Di Zenzo G.,Molecular and Cell Biology Laboratory |
Thoma-Uszynski S.,Friedrich - Alexander - University, Erlangen - Nuremberg |
Calabresi V.,Molecular and Cell Biology Laboratory |
Fontao L.,University of Geneva |
And 7 more authors.
Journal of Investigative Dermatology | Year: 2011
Bullous pemphigoid (BP), the most common autoimmune subepidermal bullous disease, is associated with an autoantibody response to BP180 and BP230, two components of junctional adhesion complexes in human skin promoting dermo-epidermal cohesion. Retrospective analyses demonstrated that these autoantigens harbor several epitopes targeted by autoaggressive B and T cells. The aim of this prospective multicenter study was to assess the evolution of IgG autoantibodies in 35 BP patients over a 12-month observation period. Epitope-spreading (ES) events were detected in 17 of 35 BP patients (49%). They preferentially occurred in an early stage of the disease and were significantly related to disease severity at diagnosis. Moreover, in three patients, spreading of IgG reactivity to intracellular epitopes of BP180 and BP230 was preceded by recognition of the BP180 ectodomain. Finally, IgG reactivity with extracellular epitopes of BP180 and intracellular epitopes of BP230 correlated with the severity of BP in disease course. These findings support the idea that IgG recognition of the BP180 ectodomain is an early and crucial event in BP disease, followed by variable intra- and intermolecular ES events, which likely shape the individual course of BP. © 2011 The Society for Investigative Dermatology.
PubMed | Molecular and Cell Biology Laboratory, Medical University of Warsaw, University of Bern, University of Lübeck and 7 more.
Type: | Journal: Journal of the American Academy of Dermatology | Year: 2016
Serologic diagnosis of autoimmune blistering disease (AIBD) usually follows a sophisticated multistep algorithm.We sought validation of a multivariant enzyme-linked immunosorbent assay (ELISA) in the routine diagnosis of AIBD.The multivariant ELISA comprising 6 recombinant immunodominant forms of major AIBD target antigens, ie, desmoglein 1, desmoglein 3, envoplakin, BP180, BP230, andtype VII collagen was applied in: (1) a cohort of well-characterized AIBD (n=173) and control sera (n=130), (2) a prospective multicenter study with 204 sera from patients with newly diagnosed AIBD with positive direct immunofluorescence microscopy, and (3) a prospective monocenter study with 292 consecutive sera from patients with clinical suspicion of AIBD in comparison with the conventional multistep diagnostic algorithm.Concordant results in the multivariant ELISA compared with direct immunofluorescence microscopy were seen in 94% of patients with pemphigus and 71% of patients with pemphigoid (Cohen value, 0.95 and 0.66) and with the conventional multistep diagnostic approach in 91% of patients with pemphigus and 88% of patients with bullous pemphigoid and 93% of autoantibody-negative sera (Cohen , 0.95, 0.84, and 0.78).IgA autoantibodies and less common target antigens were not analyzed.The multivariant ELISA is a practical, highly standardized, and widely available novel diagnostic tool for the routine diagnosis of AIBD.
News Article | February 15, 2017
LA JOLLA -- (Feb. 14, 2017) Salk Professor Tony Hunter, who holds an American Cancer Society Professorship, has been awarded $500,000 as part of the $1 million Royal Swedish Academy of Sciences' inaugural Sjöberg Prize for Cancer Research for "groundbreaking studies of cellular processes that have led to the development of new and effective cancer drugs." The prize ceremony, which is modeled after the Nobel Prize ceremony, will be held in Stockholm during the Academy's annual meeting on March 31, 2017, in the presence of His Majesty the King and Her Majesty the Queen of Sweden. "Tony is an internationally recognized leader in the field of cancer research," says Salk President Elizabeth Blackburn. "He has made enormous contributions to our understanding of cancer's basic biology and his research has led to life-saving therapies. We are delighted that his pioneering accomplishments are being honored with this important new award." Hunter studied how normal cells become tumor cells, demonstrating that a special process was necessary: tyrosine phosphorylation of proteins. His discovery led to the development of a new type of cancer pharmaceutical, tyrosine kinase inhibitors. These have revolutionized the treatment of chronic myeloid leukemia and also are of great benefit in several other forms of cancer. Hunter's work has led to a complete catalogue of the 90 human genes that encode tyrosine kinases, over half of which have become targets for the development of drugs to treat cancer and other human diseases. Currently, 26 tyrosine kinase inhibitors are FDA approved for human therapy, with many more in clinical trials. "It is a great honor to have been selected as an inaugural recipient of the Sjöberg Prize," says Hunter. "I have been fortunate to work in an inspiring and collaborative scientific community both at Salk and around the world, with excellent mentors, colleagues and students, all of whom contributed greatly to the breakthrough for which I am being honored." "I so clearly remember the floor meeting, some 40 years ago, where Tony first announced the phosphorylation of tyrosine by Rous sarcoma viral Src kinase," says Inder Verma, Salk professor. "It was thrilling at the time and now it is amazing to think that this finding has led to the making of drugs which are saving and extending the lives of cancer patients worldwide. That is the magic of basic science." The annual prize, which includes $100,000 as a personal award with $900,000 U.S. dollars designated as a grant for future research, is shared equally by the awardees. Hunter shares the honor with immunologist James P. Allison of the University of Texas MD Anderson Cancer Center, whose work on the white blood cells known as T cells led to the development of immune checkpoint therapy drugs that promote the immune response to cancer, and are now widely used in cancer therapy. Hunter, who holds the Renato Dulbecco Chair in Salk's Molecular and Cell Biology Laboratory, is also the recipient of the BBVA Foundation Frontiers of Knowledge award in biomedicine, the Royal Medal in the Biological Sciences of the Royal Society, the Wolf Prize in Medicine and the Gairdner International Award, among other prestigious honors. He is a member of the National Academy of Sciences, the National Academy of Medicine and the American Academy of Arts and Sciences. Born in 1943 in Ashford, Kent, in the United Kingdom, Hunter is also a fellow of the Royal Society of London. The Sjöberg Prize is awarded by the Sjöberg Foundation, which was established in 2016 with a donation of $2 billion Swedish krona by the late Swedish businessman Bengt Sjöberg -- one of the biggest donations in Swedish history. The annual prize totals $1 million U.S. dollars and is shared equally between the laureates, who are chosen by the Royal Swedish Academy of Sciences. The laureates will hold open lectures at the Karolinska Institutet on March 30. About the Royal Swedish Academy of Sciences: The Royal Swedish Academy of Sciences was founded in 1739 and is an independent organization that aims to promote the sciences and strengthen their influence in society. The Academy takes special responsibility for the natural sciences and mathematics, but strives to increase the exchange between different disciplines. About the Salk Institute for Biological Studies: Every cure has a starting point. The Salk Institute embodies Jonas Salk's mission to dare to make dreams into reality. Its internationally renowned and award-winning scientists explore the very foundations of life, seeking new understandings in neuroscience, genetics, immunology, plant biology and more. The Institute is an independent nonprofit organization and architectural landmark: small by choice, intimate by nature and fearless in the face of any challenge. Be it cancer or Alzheimer's, aging or diabetes, Salk is where cures begin. Learn more at: salk.edu.
News Article | December 5, 2016
LA JOLLA--(December 5, 2016) Ever since researchers connected the shortening of telomeres--the protective structures on the ends of chromosomes--to aging and disease, the race has been on to understand the factors that govern telomere length. Now, scientists at the Salk Institute have found that a balance of elongation and trimming in stem cells results in telomeres that are, as Goldilocks would say, not too short and not too long, but just right. The finding, which appears in the December 5, 2016, issue of Nature Structural & Molecular Biology, deepens our understanding of stem cell biology and could help advance stem cell-based therapies, especially related to aging and regenerative medicine. "This work shows that the optimal length for telomeres is a carefully regulated range between two extremes," says Jan Karlseder, a professor in Salk's Molecular and Cell Biology Laboratory and senior author of the work. "It was known that very short telomeres cause harm to a cell. But what was totally unexpected was our finding that damage also occurs when telomeres are very long." Telomeres are repetitive stretches of DNA at the ends of each chromosome whose length can be increased by an enzyme called telomerase. Our cellular machinery results in a little bit of the telomere becoming lopped off each time cells replicate their DNA and divide. As telomeres shorten over time, the chromosomes themselves become vulnerable to damage. Eventually the cells die. The exception is stem cells, which use telomerase to rebuild their telomeres, allowing them to retain their ability to divide, and to develop ("differentiate") into virtually any cell type for the specific tissue or organ, be it skin, heart, liver or muscle--a quality known as pluripotency. These qualities make stem cells promising tools for regenerative therapies to combat age-related cellular damage and disease. "In our experiments, limiting telomere length compromised pluripotency, and even resulted in stem cell death," says Teresa Rivera, a Salk research associate and first author of the paper. "So then we wanted to know if increasing telomere length increased pluripotent capacity. Surprisingly, we found that over-elongated telomeres are more fragile and accumulate DNA damage." Karlseder, Rivera and colleagues began by investigating telomere maintenance in laboratory-cultured lines of human embryonic stem cells (ESCs). Using molecular techniques, they varied telomerase activity. Perhaps not surprisingly, cells with too little telomerase had very short telomeres and eventually the cells died. Conversely, cells with augmented levels of telomerase had very long telomeres. But instead of these cells thriving, their telomeres developed instabilities. ""We were surprised to find that forcing cells to generate really long telomeres caused telomeric fragility, which can lead to initiation of cancer," says Karlseder, who also holds the Donald and Darlene Shiley Chair. "These experiments question the generally accepted notion that artificially increasing telomeres could lengthen life or improve the health of an organism." The team observed that very long telomeres activated trimming mechanisms controlled by a pair of proteins called XRCC3 and Nbs1. The lab's experiments show that reduced expression of these proteins in ESCs prevented telomere trimming, confirming that XRCC3 and Nbs1 are indeed responsible for that task. Next, the team looked at induced pluripotent stem cells (iPSCs), which are differentiated cells (e.g., skin cells) that are reprogrammed back to a stem cell-like state. iPSCs--because they can be genetically matched to donors and are easily obtainable--are common and crucial tools for potential stem cell therapies. The researchers discovered that iPSCs contain markers of telomere trimming, making their presence a useful gauge of how successfully a cell has been reprogrammed. "Stem cell reprogramming is a major scientific breakthrough, but the methods are still being perfected. Understanding how telomere length is regulated is an important step toward realizing the promise of stem cell therapies and regenerative medicine," says Rivera. Other authors included Candy Haggblom of the Salk Institute and Sandro Cosconati of the Second University of Naples. The work was funded by the California Institute for Regenerative Medicine training grant TG2-01158, the Salk Institute Cancer Center Core Grant (P30CA014195), the National Institutes of Health (R01GM087476, R01CA174942), the Highland Street Foundation, the Fritz B. Burns Foundation, the Emerald Foundation and the Glenn Center for Research on Aging. About the Salk Institute for Biological Studies: Every cure has a starting point. The Salk Institute embodies Jonas Salk's mission to dare to make dreams into reality. Its internationally renowned and award-winning scientists explore the very foundations of life, seeking new understandings in neuroscience, genetics, immunology and more. The Institute is an independent nonprofit organization and architectural landmark: small by choice, intimate by nature and fearless in the face of any challenge. Be it cancer or Alzheimer's, aging or diabetes, Salk is where cures begin. Learn more at: salk.edu.
Pomponi D.,Center for Molecular Allergology |
Di Zenzo G.,Molecular and Cell Biology Laboratory |
Zennaro D.,Center for Molecular Allergology |
Calabresi V.,Molecular and Cell Biology Laboratory |
And 5 more authors.
British Journal of Dermatology | Year: 2013
Background Bullous pemphigoid (BP) is an autoimmune skin disease in which patient autoantibodies react with BP180 and BP230 proteins. In addition to IgG, IgE has been shown to play a role in the disease. Objectives To evaluate the feasibility of detecting IgE and IgG against the immunodominant BP180 NC16A domain (BP180) using a microarray system. Methods BP180 was immobilized on an experimental version of the ISAC® microarray (Exp96). The BP study group and the controls were all tested on the commercial ISAC 103 version and on the Exp96. IgG and IgE were measured in a single run. BP180 IgG and IgE results were compared with those using an enzyme-linked immunosorbent assay (ELISA). Results All results obtained using the IgG ELISA on the 31 patients with BP were replicated with the ISAC IgG. Five of eight BP sera tested by ELISA showed similar results with ISAC IgE. Twenty-nine (94%) and 19 (61%) of the 31 patients with BP were IgG and IgE positive to BP180, respectively, whereas four (3%) and six (4%) of 138 normal donors were IgG and IgE positive, respectively. Interestingly, the levels of IgG against BP180 detected using the ISAC system were related to the disease severity. Patients with BP showed a peculiar profile of IgE recognition toward some groups of allergens, which was absent in a group of allergic individuals. A significant, higher prevalence of hen's egg recognition was observed in patients with BP who had specific IgE to BP180. Conclusions The present preliminary study indicates that the ISAC microarray system is suitable for detecting IgG and IgE autoantibodies in patients with BP. Notably, this system allows the assessment of IgE and IgG autoantibodies at the same time, could be employed for the detection of autoantibodies to other autoantigens, and allows profiling for specific IgE to allergens. What's already known about this topic? Bullous pemphigoid (BP) is a severe autoimmune blistering disease. IgG and IgE autoantibodies to skin BP180 and BP230 autoantigens are detected by enzyme-linked immunosorbent assay (ELISA) in patients with BP. IgE seem to play a role in a subset of patients with more severe disease. What does this study add? IgG and IgE autoantibodies in BP sera were detected using the immunodominant portion of BP180 immobilized on a microarray along with allergenic molecules, with almost overlapping results compared with ELISAs. Specific IgE allergen profiles differ in patients with BP and control groups. Specific BP180 IgG and IgE are rarely detected in an allergic population. © 2012 The Authors. BJD © 2012 British Association of Dermatologists.
News Article | April 27, 2016
Salk Institute scientists showed how an FDA-approved drug boosts the health of brain cells by limiting their energy use. Like removing unnecessary lighting from a financially strapped household to save on electricity bills, the drug--called rapamycin--prolongs the survival of diseased neurons by forcing them to reduce protein production to conserve cellular energy. Rapamycin has been shown to extend lifespan and reduce symptoms in a broad range of diseases and, at the cellular level, is known to slow down the rate at which proteins are made. But the new Salk research, published in the journal eLife, suggests that rapamycin could also target the neural damage associated with Leigh syndrome, a rare genetic disease, and potentially other forms of neurodegeneration as well. "Our study shows that protein production in neurons is one of the major utilizers of energy and that neurons of Leigh syndrome degenerate because they can't sustain a high enough level of energy," said Tony Hunter, the Renato Dulbecco Chair and American Cancer Society Professor in Salk's Molecular and Cell Biology Laboratory, who led the research. Previous studies on rapamycin, which blocks a key energy sensor in cells, found that it can alter the immune system, extend lifespan and treat disorders, including lupus and Alzheimer's disease. Researchers assumed that the drug prevented the neurodegeneration seen in Alzheimer's by encouraging cells to degrade damaged components and aggregated proteins. But recent data hinted that the drug might also have an effect on the mitochondria, organelles that act as cells' powerhouses, producing energy in the form of adenosine triphosphate (ATP). Xinde Zheng, a research associate in the Hunter lab, was already studying the properties of cells affected by Leigh syndrome, whose inherited neurodegeneration is caused by a mutation in mitochondrial DNA that reduces ATP production. Zheng wondered how rapamycin would affect the neurons plagued by the diseased mitochondria. He and Hunter teamed up with the lab of Rusty Gage, a professor in Salk's Laboratory of Genetics and holder of the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease. Zheng, together with Leah Boyer, then a researcher in Gage's lab and now director of Salk's Stem Cell Core, generated diseased neurons by taking skin cells from patients with Leigh syndrome, reprogramming them into stem cells in culture and then coaxing them to develop into brain cells in a dish. Though cells must make proteins to survive, protein production is a highly energy-consuming process and, for diseased cells, the process leaves too few energy reserves to deal with cellular stress or other demands. "Reducing protein production in aging neurons allows more energy for the cell to put toward folding proteins correctly and handling stress," said Zheng, the first author of the new paper. "The impact of our finding is that modulation of protein synthesis could be a general approach to treating neurodegeneration." In their study, the team found that Leigh Syndrome neurons decayed in the dish and showed clear signs of energy depletion. Meanwhile, Leigh syndrome neurons exposed to rapamycin had more ATP and showed less degeneration. By turning down the dial on protein production, the diseased and damaged neurons were able to survive longer. "We are surprised and delighted that rapamycin's effect to reduce protein synthesis as an energy-austerity approach may lead to a potential treatment for mitochondria-related neurodegenerative diseases," said Gage. This is a good example of the value of studying a disease in a dish, according to Hunter. "It's led to a lot of new insights into the underlying biology of this rare and understudied condition," he adds. More work is needed to determine whether the findings on rapamycin hold true in animal models of Leigh syndrome and other neurodegenerative diseases, and to ascertain how exactly rapamycin is altering the metabolism of the cells.
Cianfarani F.,Molecular and Cell Biology Laboratory |
Mastroeni S.,Clinical Epidemiology Unit |
Odorisio T.,Molecular and Cell Biology Laboratory |
Passarelli F.,Histopathology Laboratory |
And 4 more authors.
Journal of Cutaneous Pathology | Year: 2012
Background Vascular endothelial growth factor-C (VEGF-C), a lymphatic vessel growth factor, has been involved in the formation of lymph nodal metastases in different tumor types. Early evidences indicate that VEGF-C expression in human primary melanoma could be predictive of lymph nodal metastases, whereas the role of lymphangiogenesis is still controversial. Methods By immunohistochemical analysis, we investigated VEGF-C or CC chemokine receptor 7 expression, together with the lymphatic and blood vessel network, in 36 patients with primary skin melanomas and metastases at the sentinel lymph node biopsy (SLN-positive), and 26 melanoma patients with negative SLN biopsy (SLN-negative). Results We found that VEGF-C expression in primary melanoma specimens was significantly associated with SLN-positive (pâ<â0. 001), particularly in thin melanomas. An association between augmented peritumoral lymphatic vessel area and SLN-positive (pâ<â0.02) was also seen. Conversely, no association between either expression of the CC chemokine receptor 7 in the primary tumor, or intratumoral lymphatic vessel or peritumoral and intratumoral blood vessel area, and SLN-positive was found. Conclusions Our results, taking into account the expression of either VEGF-C or related histopathological markers, indicated the possibility to use VEGF-C immunohistochemistry as a marker of metastatic progression, especially in thin cutaneous melanomas. Copyright © 2012 John Wiley & Sons A/S.
Young N.P.,Molecular and Cell Biology Laboratory |
Kamireddy A.,Molecular and Cell Biology Laboratory |
Van Nostrand J.L.,Molecular and Cell Biology Laboratory |
Eichner L.J.,Molecular and Cell Biology Laboratory |
And 3 more authors.
Genes and Development | Year: 2016
Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK−/− EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. © 2016 Young et al.
Ou H.D.,Molecular and Cell Biology Laboratory |
Ou H.D.,Salk Institute for Biological Studies |
Deerinck T.J.,University of California at San Diego |
Bushong E.,University of California at San Diego |
And 4 more authors.
Methods | Year: 2015
Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and preparation of photo-oxidized samples for TEM and serial block-face scanning EM (SBEM) for large-scale volume EM data acquisition are also presented. As an example, we discuss the recent multi-scale analysis of Adenoviral protein E4-ORF3 that reveals a new type of multi-functional polymer that disrupts multiple cellular proteins. This new capability to visualize unambiguously specific viral protein structures at high resolutions in the native cellular environment is revealing new insights into how they usurp host proteins and functions to drive pathological viral replication. © 2015 Elsevier Inc.