Wibke Bechtel1 Search for this author in: * NPG journals * PubMed * Google Scholar * Scott McGoohan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisabeth M Zeisberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gerhard A Müller2 Search for this author in: * NPG journals * PubMed * Google Scholar * Hubert Kalbacher3 Search for this author in: * NPG journals * PubMed * Google Scholar * David J Salant4 Search for this author in: * NPG journals * PubMed * Google Scholar * Claudia A Müller3 Search for this author in: * NPG journals * PubMed * Google Scholar * Raghu Kalluri1 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Zeisberg1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:544–550Year published:(2010)DOI:doi:10.1038/nm.2135Received21 September 2009Accepted12 March 2010Published online25 April 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Fibrogenesis is a pathological wound repair process that fails to cease, even when the initial insult has been removed. Fibroblasts are principal mediators of fibrosis, and fibroblasts from fibrotic tissues fail to return to their quiescent stage, including when cultured in vitro. In a search for underlying molecular mechanisms, we hypothesized that this perpetuation of fibrogenesis is caused by epigenetic modifications. We demonstrate here that hypermethylation of RASAL1, encoding an inhibitor of the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney. RASAL1 hypermethylation is mediated by the methyltransferase Dnmt1 in renal fibrogenesis, and kidney fibrosis is ameliorated in Dnmt1+/− heterozygous mice. These studies demonstrate that epigenetic modifications may provide a molecular basis for perpetuated fibroblast activation and fibrogenesis in the kidney. View full text Figures at a glance * Figure 1: 5′-azacytidine ameliorates experimental renal fibrosis. () Representative photomicrographs of Masson's trichrome–stained kidney sections from mice that received folic acid (FA) (control) or folic acid and 5′-azacytidine (5′-Aza). Mice were analyzed at 0 d, 3 d, 28 d or 147 d after folic acid injection. Scale bar, 200 μm. () Interstitial fibrotic area in Masson's trichrome–stained sections. The graph summarizes average values at the indicated time points of each group. () Average serum creatinine concentrations at the indicated time points. () Representative cfonfocal photomicrographs of immunofluorescence labeling for FSP-1 (top), α-SMA (middle) or type I collagen (bottom) on kidney sections of control CD1 mice (left column), kidneys of mice after folic acid injection (middle column) and from kidneys of mice that had received both folic acid and 5′-azacytidine (right column). Scale bar, 20 μm. (–) Relative stained area in frozen sections that were labeled with antibodies to FSP-1 (), α-SMA () or type I collagen ()! . Data are presented as means ± s.e.m. *P < 0.05. * Figure 2: RASAL1 hypermethylation in fibrotic kidney fibroblasts. () Electrophoresis of PCR products using primers for methylated RASAL1 (top) and unmethylated RASAL1 (bottom). Primary fibroblasts TK188, TK261, TK270, TK274, TK110 and TK257 were isolated from fibrotic human kidneys; TK124, TK173 and TK210 were derived from nonfibrotic human kidneys. () BGS of the indicated cells. Each box is representative of the indicated cell culture; each row of dots in the boxes is representative of the RASAL1 CpG island; each dot is representative of a single CpG. Empty dots indicate unmethylated CpGs; black dots indicate methylated CpGs. Each row represents a single sequenced clone (five for each cell line). We methylated DNA with the bacterial methyltransferase SssI as positive control. () Relative activity of pGL4-luciferase plasmid containing unmethylated or methylated RASAL1 promoter. () Average RASAL1 expression (by qRT-PCR) of each fibroblast culture (normalized to TK173 nonfibrotic fibroblasts set arbitrarily to 1). AU, arbitrary units. () Rep! resentative photomicrographs of Masson's trichrome–stained kidneys of control CD1 mice and of CD1 mice 5 months after folic acid injection. Scale bars, 20 μm. () Average serum creatinine concentrations of control CD1 mice and of mice with renal fibrosis. () Relative Rasal1 expression (qRT-PCR) in kidneys of control CD1 mice and of mice that had received folic acid. Expression in control mice was set to an arbitrary value of 1. () Rasal1 methylation, as analyzed by MeDIP. The top picture shows a virtual gel of Rasal1 PCR products of captured (methylated) DNA; the bottom picture shows Rasal1 PCR products of input DNA (to control for equal loading in immunoprecipitation). () Immunofluorescence double-labeling with antibodies to Rasal1 (red) and FSP-1 (green). The arrow highlights an FSP-1+Rasal1+ fibroblast. Scale bars, 20 μm. () The average number of FSP-1+Rasal1+ cells among all FSP-1+ fibroblasts per group (top) and the number of FSP-1+ fibroblasts per visual field in e! ach group (bottom). Data are presented as means ± s.e.m. ***P! < 0.001. * Figure 3: Absence of Rasal1 hypermethylation in kidneys that do not become fibrotic upon injury. () Representative photomicrographs of periodic acid–Schiff–stained kidney before and 3, 10 and 150 d after initiation of ischemia-reperfusion injury (IRI). Scale bars, 20 μm. () Average histopathological degree of tubular injury over time. () Rasal1 methylation upon ischemia-reperfusion injury, as analyzed by MeDIP. Top, PCR analysis of immunoprecipitated methylated DNA with primers specific to Rasal1. Middle, leucine aminopeptidase (LAP) positive control. Bottom, input DNA control. () Rasal1 methylation, as analyzed in control kidneys, in fibrotic kidneys of mice that had been challenged with folic acid and in kidneys of mice that had received folic acid and had been also treated with 5′-azacytidine. The picture shows a virtual gel of methylated DNA immunoprecipitation analysis. Data are presented as means ± s.e.m. * Figure 4: Rasal1 silencing causes renal fibroblast activation via Ras hyperactivity. () Methylation of Rasal1 in primary fibroblasts isolated from fibrotic and nonfibrotic mouse kidneys, as analyzed by MeDIP. Top, sample analysis; bottom, input DNA control reaction. () Relative Rasal1 expression in control fibroblasts, fibrotic fibroblasts, control fibroblasts transfected with Rasal1-specific siRNA and fibrotic fibroblasts transfected with Rasal1-specific siRNA. () Ras activity of nonfibrotic control fibroblasts, nonfibrotic control fibroblasts transfected with Rasal1-specific siRNA, fibrotic fibroblasts and fibrotic fibroblasts transfected with Rasal1-specific siRNA after 3 d of culture in serum-free medium. () Average cell counts for nonfibrotic control fibroblasts, nonfibrotic control fibroblasts transfected with Rasal1-specific siRNA, fibrotic fibroblasts and fibrotic fibroblasts transfected with Rasal1-specific siRNA at the indicated time points. (,) Relative type I collagen and α-SMA expression, respectively, in each group listed. () Representative ph! otomicrographs of Masson's trichrome–stained kidneys of healthy CD1 mice (control), mice who had received a single injection of folic acid (24 weeks after folic acid injection) and mice who had received treatment with the Ras inhibitor FTS in addition to receiving folic acid. Scale bars, 100 μm. The bar graph summarizes the relative fibrotic area in each group. () Average serum creatinine concentrations at the indicated time points in the indicated groups. () Kidneys of three representative mice in each group were analyzed by MeDIP; the top picture shows amplified methylated Rasal1 DNA, and the bottom picture shows the control input DNA. For , and , control values were arbitrarily set to 1. Data are presented as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. * Figure 5: Dnmts in the mouse model of folic acid–induced nephropathy. () Relative expression of Dnmt1, DNmt3a and Dnmt3b in nonfibrotic and fibrotic mouse kidneys. Expression in FA-treated kidneys was normalized to kidneys from uninjected control mice, with each control value set arbitrarily to 1. () Representative photomicrographs of immunofluorescence labeling with antibodies specific to Dnmt1 (green) and FSP-1 (red). The arrows highlight nuclear DNMT1 staining in FSP-1+ fibroblasts in fibrotic kidneys (bottom). Scale bars, 20 μm. () Dnmt1 protein–Rasal1 promoter complexes were captured with Dnmt1-specific antibodies, detected by Rasal1 PCR and analyzed by electrophoresis. () Representative photomicrographs of Masson's trichrome–stained kidneys from wild-type control mice (top left), wild-type mice that had received folic acid (top right), Dnmt1+/− control mice (bottom left) and Dnmt1+/− mice that had received folic acid (bottom right). Scale bars, 200 μm. () Average fibrotic area in each of the indicated groups. () Average serum c! reatinine concentrations in each of the indicated groups. () MeDIP analysis for Rasal1 (top) and the input control DNA (bottom) of DNA isolated from kidneys of each indicated group. Data are presented as means ± s.e.m. **P < 0.01, ***P < 0.001. * Figure 6: TGF-β1–induced methylation of Rasal1 in kidney fibroblasts is mediated by Dnmt1. () Relative Rasal1 expression over time. () Rasal1 methylation in primary mouse kidney fibroblasts in response to TGF-β1 exposure for 24 h and 5 d, as indicated by the asterisks in panel . We analyzed methylation by methylation-specific PCR. Top, a virtual gel of the PCR with primers specific for methylated Rasal1; bottom, the corresponding result when primers specific for nonmethylated Rasal1. () Immunoblot with antibodies specific for Dnmt1. () Representative photomicrographs of fibroblasts labeled with antibodies to Dnmt1 (green). Fibroblasts were maintained in serum-free medium (control) or exposed to TGF-β1 for 72 h. Arrowheads highlight Dnmt1− nuclei in control fibroblasts, and arrows point to Dnmt1+ nuclei in TGF-β1–treated fibroblasts. () Rasal1 methylation in response to exposure to TGF-β1 (for 5 d) in primary fibroblasts isolated from kidneys of wild-type (WT) C57BL/6 mice and from littermate Dnmt1+/− mice. Top, MeDIP sample analysis; bottom, input DNA co! ntrol reaction. () Rasal1 methylation in control fibroblasts, fibroblasts transfected with Dnmt1-specific siRNA, TGF-β1–stimulated fibroblasts and fibroblasts transfected with Dnmt1-specific siRNA and stimulated with TGF-β1 for 5 d. () Double-immunofluorescence labeling on control mouse kidneys (top) and on kidneys that were made fibrotic by a single injection of folic acid (bottom) using antibodies to Dnmt1 (green) and phosphorylated Smad2 and Smad3 (pSmad2/3, red). Arrows highlight nuclear pSmad2/3 colocalized with nuclear Dnmt1. Scale bars, 200 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA. * Wibke Bechtel, * Scott McGoohan, * Elisabeth M Zeisberg, * Raghu Kalluri & * Michael Zeisberg * Department of Nephrology and Rheumatology, Georg-August-University Medical Center, Göttingen, Germany. * Gerhard A Müller * Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Tübingen, Germany. * Hubert Kalbacher & * Claudia A Müller * Renal Section, Boston University Medical Center, Boston, Massachusetts, USA. * David J Salant Contributions W.B. performed and designed experiments, analyzed data and edited the manuscript. S.M. performed experiments and analyzed data. E.M.Z. advised, performed experiments, analyzed data and edited the manuscript. G.A.M. and C.A.M. characterized and provided human fibroblasts. H.K. generated Rasal1 antibodies. D.J.S. provided nephrotoxic serum and edited the manuscript. R.K. advised and edited the manuscript. M.Z. designed, performed and supervised experiments, analyzed data and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Michael Zeisberg (mzeisber@bidmc.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–9 and Supplementary Tables 1–3 Additional data
• Disruption of antigenic variation is crucial for effective parasite vaccine
  Rivero FD Saura A Prucca CG Carranza PG Torri A Lujan HD - Nature Medicine 16(5):551-557 (2010)
  Nature Medicine | Article Disruption of antigenic variation is crucial for effective parasite vaccine * Fernando D Rivero1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alicia Saura1 Search for this author in: * NPG journals * PubMed * Google Scholar * Cesar G Prucca1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pedro G Carranza1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alessandro Torri1 Search for this author in: * NPG journals * PubMed * Google Scholar * Hugo D Lujan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:551–557Year published:(2010)DOI:doi:10.1038/nm.2141Received30 October 2009Accepted18 March 2010Published online25 April 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Giardia lamblia is a human intestinal pathogen. Like many protozoan microorganisms, Giardia undergoes antigenic variation, a mechanism assumed to allow parasites to evade the host's immune response, producing chronic and/or recurrent infections. Recently, we found that the mechanism controlling variant-specific surface protein (VSP) switching in Giardia involves components of the RNA interference machinery and that disruption of this pathway generates trophozoites simultaneously expressing many VSPs. Here we use these altered trophozoites to determine the role of antigenic variation in a gerbil model of giardiasis. Our results show that either primary infection with trophozoites simultaneously expressing many VSPs or immunization with purified VSPs from the transgenic cells protects gerbils from subsequent Giardia infections. These results constitute, to our knowledge, the first experimental evidence that antigenic variation is essential for parasite survival within hosts an! d that artificial disruption of this mechanism might be useful in generating vaccines against major pathogens that show similar behavior. View full text Figures at a glance * Figure 1: Expression of VSPs in wild-type G. lamblia clones and in G. lamblia with deregulated antigenic variation. (,) Representative direct immunofluorescence confocal images of trophozoites in which Dicer () or RdRP () have been knocked down. Unfixed cells expressing VSP9B10 were labeled with tetramethylrhodamine isothiocyanate (TRITC)-conjugated mAb 9B10 (red), and trophozoites expressing VSP1267 were labeled with FITC-conjugated mAb 5C1 (green). Many trophozoites expressing VSP9B10 on the surface also express VSP1267 (yellow). () Immunofluorescence image of a fixed uncloned population of the WB isolate that was stained with the CRGKA-specific mAb 12F1 (green) and counterstained with DAPI (blue). (–) Representative images of indirect immunofluorescence assays with mAbs specific for VSPs (green) counterstained with DAPI (blue), showing clonal populations of Giardia lamblia WB9B10 (), WB1267 () and GS/M-H7 (). These cells are only labeled by the specific mAb. Scale bars, 20 μm. * Figure 2: Detection and quantification of Giardia cysts in stool samples of gerbils infected with various populations of wild-type and transgenic trophozoites and challenged with WB9B10 and WB1267. Feces from individually housed gerbils were stained with a FITC-labeled mAb 7D2 specific for CWP2 and counted daily over 1 month. (–) Graphs depicting the number of cysts released per gram of feces under different conditions. () Gerbils initially infected with clonal populations of trophozoites WB9B10, WB1267 or transgenic trophozoites in which Dicer (DAS) or RdRP (RAS) were knocked down, as well as with a 1:1 mixture of both. (–) Gerbils previously infected with G. lamblia WB9B10 (), WB1267 (), DAS () and RAS () were challenged 2 months after primary infection with clonal populations of Giardia WB9B10 and WB1267. Figures represent the mean value ± s.d. of five independent experiments. * Figure 3: Serum and intestinal contents of gerbils infected with transgenic trophozoites expressing the full repertoire of VSPs are able to agglutinate different Giardia clones in vitro. Representative images of phase-contrast microscopy of clones WB9B10, WB1267 and GS/M H7 challenged with serum (left) or intestinal content (right). Each group of six images shows trophozoites incubated with serum or intestinal content from uninfected gerbils (top left), serum or intestinal content of gerbils infected with the corresponding clone (top middle), VSP-specific mAb to the corresponding clone (top right), serum or intestinal content of gerbils infected with DAS trophozoites (bottom left), serum or intestinal content of gerbils infected with RAS trophozoites (bottom middle) and serum or intestinal content of gerbils infected with an heterologous clone (bottom right). Sera and intestinal contents from infected or immunized gerbils agglutinate the trophozoites similar to the specific mAbs to VSPs, as compared to the controls. Scale bar, 25 μm. * Figure 4: Detection and quantification of Giardia cysts in stool samples of gerbils previously immunized with VSPs purified from various clonal populations of wild-type and transgenic trophozoites. Feces from individually housed gerbils were stained with FITC-labeled mAb 7D2 to CWP2 and counted daily for 1 month. Gerbils were infected with clonal populations of trophozoites of WB9B10 or WB1267. Immunizations were carried out with VSPs purified from DAS and RAS transgenic trophozoites (and a mixture of both). Graphs depict the number of cysts released per gram of feces under various conditions. (–) Gerbils previously immunized with VSPs purified from DAS (), RAS () or the mixture of both () were challenged with clones WB9B10 or WB1267. (,) Control gerbils () or those immunized with the intracellular protein BiP () were challenged with clones WB9B10 or WB1267. Figures represent the mean value ± s.d. of five independent experiments. * Figure 5: Intestinal morphology of the gerbil's upper small intestine during infection and challenge. (–) Macroscopic examination of upper small intestines from experimental gerbils. () Intestine of a gerbil during primary infection with trophozoites expressing the entire repertoire of the VSPs (DAS) 15 d after inoculation. Arrows point to an increase in size of the Peyer's patches compared to an uninfected gerbil used as control (). () Intestine of a gerbil immunized with purified VSPs obtained from DAS trophozoites. Scale bar, 0.5 cm. (–) Microscopic examination of upper small intestines from experimental gerbils. () Infected gerbil showing a clear enlargement of Peyer's patches and moderate inflammatory infiltrate in mucosa and submucosa. Asterisks depict some Giardia trophozoites at the intestinal lumen. () Control uninfected and unvaccinated gerbil. () Vaccinated gerbil showing histologically normal intestinal mucosa. Scale bars, 100 μm. Insets show a general morphology of the upper small intestine (scale bars, 200 μm). * Figure 6: VSP-specific antibodies react to heterologous isolates. () Sera from infected humans agglutinate cells and recognize the surface of the trophozoites. Sera from five infected humans (1 to 5) and a newborn Giardia-naive individual (6) were directly labeled with FITC and used to test their reactivity to VSPs on transgenic and wild-type trophozoites by indirect immunofluorescence. Results are expressed as percentage of agglutination. Error bars represent s.d. () Sera of infected humans detect many Giardia DAS proteins. Sera from five infected humans (1 to 5), a newborn Giardia-naive individual (C1) and secondary antibody only (C2), were tested for their reactivity to VSPs on DAS trophozoites by western blotting. A differential banding pattern is observed for each infected individual. () Sera from gerbils immunized with purified VSP agglutinate the cells of a new human G. lamblia isolate and recognize the surface of the trophozoites. Sera from randomly selected DAS-, RAS- and DAS + RAS–immunized gerbils and a Giardia-naive gerbil us! ed as control were directly labeled with FITC and used to test their reactivity by indirect immunofluorescence to VSPs on a new G. lamblia isolated axenized from cysts collected from an acutely infected human. The sera from immunized gerbils agglutinate the trophozoites and label the surface of the parasites in contrast to an unimmunized gerbil control. Scale bars, 20 μm. Author information * Abstract * Author information * Supplementary information Affiliations * Laboratory of Biochemistry and Molecular Biology, School of Medicine, Catholic University of Cordoba, Cordoba, Argentina. * Fernando D Rivero, * Alicia Saura, * Cesar G Prucca, * Pedro G Carranza, * Alessandro Torri & * Hugo D Lujan Contributions F.D.R. performed most of the gerbil and in vitro experiments; C.G.P. generated transgenic trophozoites and performed confocal and epifluorescence assays; A.S. and P.G.C. collaborated in gerbil experiments and developed monoclonal antibodies. A.T. collected and evaluated human samples. All authors analyzed the data. H.D.L. supervised the project and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Hugo D Lujan (hlujan@ucc.edu.ar) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–5 and Supplementary Tables 1–7 Additional data
• Memory CD4+ T cells induce innate responses independently of pathogen
  Strutt TM McKinstry KK Dibble JP Winchell C Kuang Y Curtis JD Huston G Dutton RW Swain SL - Nature Medicine 16(5):558-564 (2010)
  Nature Medicine | Article Memory CD4+ T cells induce innate responses independently of pathogen * Tara M Strutt1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * K Kai McKinstry1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * John P Dibble1 Search for this author in: * NPG journals * PubMed * Google Scholar * Caylin Winchell1 Search for this author in: * NPG journals * PubMed * Google Scholar * Yi Kuang1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jonathan D Curtis1 Search for this author in: * NPG journals * PubMed * Google Scholar * Gail Huston1 Search for this author in: * NPG journals * PubMed * Google Scholar * Richard W Dutton1 Search for this author in: * NPG journals * PubMed * Google Scholar * Susan L Swain1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:558–564Year published:(2010)DOI:doi:10.1038/nm.2142Received09 September 2009Accepted19 March 2010Published online02 May 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4+ T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4+ T cell–induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (TH1) or TH17 polarized but are independent of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4+ T cells induce an early innate response that enhances immune protection against pathogens. View full text Figures at a glance * Figure 1: Memory CD4+ T cells induce an acute increase in IICs upon influenza infection. () IIC concentrations 40 h after challenge in naive C57BL/6 mice, or mice primed with influenza A/Phil 60 d before treatment, treated with isotype, CD4- or Thy1.2-depleting antibody before influenza A/PR8 challenge (n = 5 mice per group). () IIC concentrations after bulk CD4+ T cells were isolated from naive or influenza A/PR8–primed mice (polyclonal memory) and equal numbers transferred to naive hosts or, alternatively, naive or in vivo– or in vitro–generated HNT memory cells were adoptively transferred to naive BALB/c hosts. All recipients were challenged with influenza A/PR8 and lung homogenates assessed for IIC after 40 h (n = 5 mice per group). Dotted lines in all figures represent levels of IICs in the absence of infection. Error bars indicate s.d.; *P < 0.05, **P < 0.005 (one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test). * Figure 2: Role of IFN-γ, TNF-α and CCL3 in IIC upregulation by memory CD4+ T cells. () Fold increase in IFN-γ detected in lung homogenate with transfer of TH1-polarized memory versus naive Ifng−/− CD4+ T cells to unprimed mice, on days 2 and 3 after infection with influenza A/PR8 (n = 5 mice per group). () Staining of lung cells for the indicated surface markers to identify IFN-γ–producing cells (EYFP+) after memory OT-II cells transferred to unprimed C57BL/6 Yeti hosts (n = 5 mice per group). NK, natural killer; flu, influenza. (,) Naive or TH1-polarized memory OT-II cells were transferred to WT, Ifngr−/−, Tnfrsf1ab−/− or Ccr5−/− hosts and then infected with influenza A/PR8. () Concentrations of IICs at 40 h after infection. () Viral titer on day 4 after infection (n = 5 mice per d). Error bars indicate s.d.; *P < 0.05, **P < 0.005, ***P < 0.001 (Student's t test). ND, not detected. * Figure 3: TH1- or TH17-polarization is required for enhanced IIC response and viral control. Naive, TH1-, TH17- or TH2-polarized or TH0 unpolarized memory HNT cells were transferred to BALB/c hosts, which were then infected with influenza A/PR8. () Concentrations of IICs in lungs 40 h after infection (n = 5 mice per group). () Pulmonary viral titers (n = 5 mice per group). Error bars indicate s.d.; *P < 0.05, **P < 0.005, ***P < 0.001 (one-way ANOVA followed by Bonferroni's post hoc test). * Figure 4: Recognition of antigen in the lung is sufficient for IIC upregulation. CFSE-labeled Thy-disparate naive or TH1-polarized memory HNT cells were transferred to separate hosts, which were then infected with influenza A/PR8 (n = 5 per group). () Numbers of donor cells in spleen, draining lymph node (DLN) and lung 40 h after infection. () Representative CFSE and CD69 expression of donor cells 2 and 6 d after infection. Naive or memory OT-II cells were transferred to sham-treated or splenectomized Lta−/− hosts and infected with influenza A/PR8-OVAII. () Pulmonary IIC concentrations at 40 h after infection (n = 5 mice per group). () Pulmonary viral titers (n = 5 per day). Error bars indicate s.d.; *P < 0.05, **P < 0.005, ***P < 0.001 (one-way ANOVA followed by Bonferroni's post hoc test or t test). * Figure 5: Cognate recognition of antigen on MHC class II–expressing CD11c+ cells is sufficient to induce IIC upregulation. () The ratio of viral titers on day 4 in naive C57/BL6 mice receiving naive or TH1-polarized OT-II memory cells and then infected with influenza A/PR8 or A/PR8-OVAII. (n = 5 mice per group per virus.) (,) Concentrations of IICs in lung homogenates 40 h after influenza A/PR8-OVAII infection in WT, H2-Ab1−/− or CD11c Tg.H2-Ab1−/− mice receiving TH1-polarized memory OT-II cells (n = 5 mice per group). () and pulmonary viral titers (n = 5 mice per group) (). (,) Number of CD11c+ cells in the lung 40 h after infection in mice receiving naive or memory cells () and () expression of MHC-II, CD40 and CD80 on CD11c+ cells (). () Expression of MHC-II and CD40 on DCs cultured with memory cells. Anti-CD3, CD3-specific antibody. () Cytokines detected in 48-h supernatants of naive or memory HNT cells cultured with DCs. Error bars indicate s.d.; *P < 0.05, **P < 0.005, ***P < 0.001 (one-way ANOVA followed by Bonferroni's post hoc test or t test). * Figure 6: Memory CD4+ T cells induce IIC responses independently of PAMP recognition. (,) Naive or TH1-polarized OT-II memory cells were transferred to naive WT, Ifnar2−/− or Myd88−/−;Ticam1 hosts, which were then infected with influenza A/PR8-OVAII. () Concentrations of IICs detected in lungs 40 h after infection (n = 5 mice per group). () Viral titers. () Concentrations of IICs 40 h after naive or TH1-polarized memory OT-II cells were transferred to C57BL/6 hosts, which were then administered 100 or 10 μg of soluble LPS-free OVA intranasally (i.n.) (n = 5 mice per group). () Memory OT-II cells were transferred to C57/BL6 hosts administered LPS-free OVA as in and infected with A/PR8 on the same day or 7 d later, or only infected with A/PR8, and viral titers determined (n = 5 per group). Error bars indicate s.d.; *P < 0.05, **P < 0.005, ***P < 0.001 (one-way ANOVA followed by Bonferroni's post hoc test or t test). Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Tara M Strutt & * K Kai McKinstry Affiliations * Trudeau Institute, Saranac Lake, New York, USA. * Tara M Strutt, * K Kai McKinstry, * John P Dibble, * Caylin Winchell, * Yi Kuang, * Jonathan D Curtis, * Gail Huston, * Richard W Dutton & * Susan L Swain Contributions T.M.S. and K.K.M. contributed equally to the design, processing, collection and analysis of data and, together with S.L.S., wrote the paper. S.L.S. and R.W.D. contributed to study design. J.P.D., C.W., Y.K., J.D.C. and G.H. processed and collected data. All authors discussed results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tara M Strutt (tms139@mail.usask.ca) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (864K) Supplementary Figures 1–5 Additional data
• Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy
  - Nature Medicine 16(5):565-570 (2010)
  Nature Medicine | Article Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy * Gavin M Bendle1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Carsten Linnemann1, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Anna I Hooijkaas1 Search for this author in: * NPG journals * PubMed * Google Scholar * Laura Bies1 Search for this author in: * NPG journals * PubMed * Google Scholar * Moniek A de Witte1 Search for this author in: * NPG journals * PubMed * Google Scholar * Annelies Jorritsma1 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew D M Kaiser1 Search for this author in: * NPG journals * PubMed * Google Scholar * Nadine Pouw2 Search for this author in: * NPG journals * PubMed * Google Scholar * Reno Debets2 Search for this author in: * NPG journals * PubMed * Google Scholar * Elisa Kieback3 Search for this author in: * NPG journals * PubMed * Google Scholar * Wolfgang Uckert3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ji-Ying Song4 Search for this author in: * NPG journals * PubMed * Google Scholar * John B A G Haanen1, 5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ton N M Schumacher1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:565–570Year published:(2010)DOI:doi:10.1038/nm.2128Received08 January 2010Accepted25 February 2010Published online18 April 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy–induced autoimmune pathology. View full text Figures at a glance * Figure 1: Lethal autoimmune pathology induced by OT-I TCR–modified T cells. () Experimental setup. () Cachexia in recipients of OT-I TCR–transduced (Td) T cells. Shown are pooled results of six independent experiments. Symbols represent individual mice; bars indicate group averages. For non-Td versus OT-I TCR-Td, P < 0.0001. () Kaplan-Meyer survival plot for recipients of OT-I TCR-Td T cells (n = 47; Td efficiency: 53%–72%) or non-Td T cells (n = 33). Shown are pooled results of seven independent experiments. P value of non-Td versus OT-I TCR-Td: P < 0.0001. () Bone marrow sections from recipients of OT-I TCR-Td T cells or non-Td T cells showing the marked disappearance of hematopoietic cells in recipients of OT-I TCR-Td T cells. () Histopathological scoring showing reduction in hematopoiesis in bone marrow and spleen of recipients of OT-I TCR-Td T cells. P values: OT-I TCR-Td versus non-Td bone marrow: P < 0.0001; OT-I TCR-Td versus non-Td spleen: P < 0.0001. () Histopathological scoring showing depletion of lymphocytes in lymph nodes and splee! n of OT-I TCR-Td T cell recipients. P values: OT-I TCR-Td versus non-Td lymph nodes: P < 0.0001; OT-I TCR-Td versus non-Td spleen: P < 0.0001. * Figure 2: TI-GVHD is explained by mixed-TCR dimer formation. () Kaplan-Meyer survival plot for mice receiving OT-I TCR-Td (Td efficiency: 53.17%), OT-I TCR-transgenic, or GFP-Td (Td efficiency: 73.53%) T cells. P values: OT-I Td versus OT-I transgenic: P = 0.0007; OT-I Td versus GFP Td: P = 0.0007. n = 6 mice per group. (,) Lethal TI-GVHD in mice receiving T cells transduced with only the OT-I TCR α- or β-chain. () Characterization of T cells before adoptive transfer. Plots show live-gated CD8+ cells. () Kaplan-Meyer survival plot. P values: OT-I Td versus non-Td: P < 0.0001; OT-I α-Td versus non-Td: P = 0.0004; OT-I β-Td versus non-Td: P = 0.0072. n = 7 mice per group. (,) Depletion of OT-I TCR α-chain Td T cells limits fatal TI-GVHD. () Depletion of myc-tagged OT-I TCR α-chain Td T cells in peripheral blood after tag-specific mAb administration. Flow cytometric data represent mean number of Vα2+CD8+ cells per mouse. Error bars indicate s.e.m. P values: myc-tagged OT-I α-Td and mAb day 7 versus day 14: P < 0.0001; OT-I α-Td ! and mAb day 7 versus day 14: P = 0.006. () Kaplan-Meyer survival plot for mice receiving OT-I TCR α-chain Td T cells (Td efficiency: 65%) or myc-tagged OT-I TCR α-chain Td T cells (Td efficiency: 58%). P values: OT-I α-Td versus non-Td: P = 0.0007; myc-tagged OT-I α-Td versus non-Td: P = 0.0012; OT-I α-Td and mAb versus non-Td: P = 0.0004; myc-tagged OT-I α-Td and mAb versus non-Td: P = 0.05; OT-I α-Td versus myc-tagged OT-I α-Td: P = 0.04; OT-I α-Td versus OT-I α -Td and mAb: P > 0.05; myc-tagged OT-I α-Td versus myc-tagged OT-I α-Td and mAb: P = 0.0006. n = 6 mice per group except for the myc-tagged OT-I α-Td and mAb treated group (n = 7). * Figure 3: IFN-γ−mediated pathogenesis of TI-GVHD. () Increased serum IFN-γ levels on d11 after adoptive cell transfer in recipients of OT-I TCR-Td T cells (n = 5; Td efficiency: 67%) compared to non-Td T cells (n = 6). P value of OT-I TCR-Td versus non-Td: P = 0.0003. Data are representative of two experiments. () Kaplan-Meyer survival plot showing reduced mortality in mice receiving OT-I TCR Td T cells from Ifng−/− donor mice (Td efficiency: 65%) compared to C57BL/6J donor mice (Td efficiency: 69%). P values: OT-I TCR-Td C57BL/6J versus OT-I TCR-Td Ifng−/−: P = 0.0027; OT-I TCR-Td Ifng−/− versus non-Td Ifng−/−: P = 0.4292; OT-I TCR-Td C57BL/6J versus non-Td C57BL/6J: P = 0.0008. Number of mice: non-Td C57BL/6J, n = 6; OT-I TCR-Td C57BL/6J, n = 6; non-Td Ifng−/−, n = 5; OT-I TCR-Td Ifng−/−, n = 8. * Figure 4: TI-GVHD is observed with multiple TCRs. Kaplan-Meyer survival plot showing that lethal TI-GVHD is observed in mice receiving an adoptive transfer of T cells transduced with OT-I (Td efficiency: 53%–56%), SV40IV (Td efficiency: 30%–62%), TRP2 (Td efficiency: 62%–76%), F5 (Td efficiency: 33%–37%) or pmel-1 TCR (Td efficiency: 15%–36%). P values: OT-I Td versus non-Td: P < 0.0001; SV40IV Td versus non-Td: P = 0.0014; TRP2 Td versus non-Td: P = 0.0051; F5 Td versus non-Td: P > 0.05; pmel-1 Td versus non-Td: P > 0.05. Number of mice: non-Td, n = 9; OT-I Td, n = 14; F5 Td, n = 11; TRP2 Td, n = 15; pmel-1 Td, n = 10; SV40IV Td, n = 28. Data represent cumulative results from four independent experiments. * Figure 5: TI-GVHD is observed with different strategies that promote in vivo T cell function. () Kaplan-Meyer survival plot showing lethal TI-GVHD in mice receiving low-dose IL-2 after adoptive transfer of OT-I TCR-Td T cells (Td efficiency: 42%–53%). P value of non-Td versus OT-I TCR-Td: P = 0.005. Shown are pooled results of four independent experiments (OT-I TCR-Td, n = 26; non-Td, n = 22). () Change in body weight at day 13 after adoptive cell transfer of OT-I TCR-Td T cells or non-Td T cells and treatment with low-dose IL-2. Shown are pooled results of four independent experiments. Symbols represent individual mice; bars indicate group averages. P value of non-Td versus OT-I TCR-Td: P > 0.05. (,) TI-GVHD is observed when blockade of TGF-β signaling is used to promote in vivo T cell function. () Characterization of OT-I TCR-Td T cells (Td efficiency: 53%), dnTGFβRII Td T cells, OT-I TCR/dnTGFβRII co-Td T cells (Td efficiency: 46%) and non-Td T cells before adoptive transfer. Dot plots and histograms show live-gated CD8+ cells. () Kaplan-Meyer survival plot. ! P values: OT-I/dnTGFβRII co-Td versus non-Td: P = 0.0006; OT-I/dnTGFβRII co-Td versus dnTGFβRII Td: P < 0.0001; OT-I/dnTGFβRII co-Td versus OT-I Td: P = 0.0002. Number of mice: non-Td, n = 5; OT-I Td, n = 6; dnTGFβRII Td, n = 7; OT-I/dnTGFβRII co-Td, n = 8. * Figure 6: Prevention of TI-GVHD by TCR engineering or by the use of oligoclonal T cell populations. () Kaplan-Meyer survival plot showing that TI-GVHD is prevented by TCR gene transfer into oligoclonal F5 TCR–transgenic T cells. P value: OT-I TCR-Td C57BL/6J versus OT-I TCR-Td F5 Tg: P = 0.0045. Number of mice: non-Td C57BL/6J, n = 4; OT-I TCR-Td C57BL/6J, n = 8 (Td efficiency: 65%); non-Td F5 Tg, n = 5; OT-I TCR-Td F5 Tg, n = 7 (Td efficiency: 55%). () Kaplan-Meyer survival plot showing reduced mortality with an OT-I Cys-TCR-P2A gene expression cassette compared to the OT-I TCR-IRES gene expression cassette. P values: OT-I-IRES Td versus OT-I Cys-TCR-P2A Td: P = 0.0006; OT-I Cys-TCR-P2A Td versus non-Td: P > 0.05. Number of mice: non-Td, n = 11; OT-I-IRES Td, n = 14 (Td efficiency: 42–63%); OT-I Cys-TCR-P2A Td, n = 14 (Td efficiency: 44–73%). () Change in body weight at day 13 after adoptive T cell transfer. Symbols represent individual mice, bars indicate group averages. P values: non-Td versus OT-I-IRES Td: P = 0.0031; OT-I Cys-TCR-P2A Td versus non-Td: P > 0.05; ! OT-I-IRES Td versus OT-I Cys-TCR-P2A Td: P = 0.0004. Data shown in and are cumulative results from three independent experiments. () Kaplan-Meyer survival plot showing that lethal TI-GVHD is prevented by an SV40IV Cys-TCR -P2A gene expression cassette. P value: SV40IV TCR-IRES Td versus SV40IV Cys-TCR-P2A Td: P < 0.0001. Number of mice: non-Td, n = 6; SV40IV TCR-IRES Td, n = 15 (Td efficiency: 30%–51%); SV40IV Cys-TCR-P2A Td, n = 13 (Td efficiency: 37%–41%). Data shown are cumulative results from two independent experiments. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Gavin M Bendle & * Carsten Linnemann Affiliations * Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * Gavin M Bendle, * Carsten Linnemann, * Anna I Hooijkaas, * Laura Bies, * Moniek A de Witte, * Annelies Jorritsma, * Andrew D M Kaiser, * John B A G Haanen & * Ton N M Schumacher * Laboratory of Experimental Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands. * Nadine Pouw & * Reno Debets * Max Delbrück Center for Molecular Medicine, Berlin, Germany. * Elisa Kieback & * Wolfgang Uckert * Division of Experimental Animal Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * Ji-Ying Song * Division of Medical Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. * John B A G Haanen Contributions G.M.B. designed experiments, performed experiments, analyzed and interpreted data and wrote the paper. C.L. designed experiments, performed experiments, analyzed and interpreted data and wrote the paper. A.I.H. designed experiments, performed experiments, analyzed and interpreted data. L.B. performed experiments and analyzed data. M.A.d.W. and A.J. made the initial observation of cachexia and rapid death of mice in mouse models of TCR gene therapy. A.D.M.K. performed experiments and interpreted data. N.P. and R.D. generated and provided the TRP2 TCR sequences. E.K. and W.U. provided the Tag-specific mAb and advice on in vivo depletion. J.-Y.S. performed experiments and analyzed data. J.B.A.G.H. interpreted data. T.N.M.S. designed experiments, interpreted data and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Gavin M Bendle (g.bendle@nki.nl) or * Ton N M Schumacher (t.schumacher@nki.nl) Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Methods Additional data
• Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma
  Lamprecht B Walter K Kreher S Kumar R Hummel M Lenze D Köchert K Bouhlel MA Richter J Soler E Stadhouders R Jöhrens K Wurster KD Callen DF Harte MF Giefing M Barlow R Stein H Anagnostopoulos I Janz M Cockerill PN Siebert R Dörken B Bonifer C Mathas S - Nature Medicine 16(5):571-579 (2010)
  Nature Medicine | Article Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma * Björn Lamprecht1, 2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Korden Walter3, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Kreher1, 2, 10 Search for this author in: * NPG journals * PubMed * Google Scholar * Raman Kumar4 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Hummel5 Search for this author in: * NPG journals * PubMed * Google Scholar * Dido Lenze5 Search for this author in: * NPG journals * PubMed * Google Scholar * Karl Köchert1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Mohamed Amine Bouhlel3 Search for this author in: * NPG journals * PubMed * Google Scholar * Julia Richter6 Search for this author in: * NPG journals * PubMed * Google Scholar * Eric Soler7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ralph Stadhouders7 Search for this author in: * NPG journals * PubMed * Google Scholar * Korinna Jöhrens5 Search for this author in: * NPG journals * PubMed * Google Scholar * Kathrin D Wurster1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * David F Callen4 Search for this author in: * NPG journals * PubMed * Google Scholar * Michael F Harte8 Search for this author in: * NPG journals * PubMed * Google Scholar * Maciej Giefing6, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Rachael Barlow3 Search for this author in: * NPG journals * PubMed * Google Scholar * Harald Stein5 Search for this author in: * NPG journals * PubMed * Google Scholar * Ioannis Anagnostopoulos5 Search for this author in: * NPG journals * PubMed * Google Scholar * Martin Janz1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Peter N Cockerill3 Search for this author in: * NPG journals * PubMed * Google Scholar * Reiner Siebert6 Search for this author in: * NPG journals * PubMed * Google Scholar * Bernd Dörken1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Constanze Bonifer3 Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Mathas1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:571–579Year published:(2010)DOI:doi:10.1038/nm.2129Received16 December 2009Accepted02 March 2010Published online02 May 2010 Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell–derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly.! We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications. View full text Figures at a glance * Figure 1: Lineage-inappropriate expression of CSF1R is required for survival of Hodgkin's lymphoma cells. () Analysis of CSF1R and CSF1 mRNA expression by RT-PCR in Hodgkin's and non-Hodgkin's cell lines. GAPDH expression was analyzed as a control. One representative of four independent experiments is shown. () Analysis of rhCSF-1-induced CSF1R tyrosine phosphorylation in L540Cy cells and of basal levels of CSF1R tyrosine phosphorylation in HRS cell lines following CSF1R immunoprecipitation (IP). IC, isotype control. CSF1R tyrosine phosphorylation was detected by western blotting with an antibody recognizing phosphotyrosine (p-Tyr; top). As a control, the membrane was reprobed with a CSF1R-specific antibody (bottom). One representative of three independent experiments is shown. () CSF1R ISH of a Hodgkin's lymphoma–affected lymph node, using a CSF1R antisense probe (left) or, as a control, a sense probe (right). One representative of ten samples is shown. Arrows indicate HRS cells. () [3H]-thymidine incorporation in L540Cy cells left untreated (−) or treated with the indicate! d amounts of rhCSF-1 or water control under serum-reduced conditions (1% FCS), determined after 48 h. Data of triplicates from one experiment are represented as means ± s.d. One of three experiments is shown. NS, not significant. *P < 0.001. () Growth arrest of KM-H2 cells after CSF-1 inhibition. KM-H2 cells were treated with the IgG1:Fc control or with CSF1R:Fc, as indicated. Cells treated with BSA were included as a control. After 24 h, cells were pulsed with [3H]-thymidine. Data of triplicates from one experiment are represented as means ± s.d. One of three experiments is shown. NS, not significant. *P < 0.001. () Effects of pharmacological inhibition of CSF1R activation on CSF1R tyrosine phosphorylation. L540Cy cells were left untreated (−) or stimulated with rhCSF-1 (+) without or after preincubation with the CSF1R-inhibiting compounds CYC10268, CYC12752 or CYC12200 or a DMSO control. CSF1R tyrosine phosphorylation was analyzed as described in . One representative ! of three independent experiments is shown. () Effects of pharm! acological inhibition of CSF1R activity on apoptosis. HRS (KM-H2, HDLM-2 and L540) and non-Hodgkin's (Reh and Namalwa) cell lines were treated for the indicated times with 2 μM CYC10268. The percentage of viable, annexin V-FITC/PI-negative cells is shown. One of three experiments is shown. * Figure 2: HRS cells express CSF1R from a different promoter than do myeloid cells. () To scan for 5′ ends of CSF1R transcripts, mRNA of HRS (L428, L591, L540Cy, KM-H2), myeloid (HL-60) and non-Hodgkin's (Namalwa) cell lines was amplified by quantitative RT-PCR with primers that anneal in exon 1 (−100 to −14) or 2 (+59 to +148; positions refer to the mature mRNA transcript) or in the upstream region (−169 to −78; −595 to −483; −805 to −713) of the CSF1R gene. mRNA expression is shown relative to TBP. A primer pair spanning +1 to +96 (exon 1-intron) served as a negative control. Data of triplicates from one experiment are represented as means ± s.d. One representative of two independent experiments is shown. Top, a schematic of the CSF1R gene shows the positions of the amplified regions. All numbers are given relative to the translation start site (marked as +1). Dotted line, 5′ upstream region; gray line, 5′ UTR; black bars, exons; dashed line with double slash, intron. ND, not detectable. () Gel analysis of 5′ RACE products after a! mplification by nested PCR with CSF1R-specific primers. Gray arrow, major amplification product in HL-60 cells; black arrow, major product in KM-H2 and L540Cy HRS cells. One representative of three independent experiments is shown. DNA marker, 100-bp DNA ladder; NC, water negative control. Top, a schematic of the 5′ RACE strategy. Arrows represent primer positions. For further description, refer to the legend for . () Schematic summary of the 5′ ends of the CSF1R transcripts. Gray lines represent the 5′ UTRs of CSF1R transcripts. The top and bottom transcripts correspond to those identified in HRS (noncanonical transcript) and HL-60 cells (canonical transcript), respectively. In HRS cells, regions of 975 bp and 4,849 bp are spliced out of the primary transcript. Positions of all primers used in Figures 3,4,5,6 for amplification of CSF1R mRNA transcripts are indicated relative to the CSF1R translation initiation site (primer positions −6,152, −5,090 and −143 refe! r to positions in the CSF1R genomic sequence; primer positions! +148, +720 and +1,325 refer to positions in the mature CSF1R mRNA transcript). () Genomic sequence of the CSF1R LTR and flanking sequences. The LTR region is marked with gray rectangles. The 5′ UTR of the HRS cell lines from the transcription start site (marked as +1) to the first splice site is underlined. Numbers and arrows refer to the luciferase constructs used in Figure 3. E-Box, GATA, Sp1, AP-1 and NF-κB indicate putative transcription factor binding sites; TATA indicates a TATA box. * Figure 3: The noncanonical CSF1R transcript in HRS cells initiates at an aberrantly activated LTR. () DNase I–hypersensitive site (DHS) mapping in the vicinity of the Hodgkin's lymphoma–specific CSF1R TSS. Top, map of the upstream region of the CSF1R gene. The Hodgkin's-specific TSS at position −6,197 bp is marked by a horizontal arrow. Specific restriction sites and their positions are indicated. The positions of DHSs are indicated by heavy arrows, and the position of the hybridization probe is indicated. Bottom, Southern blot analysis of DNase I–treated genomic DNA of various cell lines, as indicated. Permeabilized cells were treated without (−) or with increasing concentrations of DNase I, as indicated. Genomic DNA was digested with KpnI and analyzed by Southern blotting and indirect end-labeling with a hybridization probe abutting the restriction fragment. The arrows on the right indicate DHSs. One of two experiments is shown. () Analysis of luciferase activity of various CSF1R LTR promoter constructs. pGL2 basic (negative control), pGL2 control (positive co! ntrol; contains an SV40 promoter and an SV40 enhancer) or pGL2 LTR promoter constructs (−85 to +14; −142 to +14; −382 to +14; see also Figure 2d) were transfected into L428 and L540Cy cells. Luciferase activity is shown as fold activation compared to pGL2 basic activity (set as 1). Data of triplicates from one experiment are represented as mean ± s.d. One of three experiments is shown. *P < 0.001 for comparison to pGL2 basic. NS, not significant. () Mutational analysis of the LTR −142 to +14 promoter construct. L428 cells were transfected with unchanged or mutated LTR −142 to +14 promoter constructs (mutations of transcription factor binding sites are indicated in the schematic by black crosses), as indicated. Gray rectangles represent binding sites for transcription factors Sp1, GATA, AP-1 and NF-κB. Luciferase activity is shown as described in . One of three experiments is shown. *P < 0.001 for comparison to wild type (WT). () RT-PCR analysis of the expression! of the canonical (+720 to +1,325) and noncanonical (−5,090 ! to +148; −6,152 to −143) CSF1R transcripts in various cell lines and primary CD33-positive cells from five healthy donors (CD33_1 to CD33_5). GAPDH expression was analyzed as a control. One of three experiments is shown. () RT-PCR analysis of the canonical and noncanonical CSF1R transcripts following extraction of total RNA from frozen sections of three lymph nodes affected by Hodgkin's lymphoma (HL_1 to HL_3) and, as controls, sections of nine human tonsils (human tonsils 1 to 9). KM-H2 and Reh cells were included as controls. GAPDH expression was analyzed as a control. One of three experiments is shown. * Figure 4: Analysis of CSF1R LTR DNA methylation and CBFA2T3 expression. () Analysis of DNA methylation of two CpG elements of the CSF1R LTR by bisulfite pyrosequencing in various cell lines, primary lymphoma samples and nonmalignant primary hematopoietic cells. For each sample, the amount of DNA methylation is shown as a percentage for each individual CpG element. B-CLL/IC, B cell chronic lymphocytic leukemia/immunocytoma; DLBCL, diffuse large B cell lymphoma; MCL, mantle cell lymphoma; PBMCs, peripheral blood mononuclear cells. Methyl. control, in vitro–methylated control DNA; Pool pb, pooled DNA from PBMCs. () Analysis of canonical and noncanonical CSF1R transcripts after treatment of Reh and Namalwa cells with TSA and/or 5-aza-dC. The non-Hodgkin's cell lines Reh and Namalwa were left untreated or were treated with 5-aza-dC, TSA or 5-aza-dC and TSA together. The expression of both canonical (+720 to +1,325) and noncanonical (−5,090 to +148 and −6152 to −143) CSF1R transcripts was analyzed by RT-PCR. KM-H2 cells were used as a control.! GAPDH expression was analyzed as a control. As a negative control, water was used instead of cDNA (NC). One of three experiments is shown. () Analysis of expression of CBFA2T3 mRNA by RT-PCR (top) and protein by western blotting (WB; bottom) in various cell lines and CD19-positive human tonsilar B cells. The non-Hodgkin's cell lines and CD19-positive B cells express the two variants CBFA2T3a and CBFA2T3b. GAPDH and β-actin expression were analyzed as controls. n.s., nonspecific. One of four experiments is shown. () Methylation of the CBFA2T3A and CBFA2T3B promoters in HRS cell lines. Overview of the regions analyzed (amplicons) for DNA methylation (top) and results of bisulfite pyrosequencing in lymphoma or leukemia cell lines and controls (bottom). Top, the percentage GC content is shown in five-base windows. The exon-intron structures of CBFA2T3A and CBFA2T3B are diagrammed underneath. Bottom, the percentage of DNA methylation for each individual CpG element in each amp! licon is represented by a color code (red, CpG site fully meth! ylated (100%); green, CpG site fully unmethylated (0%)). Amplicons 1 to 3 correspond to the region of the TSS of CBFA2T3B, and amplicons 4 and 5 to the region of the TSS of CBFA2T3A. As a control, completely methylated DNA was included (Methyl. control). The CBFA2T3 mRNA expression level of the HRS cell lines analyzed is indicated on the far right. The results are shown as the mean of at least five independent experiments. * Figure 5: CBFA2T3 expression is lacking in primary HRS cells, full CSF1R LTR activation requires CBFA2T3 downregulation and active NF-κB and THE1 activation occurs in HRS cells at many genomic locations. () Immunohistochemistry (IHC) to detect CBFA2T3 expression in a germinal center in normal human tonsil tissue (Tonsil), two classical Hodgkin's lymphoma cases (HL; HRS cells are marked by arrows), and one case each of diffuse large B cell lymphoma (DLBCL), B cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL), respectively. Apart from HRS cells, nuclear staining of CBFA2T3 is observed in most cells. The IHC staining shown is representative for the respective lymphoma type. () Analysis of canonical and noncanonical CSF1R transcripts after transfection of Reh cells with shCBFA2T3, IKKβ(EE) or both. Reh cells were left untreated or transfected with a control plasmid (Mock), the shCBFA2T3 or IKKβ(EE) constructs, or both constructs. After enrichment of transfected cells, CSF1R transcripts were analyzed by RT-PCR (top). Protein expression of CBFA2T3 and IKKβ(EE) was detected by use of antibodies to CBFA2T3 or Flag, respectively (middle). CBFA2T3a and CBFA2T! 3b isoforms are not as well separated by gel electrophoresis as in Figure 4c and Supplementary Figure 7, and therefore their corresponding bands are marked as 'CBFA2T3'. GAPDH and β-actin expression were analyzed as controls. IKKβ(EE)-induced NF-κB activity was monitored by electrophoretic mobility shift assay (EMSA) (bottom). Specific NF-κB protein–DNA complexes are marked (NF-κB). SU-DHL-4 and KM-H2 cells were used as controls; in KM-H2 cells, NF-κB is constitutively activated. One of three experiments is shown. n.s., non-specific. () 3′ RACE analysis detecting multiple full-length mRNAs starting from THE1 LTRs in HRS cells. 3′ RACE was performed with forward THE1B primer_2 (Supplementary Fig. 5c) and a reverse primer recognizing a tagging sequence, as depicted in the schematic on the left. In HRS cells, bands of multiple sizes were amplified. 5′ and 3′ ends of ACTB (β-actin) were analyzed by 5′ RACE and 3′ RACE, respectively, as internal controls. As! a negative control, water was used instead of cDNA (NC). One ! of three experiments is shown. () Top, 5′ RACE of LTR RNAs performed by TD-PCR using the reverse THE1B primer_1 in combination with the reverse CSF1R primer_1 (Supplementary Fig. 5c), as depicted in the schematic on the left. The bracket to the right of the gel indicates products that fall within the predicted size range of THE1 products. The upper black arrow indicates an unexpected product that could not be re-amplified by nested PCR. As a control, the relative amounts of amplified GAPDH mRNA per μg RNA for each sample is shown. As negative control, water was used instead of cDNA (NC). Below, nested PCR of 5′ RACE TD-PCR products. Purified DNA fragments migrating within the bracketed region of predicted THE1 products (top gel) were subjected to nested PCR with primer LP25 and the reverse THE1B primer_2. This primer pair is predicted to generate THE1A or THE1B family LTR products of ~97–101 bp. DNA migrating within this size range was purified and subcloned for sequ! encing. As negative control, water was used instead of cDNA (NC). One of two experiments is shown. * Figure 6: LTR-CSF1R transcripts are expressed in anaplastic large cell lymphoma (ALCL) specimens. Total RNA was extracted from frozen lymph node tissue sections of five cases each of ALCL, follicular lymphoma (FL), B-CLL and MCL, and ten cases of DLBCL. Expression of the canonical and noncanonical CSF1R transcripts was analyzed by RT-PCR, as indicated. KM-H2 and Reh cells were used as controls. GAPDH expression was analyzed as a control. One representative of three experiments is shown. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Gene Expression Omnibus * GSE20115 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Björn Lamprecht, * Korden Walter & * Stephan Kreher Affiliations * Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. * Björn Lamprecht, * Stephan Kreher, * Karl Köchert, * Kathrin D Wurster, * Martin Janz, * Bernd Dörken & * Stephan Mathas * Hematology, Oncology and Tumor Immunology, Charité–Universitätsmedizin Berlin, CVK, Berlin, Germany. * Björn Lamprecht, * Stephan Kreher, * Karl Köchert, * Kathrin D Wurster, * Martin Janz, * Bernd Dörken & * Stephan Mathas * Section of Experimental Haematology, Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds, UK. * Korden Walter, * Mohamed Amine Bouhlel, * Rachael Barlow, * Peter N Cockerill & * Constanze Bonifer * Breast Cancer Genetics Group, Discipline of Medicine, University of Adelaide, South Australia, Australia. * Raman Kumar & * David F Callen * Institute of Pathology, Charité–Universitätsmedizin Berlin, CBF, Berlin, Germany. * Michael Hummel, * Dido Lenze, * Korinna Jöhrens, * Harald Stein & * Ioannis Anagnostopoulos * Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. * Julia Richter, * Maciej Giefing & * Reiner Siebert * Erasmus MC, University Medical Center, Department of Cell Biology, Rotterdam, The Netherlands. * Eric Soler & * Ralph Stadhouders * Cytopia Research Pty Ltd, Richmond, Victoria, Australia. * Michael F Harte * Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland. * Maciej Giefing Contributions B.L., K.W. and S.K. designed and performed experiments, interpreted data and contributed to writing of the manuscript; D.F.C., R.K., D.L. and M.F.H. gave technical support and contributed material; M.H., K.J., H.S. and I.A. performed and interpreted IHC and ISH analyses; J.R., M.G. and R. Siebert designed, performed and interpreted bisulfite pyrosequencing and FICTION analyses; M.A.B., K.D.W., R.B., E.S. and R. Stadhouders performed experiments; P.N.C. designed TD-PCR experiments and interpreted data; K.K. analyzed microarray data and performed real-time PCR analyses; M.J. and B.D. interpreted data and contributed to the writing of the manuscript; C.B. and S.M. designed research, interpreted data, wrote the manuscript and supervised the project. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Stephan Mathas (stephan.mathas@charite.de) or * Constanze Bonifer (c.bonifer@leeds.ac.uk) Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–12, Supplementary Tables 1–5 and Supplementary Methods Additional data
• PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2
  Aikawa Y Katsumoto T Zhang P Shima H Shino M Terui K Ito E Ohno H Stanley ER Singh H Tenen DG Kitabayashi I - Nature Medicine 16(5):580-585 (2010)
  Nature Medicine | Letter PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2 * Yukiko Aikawa1 Search for this author in: * NPG journals * PubMed * Google Scholar * Takuo Katsumoto1 Search for this author in: * NPG journals * PubMed * Google Scholar * Pu Zhang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Haruko Shima1 Search for this author in: * NPG journals * PubMed * Google Scholar * Mika Shino1 Search for this author in: * NPG journals * PubMed * Google Scholar * Kiminori Terui3 Search for this author in: * NPG journals * PubMed * Google Scholar * Etsuro Ito3 Search for this author in: * NPG journals * PubMed * Google Scholar * Hiroaki Ohno4 Search for this author in: * NPG journals * PubMed * Google Scholar * E Richard Stanley5 Search for this author in: * NPG journals * PubMed * Google Scholar * Harinder Singh6 Search for this author in: * NPG journals * PubMed * Google Scholar * Daniel G Tenen2, 7 Search for this author in: * NPG journals * PubMed * Google Scholar * Issay Kitabayashi1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:580–585Year published:(2010)DOI:doi:10.1038/nm.2122Received11 January 2010Accepted18 February 2010Published online25 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer1, 2. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony–stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1Rhigh cells), but not those expressing low amounts of CSF1R (CSF1Rlow cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlle! d by the CSF1R promoter, we cured AML by ablation of CSF1Rhigh cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2–induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1Rhigh cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach. View full text Figures at a glance * Figure 1: CSF1Rhigh cells show potent leukemia-initiating activity. () FACS analysis of bone marrow cells from mice with MOZ-TIF2–induced AML for expression of GFP and CSF1R. The red and black boxes signify CSF1Rhigh and CSF1Rlow cell fractions, respectively. () Immunoblot analysis of MOZ-TIF2 expression in CSF1Rhigh and CSF1Rlow cell populations (sorted by flow cytometry) with a MOZ-specific antibody. MW, molecular weight; BM, bone marrow. (,) Leukemia-free survival after the indicated numbers of flow-sorted CSF1Rhigh () and CSF1Rlow () cells were transplanted into sublethally irradiated mice. n = 6, P = 0.0001 (1 × 104, 1 × 103 and 1 × 102) and 0.3173 (1 × 101) (CSF1Rhigh versus CSF1Rlow cells). () FACS analysis of Mac-1 and CSF1R expression in bone marrow cells from mice with MOZ-TIF2–induced AML. The red and blue boxes signify CSF1Rhigh and CSF1Rlow cell fractions, respectively. (–) CSF1Rhigh and CSF1Rlow cells were sorted and analyzed for morphology by staining with May-Giemsa (), colony-forming activity in methylcellulose med! ium () and levels of total and phosphorylated STAT5, phosphorylated ERK and PU.1 (). Scale bars represent 10 μm in . The error bars represent s.d. in . () FACS analysis of CSF1R expression in bone marrow cells from an individual with AML with a t(8;16) translocation; the cells were cultured for 3 d in 10 ng ml−1 human M-CSF. () RT-PCR analysis of MOZ-CBP transcripts in CSF1Rhigh and CSF1Rlow cells of the individual with t(8;16) AML. The results are representative of 25 (,), four (), three (,,–) and two (,) independent experiments. * Figure 2: Cure of AML by ablation of CSF1Rhigh cells. () Top, structure of the CSF1R promoter-EGFP-NGFR-FKBP-Fas suicide construct. Bottom, schematic showing the activation of the NGFR-FKBP-Fas fusion protein: in transgenic mice carrying this suicide construct, ablation of cells expressing high levels of CSF1R can be induced by exposure to the AP20187 dimerizer. () FACS analysis of GFP and CSF1R expression in bone marrow cells of mice with AML 2 months after the transplantation of MSCV-MOZ-TIF2-IRES-GFP–transfected bone marrow cells derived from transgenic mice into lethally irradiated C57BL/6 mice. The red boxes signify CSF1Rhigh and CSF1Rlow cell fractions. (–) Bone marrow cells (1 × 105) of primary transplanted mice with AML, generated as in , were transplanted into sublethally irradiated C57BL/6 mice. Administration of AP20187 or solvent (control) to the secondary transplanted mice was started by intravenous injection 3 weeks after transplantation. Expression of GFP and CSF1R in bone marrow cells () and spleen sizes ()! were analyzed 4 weeks after transplantation. Scale bars, 1 cm. () Leukemia-free survival of the untreated (n = 6) and AP20187-treated (n = 6) secondary transplanted mice. P < 0.0001. The results are representative of five (), four () and three (,) independent experiments. * Figure 3: The requirement for CSF1R in MOZ-TIF2–induced AML. () Leukemia-free survival after fetal liver cells of E16.5 Csf1r+/+ and Csf1r−/− mouse embryo littermates were infected with the MOZ-TIF2-IRES-GFP virus and transplanted into irradiated mice. n = 8, P < 0.0001. () Leukemia-free survival after fetal liver cells of Csf1r+/+ mice were infected with CSF1R− or MOZ-TIF2–encoding viruses, or both, and transplanted into irradiated mice. n = 5. (–) Bone marrow cells (1 × 105) expressing MOZ-TIF2 (,) or N-Myc () from mice with AML were transplanted into irradiated mice. Imatinib mesilate (100 mg per kg body weight) and Ki20227 (20 mg per kg body weight) were administered twice daily. The micrographs depict spleen sizes of the mice transplanted with MOZ-TIF2–expressing cells, analyzed three weeks after transplantation (). Scale bars, 1 cm. (,) Leukemia-free survival of the control and drug-treated mice was analyzed. In , n = 8, P < 0.0001 (control versus + Ki20227 and control versus + imatinib). In , n = 8, P = 0.3825 (con! trol v.s. + Ki20227) and 0.4051 (control versus + imatinib). * Figure 4: PU.1-dependent upregulation of CSF1R by MOZ-fusion proteins. () Schematic diagram indicating protein-interacting domains in MOZ and 1. H15, histone H1- and H5-like domain; PHD, PHD-finger domain; PHD, PHD-finger domain; HAT, histone acetyltransferase catalytic domain; PQ, proline- and glutamine-rich domain; M, methionine-rich domain; DE, aspartic acid– and glutamic acid–rich domain; Q, glutamine-rich domain; PEST, proline-, glutamic acid–, serine- and threonine-rich domain; ETS, Ets DNA-binding domain. () Effects of MOZ, MOZ-CBP and MOZ-TIF2 on AML1- and 1-mediated transcription of the CSF1R promoter. Osteocarcinoma SaOS2 cells were transfected with the CSF1R-luciferase construct and the indicated effector constructs encoding AML1 or 1 together with MOZ, MOZ-CBP or MOZ-TIF2. Luciferase activity was analyzed 24 h after transfection. Error bars represent s.d. *P < 0.01 and **P < 0.005 (comparison to 1 only). The results are representative of six independent experiments in which three samples were tested for each group in each expe! riment. () 1-dependent activation of CSF1R promoter. SaOS2 cells were transfected with the wild-type (WT) CSF1R-luciferase construct or its mutant lacking the 1-binding site (d1), together with the indicated effectors. Error bars represent s.d. *P < 0.01 and **P < 0.005 (comparison to 1 only). The results are representative of three independent experiments in which three samples were tested for each group in each experiment. () FACS analysis of CSF1R expression in PUER cells infected with MSCV-GFP (top) or MSCV-MOZ-TIF2-IRES-GFP (bottom) retroviruses and exposed to 100 nM 4-HT for 0, 2 or 5 d. Population (%) of CSF1Rhigh and CSF1Rlow cells were indicated. The results are representative of three independent experiments. The horizontal lines and the numbers above the graphs indicate CSF1Rhigh (right) and CSF1Rlow (left) cell fractions and their populations (%), respectively. (,) Leukemia-free survival after fetal liver cells of E12.5 Sfpi1+/+ and Sfpi1−/− mouse embryo lit! termates were infected with either MOZ-TIF2− () or N-Myc− ! () encoding viruses and transplanted into irradiated mice. () Leukemia-free survival after fetal liver cells of Sfpi1−/− mice were infected with 1− or MOZ-TIF2–encoding viruses, or both, and transplanted into irradiated mice. In , n = 8, P < 0.0001; in , n = 4, P = 0.0943; in , n = 5, P = 0.0001 (1 + MOZ-TIF2 versus either 1 or MOZ-TIF2). () Fetal liver cells of E14.5 Sfpi1flox/flox ER-Cre mice were infected with the MOZ-TIF2–encoding virus and transplanted into irradiated mice, which developed AML. The bone marrow cells of these mice were then transplanted into sublethally irradiated wild-type mice. Tamoxifen or solvent (control) was administered to the secondary transplanted mice every 2 d by intravenous injection starting 17 d after transplantation, when GFP+ cells were detected in peripheral blood. Leukemia-free survival of the secondary transplanted mice is shown. n = 5, P = 0.0018. () Model for transcriptional regulation by normal and fusion MOZ proteins. MO! Z fusion proteins stimulate constitutive CSF1R expression to induce leukemia (left). Normal MOZ protein controls CSF1R expression by binding to 1 to regulate normal hematopoiesis (right). Author information * Author information * Supplementary information Affiliations * Molecular Oncology Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan. * Yukiko Aikawa, * Takuo Katsumoto, * Haruko Shima, * Mika Shino & * Issay Kitabayashi * Harvard Stem Cell Institute, Boston, Massachusetts, USA. * Pu Zhang & * Daniel G Tenen * Department of Pediatrics, Hirosaki University School of Medicine, Hirosaki, Japan. * Kiminori Terui & * Etsuro Ito * Pharmacological Research Laboratories, Research Division, Kyowa Hakko Kirin, Gunma, Japan. * Hiroaki Ohno * Albert Einstein College of Medicine, Bronx, New York, USA. * E Richard Stanley * Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. * Harinder Singh * Cancer Science Institute, National University of Singapore, Singapore. * Daniel G Tenen Contributions Y.A., I.K., T.K. and M.S. conducted experiments in AML mice. Y.A., H. Shima and I.K. performed western blotting, immunoprecipitation, GST pull down, ChIP and reporter assays. P.Z. and D.G.T. conducted experiments in PU.1-deficient mice. E.R.S. designed and performed experiments in CSF1R-deficient mice. K.T. and E.I. analyzed expression of CSF1R in human AML cells. H. Singh designed and performed experiments in PUER cells. H.O. prepared Ki20227. I.K. and Y.A. analyzed data and edited the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Issay Kitabayashi (ikitabay@ncc.go.jp) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (4M) Supplementary Figures 1–12 Additional data
• A CD8+ T cell transcription signature predicts prognosis in autoimmune disease
  - Nature Medicine 16(5):586-591 (2010)
  Nature Medicine | Letter A CD8+ T cell transcription signature predicts prognosis in autoimmune disease * Eoin F McKinney1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul A Lyons1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Edward J Carr1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jane L Hollis2 Search for this author in: * NPG journals * PubMed * Google Scholar * David R W Jayne2 Search for this author in: * NPG journals * PubMed * Google Scholar * Lisa C Willcocks1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Koukoulaki1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Alvis Brazma3 Search for this author in: * NPG journals * PubMed * Google Scholar * Vojislav Jovanovic4 Search for this author in: * NPG journals * PubMed * Google Scholar * D Michael Kemeny4 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J Pollard5 Search for this author in: * NPG journals * PubMed * Google Scholar * Paul A MacAry4 Search for this author in: * NPG journals * PubMed * Google Scholar * Afzal N Chaudhry2 Search for this author in: * NPG journals * PubMed * Google Scholar * Kenneth G C Smith1, 2 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:586–591Year published:(2010)DOI:doi:10.1038/nm.2130Received08 July 2009Accepted05 March 2010Published online18 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of potentially toxic immunosuppressive therapy. Gene expression–based biomarkers facilitating such tailoring of chemotherapy in cancer, but not autoimmunity, have been identified and translated into clinical practice1, 2. We show that transcriptional profiling of purified CD8+ T cells, which avoids the confounding influences of unseparated cells3, 4, identifies two distinct subject subgroups predicting long-term prognosis in two autoimmune diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a chronic, severe disease characterized by inflammation of medium-sized and small blood vessels5, and systemic lupus erythematosus (SLE), characterized by autoantibodies, immune complex deposition and diverse clinical manifestations ranging from glomerulonephritis to neurological dysfunction6. We show t! hat the subset of genes defining the poor prognostic group is enriched for genes involved in the interleukin-7 receptor (IL-7R) pathway and T cell receptor (TCR) signaling and those expressed by memory T cells. Furthermore, the poor prognostic group is associated with an expanded CD8+ T cell memory population. These subgroups, which are also found in the normal population and can be identified by measuring expression of only three genes, raise the prospect of individualized therapy and suggest new potential therapeutic targets in autoimmunity. View full text Figures at a glance * Figure 1: T cell gene expression identifies a previously unrecognized subgroup of subjects with AAV at increased risk of relapsing disease. () Supervised hierarchical clustering of expression data from 925 genes with a more than twofold difference in expression between the subgroups defined in Supplementary Figure 1 (false discovery rate (FDR); P < 0.05) clusters the initial cohort of individuals with AAV (v8.1i, n = 32) into two distinct subgroups. () Hierarchical clustering of an independent validation cohort of subjects with AAV (v8.1v, n = 27) using expression data for the same gene list defined in , also identifying two distinct subgroups. Hierarchical clustering was performed by uncentered correlation and average linkage. () Survival curve showing shorter time to first flare in group v8.1 after induction therapy. Flare-free survival is represented as the proportion of all individuals reaching each time point, with the number of people remaining at risk within each cohort detailed at the bottom. Significance was measured with the log-rank test. () Flare frequency during follow up out to 1,000 d. Flare rate ! normalized to duration of follow up performed at day 500 (mean flare rate 0.91 per year for group v8.1 versus 0.17 per year for group v8.2, P = 0.01) and at day 1,000 (mean flare rate 0.81 per year for group v8.1 versus 0.15 per year for group v8.2, P = 0.004). * Figure 2: The CD8+ T cell signature that predicts prognosis in AAV defines analogous subgroups in SLE. Consensus subgroups in a cohort of 26 subjects with SLE were defined by unsupervised hierarchical clustering. () Supervised hierarchical clustering of the SLE cohort using 1,913 genes that best define the subgroups (more than twofold statistically significant differential expression, FDR P < 0.05). One subject (black asterisks) underwent repeat analysis at the time of a second disease flare 14 months after enrollment and remained in subgroup s8.1 despite changes in therapy. () Hierarchical clustering of AAV samples using expression data for the same genes as in . Gene lists defining the two subgroups in vasculitis (n = 1,228) and SLE (1,913) cohorts were highly overlapping (overlap of 639 genes, hypergeometric P < 1 × 10−300). () Kaplan-Meier plot showing shorter time to first flare in group s8.1. Significance was measured with the log-rank test. () Flare frequency in SLE subgroups when followed out to 1,000 d. Red asterisk, mean flare rate normalized per unit time of fol! low up with Mann-Whitney U test of significance performed at day 500 (flare rate of 0.86 per year in s8.1 versus 0.08 per year in s8.2, P = 0.0006) and at day 1,000 (flare rate of 0.81 per year in s8.1 versus 0.09 per year in s8.2, P = 0.001). * Figure 3: Similar subgroups can be identified in a healthy population, and the defining signature is composed of genes whose expression predominantly conforms to a bimodal distribution. Consensus subgroups in a cohort of 22 healthy European control subjects, age- and sex-matched to the AAV cohort, were defined by unsupervised hierarchical clustering as for the disease cohorts. () Hierarchical clustering of the 944 genes best defining the subgroups (more than twofold statistically significant differential expression, FDR P < 0.05). () Unsupervised hierarchical clustering of a pooled CD8+ T cell expression data set including all samples (AAV, SLE and control). (,) Hierarchical clustering of CD8+ T cell expression data from a cohort of 18 healthy European () and 25 Singaporean () control subjects derived from the Affymetrix Gene 1.0 ST Array platform. The 780 genes used represent the subset of genes from that could be mapped onto Affymetrix feature names using common Entrez identification numbers. Unsupervised de novo clustering produced the same subgroups (data not shown). () Matched expression density distributions for genes differentially expressed (FDR P
• Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions
  Xu ZZ Zhang L Liu T Park JY Berta T Yang R Serhan CN Ji RR - Nature Medicine 16(5):592-597 (2010)
  Nature Medicine | Letter Resolvins RvE1 and RvD1 attenuate inflammatory pain via central and peripheral actions * Zhen-Zhong Xu1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ling Zhang1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Tong Liu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jong Yeon Park1 Search for this author in: * NPG journals * PubMed * Google Scholar * Temugin Berta1 Search for this author in: * NPG journals * PubMed * Google Scholar * Rong Yang2 Search for this author in: * NPG journals * PubMed * Google Scholar * Charles N Serhan2, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Ru-Rong Ji1, 3 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:592–597Year published:(2010)DOI:doi:10.1038/nm.2123Received21 December 2009Accepted19 February 2010Published online11 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Inflammatory pain, such as arthritis pain, is a growing health problem1. Inflammatory pain is generally treated with opioids and cyclooxygenase (COX) inhibitors, but both are limited by side effects. Recently, resolvins, a unique family of lipid mediators, including RvE1 and RvD1 derived from omega-3 polyunsaturated fatty acid, have shown marked potency in treating disease conditions associated with inflammation2, 3. Here we report that peripheral (intraplantar) or spinal (intrathecal) administration of RvE1 or RvD1 in mice potently reduces inflammatory pain behaviors induced by intraplantar injection of formalin, carrageenan or complete Freund's adjuvant (CFA), without affecting basal pain perception. Intrathecal RvE1 injection also inhibits spontaneous pain and heat and mechanical hypersensitivity evoked by intrathecal capsaicin and tumor necrosis factor-α (TNF-α). RvE1 has anti-inflammatory activity by reducing neutrophil infiltration, paw edema and proinflammatory cyto! kine expression. RvE1 also abolishes transient receptor potential vanilloid subtype-1 (TRPV1)- and TNF-α–induced excitatory postsynaptic current increases and TNF-α–evoked N-methyl-D-aspartic acid (NMDA) receptor hyperactivity in spinal dorsal horn neurons via inhibition of the extracellular signal–regulated kinase (ERK) signaling pathway. Thus, we show a previously unknown role for resolvins in normalizing the spinal synaptic plasticity that has been implicated in generating pain hypersensitivity. Given the potency of resolvins and the well-known side effects of opioids and COX inhibitors, resolvins may represent new analgesics for treating inflammatory pain. View full text Figures at a glance * Figure 1: Preemptive spinal (intrathecal) administration of RvE1 reduces the second phase of formalin-induced inflammatory pain. (,) Reduction of the second phase of formalin-induced spontaneous pain by RvE1, morphine and the COX-2 inhibitor NS-398. () Time course. *P < 0.05 (vehicle versus RvE1). () First and second phase. *P < 0.05 versus vehicle, n = 5–8 mice. () Dose-response curve of percentage inhibition (versus vehicle control) of RvE1, morphine and NS-398 on the second phase of formalin-induced pain. n = 5–8 mice. () Reversal of RvE1-mediated inhibition of second phase pain by pertussis toxin (PTX) but not naloxone. NS, not significant. *P < 0.05, n = 5–7 mice. () Dose-dependent reduction of second-phase pain by the ChemR23 agonist chemerin. *P < 0.05 versus vehicle, n = 6 mice. () Expression of ChemR23 mRNA in the DRG and spinal cord dorsal horn, as revealed by in situ hybridization. Scale bars, 50 μm. () Colocalization of ChemR23 with TRPV1 in a cultured DRG neuron (top) and with NeuN in the superficial dorsal horn (bottom), as demonstrated by double immunostaining. Scale bars, 25 μm! . All data are means ± s.e.m. * Figure 2: Central and peripheral actions of resolvins on persistent inflammatory pain and inflammation. (–) Effects of intrathecal administration of resolvins on day 3 after CFA treatment on CFA-evoked heat hyperalgesia. () Development of heat hyperalgesia 3 d after CFA injection. () Acute effects (15–45 min) of RvE1, RvD1, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). () Persistent effects (1–6 h) of RvE1, NS-398 and 19-pf-RvE1. () Lack of effect of RvE1 on basal pain thresholds in naive mice. PWL, paw withdrawal latency; MPE, maximum possible antihyperalgesic effect. *P < 0.05 versus baseline (BL, ) or vehicle (–); #P < 0.05, n = 4–7 mice. (–) Quantification of carrageenan (CRG)-elicited heat hyperalgesia (), paw edema (), neutrophil infiltration () and expression of proinflammatory cytokines and chemokines () in the inflamed paw after intraplantar pretreatment of resolvins. Edema, neutrophil infiltration and cytokine expression at the protein level were examined by paw volume (), myeloperoxidase (MPO) activity () and cytokine array (), respectively! , at 4 h (,) or 2 h () after carrageenan injection. *P < 0.05 versus vehicle (,) or naive (,), #P < 0.05, versus CRG (), n = 3–6 mice. All data are means ± s.e.m. * Figure 3: Spinal administration of RvE1 reduces heat hyperalgesia and spontaneous pain by blocking TRPV1 and TNF-α signaling in DRG neurons and spinal presynaptic terminals. () CFA-induced heat hyperalgesia and formalin-induced second-phase pain in wild-type and Tnfrsf1a−/−;Tnfrsf1b−/− mice. () TNF-α–induced heat hyperalgesia and formalin-induced second-phase spontaneous pain in wild-type and Trpv1−/− mice. () Prevention of TNF-α–induced heat hyperalgesia by RvE1. *P < 0.05 versus wild-type control (,) or vehicle (), n = 4–6 mice. () Effects of RvE1 and capsazepine (CZP, 10 μM) on TNF-α–induced increase in sEPSC frequency in spinal cord lamina II neurons. Bottom, quantification of sEPSC frequency and amplitude. () Effects of RvE1, PTX (0.5 μg ml−1), and MEK inhibitor PD98059 and U0126 (1 μM) on capsaicin-induced increase in sEPSC frequency in lamina II neurons. Bottom, quantification of sEPSC frequency and amplitude. *P < 0.05 versus baseline; #P < 0.05 versus TNF-α () or capsaicin (); $P < 0.05; n = 5–10 neurons. () Spontaneous pain induced by intrathecal capsaicin and its prevention by RvE1. *P < 0.05 versus RvE1! , n = 6 mice. () ERK phosphorylation in cultured DRG neurons after TNF-α and capsaicin with or without RvE1. Scale bar, 100 μm. Bottom, percentage of pERK-positive neurons in DRG cultures. *P < 0.05, n = 4 cultures from separate mice. () Schematic of RvE1-induced inhibition of inflammatory pain (heat hyperalgesia) via presynaptic mechanisms. * Figure 4: Spinal RvE1 administration attenuates mechanical allodynia and blocks TNF-α signaling in postsynaptic dorsal horn neurons. () Mechanical allodynia after intrathecal TNF-α and reduction of the allodynia by RvE1 pretreatment. *P < 0.05 versus vehicle control, n = 5 mice. () TNF-α–induced mechanical allodynia in wild-type and Trpv1−/− mice. n = 5 mice. () NMDA-evoked currents in lamina II neurons by TNF-α and effects of RvE1 on the currents. *P < 0.05 versus baseline, #P < 0.05, n = 6 neurons. () ERK phosphorylation in superficial dorsal horn neurons of spinal cord slices after perfusion of TNF-α and effects of RvE1 on the phosphorylation. White line, border of the dorsal horn gray matter. *P < 0.05, n = 4 slices from separate mice. Scale bar, 50 μm. () Effects of the MEK inhibitors PD98059 (1 μM) and U0126 (1 μM) and capsazepine (CZP, 10 μM) on NMDA-evoked currents after TNF-α stimulation. *P < 0.05 versus corresponding baseline; #P < 0.05, versus TNF-α; n = 5–10 neurons. () Schematic of RvE1-induced inhibition of inflammatory pain (mechanical allodynia) via postsynaptic mechanis! ms. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Zhen-Zhong Xu, * Ling Zhang, * Charles N Serhan & * Ru-Rong Ji Affiliations * Sensory Plasticity Laboratory, Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Zhen-Zhong Xu, * Ling Zhang, * Tong Liu, * Jong Yeon Park, * Temugin Berta & * Ru-Rong Ji * Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA. * Rong Yang & * Charles N Serhan Contributions R.-R.J. and C.N.S. formulated the hypotheses, designed and supervised the project and prepared the manuscript; R.-R.J. designed most experiments; Z.-Z.X., T.L. and J.Y.P. conducted behavioral studies; L.Z. performed electrophysiological studies; Z.-Z.X. and L.Z. performed immunohistochemistry; Z.-Z.X. performed siRNA-mediated knockdown, cytokine arrays and western blotting; T.B. performed in situ hybridization; R.Y. prepared resolvins and their analogs. Competing financial interests Resolvins are biotemplates for stable analogs. Patents on these are awarded and assigned to the Brigham and Women's Hospital, and C.N.S. is the inventor. These patents are licensed for clinical development. Corresponding authors Correspondence to: * Ru-Rong Ji (rrji@zeus.bwh.harvard.edu) or * Charles N Serhan (cnserhan@zeus.bwh.harvard.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (812K) Supplementary Figures 1–7 and Supplementary Methods Additional data
• Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive–like behaviors in mice
  Shmelkov SV Hormigo A Jing D Proenca CC Bath KG Milde T Shmelkov E Kushner JS Baljevic M Dincheva I Murphy AJ Valenzuela DM Gale NW Yancopoulos GD Ninan I Lee FS Rafii S - Nature Medicine 16(5):598-602 (2010)
  Nature Medicine | Letter Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive–like behaviors in mice * Sergey V Shmelkov1, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Adília Hormigo1, 2, 3, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Deqiang Jing4, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Catia C Proenca4, 5, 9 Search for this author in: * NPG journals * PubMed * Google Scholar * Kevin G Bath4 Search for this author in: * NPG journals * PubMed * Google Scholar * Till Milde1 Search for this author in: * NPG journals * PubMed * Google Scholar * Evgeny Shmelkov1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jared S Kushner1 Search for this author in: * NPG journals * PubMed * Google Scholar * Muhamed Baljevic1 Search for this author in: * NPG journals * PubMed * Google Scholar * Iva Dincheva4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew J Murphy7 Search for this author in: * NPG journals * PubMed * Google Scholar * David M Valenzuela7 Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas W Gale7 Search for this author in: * NPG journals * PubMed * Google Scholar * George D Yancopoulos7 Search for this author in: * NPG journals * PubMed * Google Scholar * Ipe Ninan8 Search for this author in: * NPG journals * PubMed * Google Scholar * Francis S Lee4, 6 Search for this author in: * NPG journals * PubMed * Google Scholar * Shahin Rafii1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorsJournal name:Nature MedicineVolume:16,Pages:598–602Year published:(2010)DOI:doi:10.1038/nm.2125Received29 October 2009Accepted22 February 2010Published online25 April 2010 Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions, and it often encompasses anxiety and depressive symptoms1, 2. Recently, the corticostriatal circuitry has been implicated in the pathogenesis of OCD3, 4. However, the etiology, pathophysiology and molecular basis of OCD remain unknown. Several studies indicate that the pathogenesis of OCD has a genetic component5, 6, 7, 8. Here we demonstrate that loss of a neuron-specific transmembrane protein, SLIT and NTRK-like protein-5 (Slitrk5), leads to OCD-like behaviors in mice, which manifests as excessive self-grooming and increased anxiety-like behaviors, and is alleviated by the selective serotonin reuptake inhibitor fluoxetine. Slitrk5−/− mice show selective overactivation of the orbitofrontal cortex, abnormalities in striatal anatomy and cell morphology and alterations in glutamate receptor composition, which contribute to de! ficient corticostriatal neurotransmission. Thus, our studies identify Slitrk5 as an essential molecule at corticostriatal synapses and provide a new mouse model of OCD-like behaviors. View full text Figures at a glance * Figure 1: Targeted inactivation of Slitrk5 in mice and its expression pattern in the mouse brain. () Genomic structure and the design of the Slitrk5-knockout, lacZ–knock-in mouse. The entire open reading frame (ORF) is localized to exon 2 (Ex2); exon 1 (Ex1) is noncoding. The Slitrk5-encoding region was replaced with lacZ downstream of the signal sequence cleavage site. WT, wild-type; KO, knockout. () X-gal staining of mouse brain tissue, showing ubiquitous expression of lacZ in the gray matter of the various parts of the brain, including cortex and striatum. Cx, cortex; St, striatum; Hp, hippocampus; cc, corpus callosum; Th, thalamus; Cbl, cerebellum. The higher magnification image shows the distribution of lacZ-expressing cells in the striatum of the Slitrk5-knockout, lacZ–knock-in mouse. () Immunostaining of cortex and striatum with antibodies to β-galactosidase (anti–β-gal) and NeuN (anti-NeuN), indicating that the majority of neurons express Slitrk5. * Figure 2: Facial lesions, OCD-like behavior and its alleviation with fluoxetine treatment in Slitrk5-knockout mice. () Phenotypic characteristic of Slitrk5−/− mice: excessive grooming leads to severe facial lesions. () Time spent grooming in Slitrk5−/− mice (n = 9) compared to their wild-type littermates (n = 8) before and after treatment with fluoxetine. Error bars depict the s.e.m. () Anxiety-related behavior of Slitrk5−/− and WT mice in the open-field test. Percentage of time spent in the center and entries into the center of the open field are shown. All open-field results are presented as means ± s.e.m. determined from analysis of 20 mice per genotype. * Figure 3: Metabolic changes in the cortex and anatomical defects in the striatum of Slitrk5−/− mice. () Expression of FosB in orbitofrontal cortex by immunostaining for FosB (red) and with DAPI (blue). The top images show the distribution of FosB expression in the various layers of orbitofrontal cortex. The bottom images show a higher magnification of layer II of FosB immunoreactivity in nuclei. () Quantification of FosB expression in all layers of the orbitofrontal cortex. () Cavalieri estimation of striatal volume in Slitrk5−/− and WT mice. () Examples of Golgi staining and Neurolucida reconstruction of striatal medium spiny neurons in WT and Slitrk5−/− mice. () Sholl analysis of striatal medium spiny neurons in WT and Slitrk5−/− mice. All results are presented as means ± s.e.m.; 40 neurons per genotype. () Fractal dimension analysis of striatal medium spiny neurons in Slitrk5−/− and WT mice. All results are presented as means ± s.e.m.; 40 neurons per genotype. * Figure 4: Deficiency in corticostriatal transmission in Slitrk5−/− mice is mediated by changes in glutamate receptor composition. () Immunostaining of primary striatal rat neurons (infected with Flag-Slitrk5 lentivirus and transfected with PSD95 fused to mCherry (PSD95-cherry)) in culture with cortical neurons (isolated from transgenic mice that ubiquitously express green fluorescent protein) with Flag-specific antibody (anti-Flag). The arrow points to a magnified area (bottom images) that represents the synapses between cortical and striatal neurons. () Western blot analysis of NMDA and AMPA receptor subunits in the striatum of 5-month-old Slitrk5−/− and WT mice. The protein amounts are adjusted to the expression of actin. () Population spike amplitude in Slitrk5−/− mice (n = 11, from four mice) and matched WT mice (n = 9, from four mice). The population spike amplitude is significantly lower in Slitrk5−/− mice, P < 0.01, repeated-measures analysis of variance. The inset shows examples of corticostriatal population spike amplitudes in Slitrk5−/− mice and matched WT mice. () Average pai! red-pulse ratios of the population spike in Slitrk5−/− mice (n = 17, from five mice) and matched WT mice (n = 17, from five mice). There is no significant difference in the paired-pulse ratio between Slitrk5−/− mice and wild-type mice. Author information * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sergey V Shmelkov, * Adília Hormigo, * Deqiang Jing & * Catia C Proenca Affiliations * Howard Hughes Medical Institute, Ansary Stem Cell Institute and Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, USA. * Sergey V Shmelkov, * Adília Hormigo, * Till Milde, * Evgeny Shmelkov, * Jared S Kushner, * Muhamed Baljevic & * Shahin Rafii * Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Adília Hormigo * Department of Neurology, Weill Cornell Medical College, New York, New York, USA. * Adília Hormigo * Department of Psychiatry, Weill Cornell Medical College, New York, New York, USA. * Deqiang Jing, * Catia C Proenca, * Kevin G Bath, * Iva Dincheva & * Francis S Lee * Gulbenkian PhD Programe in Biomedicine, Instituto Gulbenkian de Ciência, Oeiras, Portugal. * Catia C Proenca * Department of Pharmacology, Weill Cornell Medical College, New York, New York, USA. * Iva Dincheva & * Francis S Lee * Regeneron Pharmaceuticals, Tarrytown, New York, USA. * Andrew J Murphy, * David M Valenzuela, * Nicholas W Gale & * George D Yancopoulos * Department of Psychiatry, New York University Langone Medical Center, New York, New York, USA. * Ipe Ninan Contributions S.V.S. conceived of and designed the study, performed experiments, analyzed data and wrote the manuscript; A.H., D.J, C.C.P. and K.G.B. designed and performed experiments, analyzed data and assisted in writing the manuscript; T.M., E.S., J.S.K., M.B. and I.D. performed experiments and analyzed data; A.J.M., D.M.V., N.W.G. and G.D.Y. designed and generated the Slitrk5−/− mice; I.N. designed, performed and analyzed electrophysiology experiments; F.S.L. and S.R. conceived of and designed the study and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Francis S Lee (fslee@med.cornell.edu) or * Shahin Rafii (srafii@med.cornell.edu) Supplementary information * Author information * Supplementary information PDF files * Supplementary Text and Figures (856K) Supplementary Figures 1–9 and Supplementary Methods Additional data
• Multispectral scanning during endoscopy guides biopsy of dysplasia in Barrett's esophagus
  Qiu L Pleskow DK Chuttani R Vitkin E Leyden J Ozden N Itani S Guo L Sacks A Goldsmith JD Modell MD Hanlon EB Itzkan I Perelman LT - Nature Medicine 16(5):603-606 (2010)
  Nature Medicine | Technical Report Multispectral scanning during endoscopy guides biopsy of dysplasia in Barrett's esophagus * Le Qiu1 Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas K Pleskow2 Search for this author in: * NPG journals * PubMed * Google Scholar * Ram Chuttani2 Search for this author in: * NPG journals * PubMed * Google Scholar * Edward Vitkin1 Search for this author in: * NPG journals * PubMed * Google Scholar * Jan Leyden2 Search for this author in: * NPG journals * PubMed * Google Scholar * Nuri Ozden2 Search for this author in: * NPG journals * PubMed * Google Scholar * Sara Itani1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lianyu Guo1 Search for this author in: * NPG journals * PubMed * Google Scholar * Alana Sacks2 Search for this author in: * NPG journals * PubMed * Google Scholar * Jeffrey D Goldsmith3 Search for this author in: * NPG journals * PubMed * Google Scholar * Mark D Modell1 Search for this author in: * NPG journals * PubMed * Google Scholar * Eugene B Hanlon1, 4 Search for this author in: * NPG journals * PubMed * Google Scholar * Irving Itzkan1 Search for this author in: * NPG journals * PubMed * Google Scholar * Lev T Perelman1 Search for this author in: * NPG journals * PubMed * Google Scholar * Affiliations * Contributions * Corresponding authorJournal name:Nature MedicineVolume:16,Pages:603–606Year published:(2010)DOI:doi:10.1038/nm.2138Received08 July 2009Accepted11 December 2009Published online11 April 2010 Abstract * Abstract * Author information * Supplementary information Article tools * Full text * 日本語要約 * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Esophageal cancer is increasing in frequency in the United States faster than any other cancer. Barrett's esophagus, an otherwise benign complication of esophageal reflux, affects approximately three million Americans and precedes almost all cases of esophageal cancer. If detected as high-grade dysplasia (HGD), most esophageal cancers can be prevented. Standard-of-care screening for dysplasia uses visual endoscopy and a prescribed pattern of biopsy. This procedure, in which a tiny fraction of the affected tissue is selected for pathological examination, has a low probability of detection because dysplasia is highly focal and visually indistinguishable. We developed a system called endoscopic polarized scanning spectroscopy (EPSS), which performs rapid optical scanning and multispectral imaging of the entire esophageal surface and provides diagnoses in near real time. By detecting and mapping suspicious sites, guided biopsy of invisible, precancerous dysplasia becomes practic! able. Here we report the development of EPSS and its application in several clinical cases, one of which merits special consideration. View full text Figures at a glance * Figure 1: Clinical EPSS instrument. The EPSS instrument is shown in the endoscopy suite before the clinical procedure, with the scanning probe inserted into the working channel of an endoscope. The insets show details of the scanning probe tip and the control box. * Figure 2: EPSS scanning esophageal epithelium during screening endoscopy. () Illustration depicting the probe tip extended from the endoscope working channel during the scan; arrows indicate linear and rotary motions of the probe tip before and during each scan, respectively. () Frame capture, obtained and shown via the EPSS user interface, of an image acquired by the endoscope video channel showing the actual EPSS probe tip during scanning of the esophageal epithelium of a patient with Barrett's esophagus during a clinical procedure. The scanning illumination spot is seen on the esophagus wall at the upper right of the image. The EPSS probe tip diameter is 2.5 mm. * Figure 3: EPSS spectra acquired during routine screening endoscopy. () Parallel (solid red line) and perpendicular (dotted blue line) polarization spectra collected with the EPSS instrument from a single spatial location in a subject with Barrett's esophagus. () Parallel polarization spectra from ten different locations in the same subject. AU, arbritary units. * Figure 4: Pseudo-color maps highlighting areas suspicious for dysplasia in five subjects. Maps produced from EPSS data are overlaid with circles indicating biopsy sites and confirmed pathology. The vertical axis indicates the angle of rotation (°) from the start of each rotary scan; the horizontal axis indicates the distance (cm) from upper incisors. Blue and green map areas and red and pink map areas represent epithelium unlikely for dysplasia and suspicious for dysplasia, respectively, as determined by EPSS. Red, pink and green circles indicate biopsy sites of HGD, LGD and nondysplastic Barrett's esophagus, respectively, as determined by pathology. () EPSS maps, biopsy sites and pathology for subjects A–E. () Biopsies taken during the initial and follow-up endoscopy procedures for subject A, overlaid on the EPSS map acquired during the initial procedure. Three follow-up biopsies were guided by the EPSS map, and pathology was confirmed HGD for each (indicated at 360°). * Figure 5: HRE with NBI image of a location with invisible HGD. Video capture was acquired in subject A at one of the locations where invisible dysplasia was missed by visual examination by HRE with NBI, but located by EPSS, and later confirmed by pathology. The site is marked by an arrow. Note that the site is visually indistinguishable from the surrounding nondysplastic Barrett's esophagus tissue. Author information * Abstract * Author information * Supplementary information Affiliations * Biomedical Imaging and Spectroscopy Laboratory and Department of Obstetrics, Gynecology and Reproductive Biology, Harvard University and Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA. * Le Qiu, * Edward Vitkin, * Sara Itani, * Lianyu Guo, * Mark D Modell, * Eugene B Hanlon, * Irving Itzkan & * Lev T Perelman * Division of Gastroenterology, Department of Medicine, Harvard University and Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA. * Douglas K Pleskow, * Ram Chuttani, * Jan Leyden, * Nuri Ozden & * Alana Sacks * Department of Pathology, Harvard University and Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA. * Jeffrey D Goldsmith * Medical Research Service and Geriatric Research Education and Clinical Center, Department of Veterans Affairs, Bedford, Massachusetts, USA. * Eugene B Hanlon Contributions L.Q., E.V., M.D.M., E.B.H., I.I. and L.T.P. developed and evaluated the method; S.I., L.Q. and E.V. contributed codes for instrument control; D.K.P., R.C., J.D.G., J.L., N.O., L.G., L.Q. and A.S. performed clinical procedures; L.Q., D.K.P., R.C., E.B.H., I.I. and L.T.P. contributed to the writing of the manuscript; E.B.H., I.I., D.K.P., R.C. and L.T.P. designed and planned the project. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Lev T Perelman (ltperel@bidmc.harvard.edu) Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Video 1 (9M) This video shows in real time the EPSS probe scanning a 2-cm section of esophagus during an endoscopy screening procedure. The regions of Barrett's esophagus, distributed in a diffuse pattern, appear darker in the video, which was acquired by the NBI mode of the endoscope. PDF files * Supplementary Text and Figures (392K) Supplementary Figures 1–3 and Supplementary Methods Additional data
• Kynurenine is an endothelium-derived relaxing factor produced during inflammation
  - Nature Medicine 16(5):607 (2010)
  Nature Medicine | Corrigendum Kynurenine is an endothelium-derived relaxing factor produced during inflammation * Yutang Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Hanzhong Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Gavin McKenzie Search for this author in: * NPG journals * PubMed * Google Scholar * Paul K Witting Search for this author in: * NPG journals * PubMed * Google Scholar * Johannes-Peter Stasch Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Hahn Search for this author in: * NPG journals * PubMed * Google Scholar * Dechaboon Changsirivathanathamrong Search for this author in: * NPG journals * PubMed * Google Scholar * Ben J Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Helen J Ball Search for this author in: * NPG journals * PubMed * Google Scholar * Shane R Thomas Search for this author in: * NPG journals * PubMed * Google Scholar * Vimal Kapoor Search for this author in: * NPG journals * PubMed * Google Scholar * David S Celermajer Search for this author in: * NPG journals * PubMed * Google Scholar * Andrew L Mellor Search for this author in: * NPG journals * PubMed * Google Scholar * John F Keaney Jr Search for this author in: * NPG journals * PubMed * Google Scholar * Nicholas H Hunt Search for this author in: * NPG journals * PubMed * Google Scholar * Roland Stocker Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:607Year published:(2010)DOI:doi:10.1038/nm0510-607a Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.16, 279–285 (2010), published online 28 February 2010; corrected after print 6 May 2010 In the version of this article initially published, the symbol key in Figure 2g,h is incorrect. The correct symbol key is open circles for Ido1−/− and filled circles for WT. The error has been corrected in the HTML and PDF versions of the article. Additional data
• Obesity: stressing about unfolded proteins
  - Nature Medicine 16(5):607 (2010)
  Nature Medicine | Corrigendum Obesity: stressing about unfolded proteins * Ronald C Wek Search for this author in: * NPG journals * PubMed * Google Scholar * Tracy G Anthony Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:607Year published:(2010)DOI:doi:10.1038/nm0510-607b Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.16, 374–376 (2010); published online 7 April 2010; corrected after print 6 May 2010 In the version of this News and Views initially published, the last name of one of the authors whose work was being discussed, Jonathon N. Winnay, was incorrectly spelled as 'Winney'. The error has been corrected in the HTML and PDF versions of the article. Additional data
• Rebuilding Humpty Dumpty with a serotonin inhibitor
  - Nature Medicine 16(5):607 (2010)
  Nature Medicine | Erratum Rebuilding Humpty Dumpty with a serotonin inhibitor * Ego Seeman Search for this author in: * NPG journals * PubMed * Google ScholarJournal name:Nature MedicineVolume:16,Page:607Year published:(2010)DOI:doi:10.1038/nm0510-607c Article tools * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Nat. Med.16, 264–265 (2010); published online 5 March 2010; corrected after print 1 April 2010 In the version of this article initially published, several articles were eliminated from the reference list during layout, and several typographical errors were introduced. These errors have been corrected in the HTML and PDF versions of the article. Additional data