Department of Microbiology, Tumor and Cell Biology Scientific Report 2007–2009 2 Department of Microbiology, Tumor and Cell Biology – MTC Contents Looking Beyond Tomorrow – Moving into the Future.............................4 MTC 15 Years – Where We Came From...............................................6 Newcomers A Long and Winding Road to “Chromosomics”...............................8 Bringing a Touch of Chemistry to MTC..........................................10 Tracking Down Fresh Footprints of the Myc Tumor Protein..........11 The Secret Garden of Rho GTPases............................................12 Awards, Major Grants and Appointments............................................14 Scientists in Focus From the Clinics to Basic Science.................................................18 Towards an Adjunct Professorship in Parasitology ......................19 Hans G Boman in Memoriam .............................................................20 High Impact Publications 2007–2009.................................................23 Immunology Research........................................................................28 Companies Founded by MTC Scientists Intervacc AB.................................................................................84 DILAFOR AB................................................................................84 Future Faculty.....................................................................................85 Equality and Diversity Group..............................................................85 Undergraduate Education...................................................................86 Training for the Future – PhD Studies.................................................87 Summer Research Schools................................................................88 Life as a PhD Student.........................................................................89 Doctoral Theses 2007–2009...............................................................90 Core Facilities MTC FACS Facility.......................................................................94 KI Visualization Facility (KIVIF).....................................................95 MTC Research Facility ................................................................96 Infection Biology Research.................................................................38 HIV-laboratory .............................................................................97 Cell and Tumor Biology Research.......................................................56 Interactions with the Outside World.....................................................98 What Happens Down in the Cellar? Lennart Nilsson at MTC..............68 Georg and Eva Klein Foundation........................................................99 Foreign Adjunct Professors MTC Organization............................................................................100 p53 Forever – Focus on this Fascinating Protein..........................70 MTC – Not Only a Scientific Experience!......................................71 Innovating Vaccines by Translational Entrepreneurship...............72 Our valuable Collaboration with the Karolinska Institutet..............73 MTC International Economy..........................................................................................101 Contributors and List of Financiers...................................................102 Location of MTC...............................................................................103 Evaluation of HIV Vaccines in Tanzania........................................74 Scientific Alliances in Africa .........................................................75 The Singapore Partnership for the Future....................................76 International networks for high-quality graduate education..........77 Scientific Networks KICancer......................................................................................78 Editor-in-Chief: Marie Arsenian Henriksson Scientific Editors: Mikael Jondal and Ute Römling Bibliometric Analysis: Catharina Rhen Text Editor: Anna Lögdberg Photo: Fredrik Persson and John Sennett KI-Infection...................................................................................79 Artistic Contributions: Ewert Linder KI Inflammation and Immunology.................................................79 Layout: Björn Lundquist, Malmö Print: Edita Västra Aros Emerging Collaborations The Royal Institute of Technology (KTH)......................................80 Center for Infectious Medicine (CIM)............................................81 Translational Research.......................................................................82 Cover photo by Lennart Nilsson/Scanpix: Malaria parasites have multiplied inside two red blood cells. One has burst open. Department of Microbiology, Tumor and Cell Biology – MTC 3 Head of Department Looking Beyond Tomorrow – Moving into the Future It all starts with an open mind, a new idea. A mixture of creativity and originality in combination with intellectual courage, hard and committed work as well as the intuition of a single person. Needless to say, the individual scientist is the driving force and the key to successful medical research. Equally important is the critical mass of fellow scientists in related or different areas while the infrastructure and state-of-the-art equipment are the essential pre-requisites enabling ideas to be tested experimentally. Karolinska Institutet (KI) has a strong tradition of cross-fertilization between disciplines. These interactions permeate all research at the Department of Microbiology, Tumor and Cell Biology (MTC), which is characterized by collaborations between preclinical and clinical scientists in dynamic national and international interactions. ment policy with a transparent procedure to guarantee the next generation of scientists. During the last three years MTC has recruited several new professors, senior lecturers, adjunct and foreign adjunct professors as well as scientists with external senior research positions. It is particularly rewarding that several researchers at early stages in their careers have joined the department. As science is built on individuals we will pursue recruitments at all levels with a focus on young and promising scientists. We will deepen the interaction with the clinic in parallel with building of the new university hospital, NKS (Nya Karolinska Solna). A significant part of the collaboration with the clinic occurs with the unit of Clinical Microbiology, which is part of MTC but located at the Karolinska University Hospital Solna. This natural connection to the hospital has been very fruitful and is now being Our focus is the fight against infections and intensified. At present several clinical trials cancer. The merge of immunology, infection are being performed in biology and cell and tuthe oncology field. These mor biology provides us “We have students and and other contacts will with a unique platform researchers from all over the be developed further to where fruitful interacglobe who contribute to our allow the translation of tions and creative ideas intellectual platform built basic research to mediarise and flourish. As on creativity, originality, cal care as well as into illustrated on page 26, diversity and equlity.” commercial products these interactions are and business. Scientists also revealed by biblioat MTC are founders metric analysis. It is our and board members of several start-up comfuture mission to maintain MTC as a frontpanies, including Aprea, Dilafor, Imed, Index line department at the international level in and Intervacc, located within the Karolinska science and education. To achieve this, we will Institutet Science Park. continue to carry out a pro-active recruit- 4 Department of Microbiology, Tumor and Cell Biology – MTC The scientific infrastructure is of fundamental importance when facing international competition. We have made strategic investments in our FACS facility, atomic force microscopy and live cell imaging equipment. These long-term commitments in infrastructure will continue. Moreover, maintaining a top-notch administration and technical support are necessary pre-requisites to meet future needs. It is also rewarding that the new Science for Life Laboratory (SciLifeLab), a national resource center dedicated to large scale research in molecular biosciences and medicine, is situated close to MTC. Our projects will be greatly facilitated through collaborations and discussions with the SciLifeLab scientists and will benefit by being integrated into their large scale stateof-the-art technology platforms. During the recent years we have increased our interactions and collaborations with the Royal Institute of Technology (KTH) both with respect to research collaborations as well as technical developments. These strategic contacts will be even more important in the future. International collaborations already established by individual researchers, as well as interactions formally established with Southern China, Vietnam, Uganda, and Singapore, will be further developed. Several MTC scientists are involved in the unique, international research education collaboration between KI and Makerere University in Kampala, Uganda. The collaboration with Chinese cancer research started during the ’90s and has generated many contacts and projects of common interest. The collaboration started at the Sun Yat Sen Cancer Center in Guangzhou (Canton), but has been expanded to several regions in China. MTC has also a fruitful interaction with Hanoi Medical University in Vietnam through the research education program “Common Diseases in Vietnam”. Several PhD students and post-docs from Vietnam have been working at MTC during the recent years. A*STAR is our foremost partner in Singapore, through which education of both Swedish and Singaporean students are financed. All of these collaborations have generated novel knowledge and several joint publications throughout the years. Training of the next generation of scientists is one of the most important missions of any university. Subsequently, research education both for PhD students and post-docs is the core of our activities and builds on learning the trade in the individual research project, like in the former guilds. It is of fundamental importance that we keep this tradition alive, while ensuring a high quality education. The intensive seminar series that take place everyday at MTC play a central role in our scientific life. They focus on our three areas of research; immunology, infection biology and tumor cell biology and function as a platform for communication and discussion of new scientific developments. In combination with the many international conferences that are being organized by MTC scientists, the seminars constitute an ideal educational forum and for spreading knowledge. It is our challenge to maintain MTC as a prominent institution where scientists open up new fields of investigation and make discoveries of fundamental importance. Key words are multidisciplinary, bridging, translational, national and international collaboration. The power and greatest asset of MTC is each and every individual person working at the department. We have students and researchers from Africa, Asia, Australia, Europe and the Americas who contribute to the intellectual scientific platform built on creativity, originality, diversity and equality. It is a joy to foster this spirit to meet future challenges. Marie Arsenian Henriksson Department of Microbiology, Tumor and Cell Biology – MTC 5 Inauguration of MTC by the Minis ter of Education and KI Preside Per Unckel nt Bengt Samu elsson (right). 3. Group Leaders of MTC in 199 MTC 15 Years – Where We Came From We celebrated our 15th BIRTHDAY in May 2008 with an international top symposium and a warm and cheerful party at the Medical Union (MF) venue. MTC was born on the first of July 1993 as a result of the launching of the “KI 93” – a major reform at the Karolinska Institutet. More than 100 departments were fused over night to become 30 “storinstitutioner” – BIG departments. For our department this was unusually timely. The construction of our new buildings was already on its way at the site where they stand today. The decision to create a totally new department was made after decades of discussion about expanding the facilities of the Tumor Biology Department successfully lead by George and Eva Klein, the founders of Tumor Biology at KI and vague plans for a new KI Microbiology Center. In 1989 this “long-bench” process transformed into accelerated planning partly due to a new Swedish government with an interest in reforming the university system. In particular it was due to a strong push from inside KI from some colleagues, in particular the KI president, professor Bengt Samuelsson, Hans Wigzell, professor of immunology, who later became the president of KI, and the dean, Erling Norrby, professor of virology. Until the last minute it was uncertain whether this new center should be located on the KI campus area in Solna or on the KI area in Huddinge. The construction of new buildings was initially independent of the KI 93 reorganization, but in the end these two events converged. Integrated into the global reor- 6 Department of Microbiology, Tumor and Cell Biology – MTC ganization of KI, the fusion of five departments, the Departments of Bacteriology, Infectious Disease Protection, Immunology, Tumor Biology and Virology in a new building around the corner went smooth, although these departments were previously spread all over the KI campus and the Department of Virology was even located outside the campus at the former State Bacteriological Laboratory (SBL) in Huvudsta. Organization of MTC Before moving into the new buildings we joined forces in these five departments and formed a dozen working groups, made up by appropriate staff members, to plan for everything from a joint economy, to heavy equipment, clean water and common facilities as well as joint shops, like the “Boutique Plastique” where researchers can buy plastic material 24 hours a day. We could then move into our new houses in February 1994 with a new organization in place. The overriding concept was “The Research Group in the Middle”, i.e. an effort to plan everything from laboratories and offices to economy in order to support the everyday work of the research groups. After all, the creativity and production of the research groups was the basis for generating a solid economy – pretty obvious today but not in those days when university departments were organized as public authorities based on state funding. As a result, MTC developed a highly research oriented environment. The total budget in 1993–94 was around 50 million Swedish crowns and has more than trippled over 15 years. Thirty research groups were identified within the old departments. To qualify as a group leader he/she must have a tenured or 4–6 year research council position. As a result several young scientists including women were identified as leaders at the same level as the most established professors. They all joined in the MTC faculty group (“forskarkollegiet” in Swedish) which started off as a consensus group for forming continued strategies. Interdisciplinary build up The diversity and flow of research at MTC is also reflected in its buildings. During the whole ’90s the MTC buildings were continuously improved. After moving into the new A, B, C and D-buildings, part of the old bacte- The builders of MTC Alex Feldötö and Greger Blomquist. er. MTC millenium calend riology houses were replaced by the new G-wing in 1996. In the last phase, the teaching building (E) and the bacteriology building (F) were renovated, ready for use in 1998. During these events, research groups had to move around a lot, some groups as much as three times! In spite of this inconvenience, this continuous change also created new constellations of research groups with new neighbours melting together our 4–5 disciplines. The interdisciplinary build up by MTC became its hallmark that has generated a lot of novel collaborations and new projects, although one could always have wished for more efforts in this direction. Due to the large number of groups which were very successful in being awarded external research grants, the economy of the department became quite stable. Consequently, there He did everyth ing himself – th e first head of MTC, professo r Ingemar Ernb erg, appearing as the depa rtmental Lucia . was money to buy state-of-the-art equipment which was the basis to form new core facilities within the department. By the millennium shift we had a positive balance and funding for new investments. This development did not occur without some pain, however, as during the ’90s the organization of MTC had to be dramatically restructured. The key concept was to invest state funds in the research groups that in turn could generate more money through external grants. This process required the dramatic reduction of supportive personnel such as technicians (BMAs), administrators and engineers. Altogether 35 persons had to leave MTC during 1995-97. With reforming the KI economy and introducing a new rental system MTC again went through economic harsh times shortly after the beginning of the new millennium. However, with a strong management the economy has improved and MTC is thriving again. Added value of neighbourship One important development for MTC was the move of SBL in Huvudsta to the KI Campus, next door to MTC. SBL was renamed the Center for Infectious Disease Control (SMI) in 1997. Along with this fusion came a donation from the Swedish government for six research chairs in clinical immunology, virology, bacteriology, parasitology, vaccinology and epidemiology. Most of these professors became affiliated with MTC through their research and PhD education. Over the years significant efforts were made to optimize the added value of this neighbourship. In addition, later The European Center for Disease Control (ECDC) was placed close to MTC and SMI; all three insti- tutions together form an impressive cluster of knowledge in the area of microbiology. A new generation of scientists at MTC By the end of the ’90s there had been a major turnover of research groups. Rolf Kiessling, Klas Wiman, Eva-Maria Fenyö, Lars Holmgren, Hans-Gustav Ljunggren and Joakim Dillner were some who left for chairs in other departments and universities. A whole generation of modern cell biologists and microbiologists joined MTC. Newcomers were Staffan Normark, Yihai Cao and Mikael Rhen, followed by Ute Römling, Sven Pettersson and Markus Maeurer in the early 2000s and recently by Lars-Gunnar Larsson, Pontus Aspenström, Rolf Ohlsson and Sonia Laín. Thus the turnover in the faculty has been vivid and healthy. It is also worth to recognize the balancing input by our professor emeriti like Carl-Ivar Brändén (1998 –2003) and Hans G Boman (1997–2007). Recently professor David Lane joined the team as a foreign adjunct professor from the UK and Singapore. By 2000, professor Ingemar Ernberg (1993–2000) handed over the department chair to professor Klas Kärre (2000–2002) followed by professor Mats Wahlgren (2003– 2006) and associate professor Marie Arsenian Henriksson (2006–). MTC moved from creation to consolidation. The Microbiology and Tumor Biology Center (MTC) became The Department of Microbiology, Tumor and Cell Biology (MTC) well poised to supply a creative research environment for young and senior scientist to make important contributions to human health in the future! Ingemar Ernberg Department of Microbiology, Tumor and Cell Biology – MTC 7 Newcomers Rolf Ohlsson A Long and Winding Road to “Chromosomics” as a scientist you have to show passion for your work. However, this passion must be something extraordinary to be able to buffer against the disappointments and setbacks that science always has in preparation for each one of us. It was ominously Edgar Allan Poe who wrote the following (abbreviated): “From childhood hours I could not bring my passion from a common spring”. My own outlandish passion and curiosity for science has brought both pros and cons to me. During my career I have thus worked in many different disciplines, including pathology, genetics, developmental biology, cell biology, evolution and so on. Although each research interest usually had to be built up from scratch, which slowed down short-term progress, my search for creative space on the interface between different research areas benefits from insights into several different disciplines. My first wobbling steps in the academic world started with a BSc in Zoology and Chemistry at Lund University. After my PhD in Biochemistry in the stone ages, 1976, in Lund, I went for postdoctoral research at the National Institute of Medical Research in Mill Hill, London. I returned to Sweden as a postdoc at Uppsala University to work on adenovirus mRNA processing. I took up my first position as an independent scientist as a guest professor at Umeå university. Against all the Myc oncogene and cell proliferation in odds, one of the most successful periods in my normal cells1 and the first identification of a scientific life was completed in a 4 m2 lab, sharnormal cell with autocrine growth control2. ing desk with another scientist. This progress From the discovery of genomic imprinting in opened up an opportunity to take up a posihumans in 19933 to the discovery of systemic tion at Karolinska Instiloss of imprinting in tutet, first as a senior sciencolon cancer patients “Of course we hope to tist funded by the Swedish in 19984, I have more one day be able to use Cancer Society and later as recently established the acquired information to a professor funded by the link between chromatin not only diagnose early Swedish Research Couninsulators and genomic stages of cancer, but cil for Natural Sciences. imprinting5. Thus, the also to design cancer During this time, my intertargeted destruction of a treatments.” est in genomic imprinting chromatin insulator at an was initiated. After 16 years imprinted locus perturbs as a professor in Developmental biology at stem cell populations in colon crypts to Uppsala University and one wonderful year at predispose to cancer6,7. This feature involves MTC I am still on this track. a novel epigenetic mark, poly(ADP-ribosyl) Among my best results I would rate as ation8, which has most recently been incorpobeing one of the first to make the link between rated into the context of higher order chro- If you want to make it 8 Department of Microbiology, Tumor and Cell Biology – MTC matin conformation analyses 9,10,11. The most recent work, since we came to MTC, places such chromosomal networks in the context of nuclear architecture and describes the transfer of epigenetic states between chromosomes12,13,14. Strong scientific competence at MTC There are many reasons why I chose MTC as my new address. Apart from wanting to be part of the same institution that hosts a legend, professor Georg Klein, I was attracted by the strong scientific competence in cell and molecular biology, particularly in cancer pathways. The reason is that our work on chromosomal networks point in these directions. I very much look forward to several joint interactions with MTC scientists and hope that MTC scientists will share my feeling for many interfaces of common interests. Newcomers We do have some know-how on chromatin, epigenetics and nuclear architecture that MTC scientists might be interested in. Since my wonderful group moved to MTC, we have further penetrated the network of physically interacting chromosomes in human stem cells as well as cancer cells. We have uncovered links to cell signaling pathways and to apoptosis within these networks and we are starting to think of our projects in term of systems biology and nuclear architecture, which are two features not commonly associated with each other. Our vision is not only to understand epigenetics, genomics, and nuclear architecture, I call this interface “chromosomics”, but to take this interface to stem/progenitor cells in particular and explore the early stages of what can go wrong in cancer. To this end, we have established a large national and international network of friends and colleagues who have complementing objectives. Of course we hope one day to be able to use acquired information not only to diagnose early stages of cancer, but also to design cancer treatments. Key publications 1. Pfeifer S, Goustin AS, Rydnert J, Wahlström T, Bjersing L and Ohlsson R. Spatial and temporal pattern of cellular myc oncogene expression in developing human placenta: implications for embryonic cell proliferation. Cell 38, 585, 1984. 2. Goustin AS, Betsholtz C, Pfeifer S, Persson H, Rydnert J, Bywater M, Heldin C-H, Westermark B and Ohlsson R. Co-expression of the sis and myc protooncogenes in early human placenta suggests autocrine regulation of trophoblast growth. Cell 41, 301, 1985. 3. Ohlsson R, Nyström A, Pfeifer-Ohlsson S, Hedborg F, Schofield P, Flam F and Ekström T. IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome. Nat Genet 4, 94, 1993. 4. Cui H, Horon IL, Ohlsson R, Hamilton SR and Feinberg AP. Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability. Nat Med 4, 1276, 1998. The image shows a physical network of interacting chromatin regions from different chromosomes. The data were generated from a library of interacting sequences in human embryoid bodies obtained by the circular chromosome conformation capture method that the Ohlsson laboratory has invented. There is a central node, representing a particular region on chromosome 2, which is connected to other nodes and so-called outliers. We hypothesize that this network represents an overall regulatory feature of gene expression that connects different modules of biological processes, such as different cell signaling pathways, to coordinate and diversify the expressivity of the genome. 5. Pant V, Mariano P, Kanduri C, Mattsson A, Lobanenkov V and Ohlsson R. The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes Dev 17, 586, 2003. 6. Sakatani T, Kaneda A, LacobuzioDonahue C, Carter M, Okano H, Ko MS, Ohlsson R, Longo DL and Feinberg AP. LOI of Igf2 alters maturation of intestinal epithelium and increases adenoma formation in Min mice. Science 307, 1976, 2005. 7. Feinberg A, Ohlsson R and Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet 7, 21, 2006. 8. Yu WQ, Ginjala V, Pant V, Chernukhin I, Whitehead J, Mukhopadhyay R, Kanduri C, Oshimura M, Feinberg AP, Lobanenkov V, Klenova E and Ohlsson R. Poly(ADP-ribosyl)ation regulates CTCF-dependent chromatin insulation. Nat Genet 36, 1105, 2004. 9. Göndör A and Ohlsson R. Transcription in the loop. Nat Genet 38, 1229, 2006. 10.Zhao Z, Tavoosidana G, Sjölinder M, Göndör A, Mariano P. Wang S, Kanduri C, Lezcano M, Singh Sandhu K, Singh U, Pant V, Tiwari V, Kurukuti S and Ohlsson R. Circular chromosomal conformation capture (4C) uncovers epigenetically regulated networks of intra- and interchromosomal complexes. Nat Genet 38, 1341, 2006. 11.Ohlsson R. Widespread monoallelic expression. Science 318, 1077, 2007. 12. Göndör A and Ohlsson R. Replication timing and epigenetic reprogramming of gene expression: a two-way relationship? Nat Rev Genet 10, 269, 2009. 13. Göndör A and Ohlsson R. Chromatin crosstalk in three dimensions. Nature 461, 212, 2009. 14.Sandhu KS, Shi C, Sjölinder M, Zhao Z, Göndör A, Liu L, Tiwari VK, Guibert S, Emilsson L, Imreh MP and Ohlsson R. Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development. Genes Dev 23, 2598, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 9 Newcomers Sonia Laín Bringing a Touch of Chemistry to MTC My research career started in 1988 in Madrid where, after obtaining a University degree in biology, I carried out my PhD thesis work on molecular biology aspects of a poliovirus-like RNA virus infecting plants. In 1992 I was awarded an EMBO fellowship to work at the University of Gothenburg in the Epstein-Barr Virus (EBV) field, a main topic of research at the former Tumor Biology department at the Karolinska Institutet (KI) which is now part of MTC. Therefore, very early on I became familiar with the exciting EBV research by Georg and Eva Klein as well as by other Tumor Biology members, many of whom are currently at MTC. After three years in Sweden, I moved to Dundee in Scotland, where I began studies on central tumor suppressor proteins such as p53. Although I maintain a strong interest in basic research, I quickly realized the importance of using the information and reagents we were gathering for the discovery of therapeutics with antitumor activity. My interest in using p53 to discover small molecules with therapeutic potential began when I found the Streptomyces metabolite leptomycin B to be one of the most potent activators of p531 and established the basis for a Phase I/II trial with this compound through our interaction with clinicians. Fruitful and ongoing collaboration My research on drug discovery was broadened when, together with professor David Lane, one of the discoverers of p53 (and adjunct Professor at MTC-KI) we obtained substantial funding to perform compound screens using a robust p53 activation cellbased assay2. More importantly, this work led to a fruitful and ongoing collaboration with the chemistry lab of Dr Nick Westwood in St Andrews (Scotland). By mid-2004 we had screened 34,000 compounds and identified a large number of hits that we classified according to carefully chosen criteria. It was exciting to see that two of these small molecules showed the capacity to reduce tumor growth when tested in animals3,4 as this validated our selection procedures. New screens based on our protocols are currently underway. Another important aspect of this work was that we were able to apply ground-breaking technologies to solve the so-called “target identification problem” of elucidating the mechanism of action of small molecules in cells4. For example, we showed that one of our families of hit molecules includes inhibitors of the protein deacetylase activity of sirtuins, an extremely interesting group of enzymes linking metabolism with gene expression. These enzymes have been implicated in the ageing process as well as in a variety of illnesses including cancer and infectious diseases. In the fall of 2008 I started a research lab at MTC with five members so far constituting expertise in cell biology and chemistry. Our plan is to continue with our approaches for drug discovery focusing primarily on our previously identified sirtuin inhibitors4, 5 as well as on hit compounds from new screens. Chemical biology is rapidly becoming popular in academia. Here, MTC can be regarded as pioneering and I see, that there are many opportunities for cross-fertilization between research groups. We are keen to share our best-characterised molecules, compound collections and experience in chemical biology as well as to actively participate in training in this highly exciting novel research area. Our general goal is to develop well-characterised compounds that constitute high quality tools for research and/or potential therapeutics. This in-depth knowledge will help to In the primary assay to screen for compounds affecting p53 activity, a p53 reporter mammalian cell line is seeded into wells of 96 well plates and each well is treated with a compound. Activation of p53’s transcriptional function is measured by colourimetric analysis of β-galactosidase reporter activity expressed from the p53-dependant lacZ gene. 10 Department of Microbiology, Tumor and Cell Biology – MTC recognise clinical situations where they are beneficial as well as to assess potential risks in future clinical trials. Our hit compounds are of interest for the treatment and understanding of diseases other than cancer and in this regard, working at MTC opens up the possibility to test our hit compounds in collaboration with experts in a range of disease models. Key publications 1. Laín S and Lane D. Improving cancer therapy by non-genotoxic activation of p53. Eur J Cancer 39, 1053, 2003. 2. Berkson R, Hollick J, Westwood N, Woods J, Lane D and Laín S. A pilot screening programme for small molecule activators of p53. Int J Cancer 115, 701, 2005. 3. Laín S, Hollick JJ, Campbell J, Staples O, Higgins M, Aoubala M, McCarthy A, Appleyard V, Murray KE, Thompson A, Mathers J, Holland SJ, Stark MJR, Pass G, Woods J, Lane DP and Westwood NJ. Discovery, in vivo activity and mechanism of action of a small-molecule p53 activator. Cancer Cell 13, 454, 2008. 4. Staples OD, Hollick JJ, Campbell J, Higgins M, McCarthy A, Appleyard V, Murray KE, Thompson A, Ronseaux S, Lane DP, Westwood NJ and Laín S. Characterisation, chemical optimisation and anti-tumor activity of tubulin poisons identified by a p53-based phenotypic screen. Cell Cycle 7, 3417, 2008. 5. Medda R, Russell RJM, Higgins M, McCarthy AR, Campbell J, Slawin AMZ, Lane DP, Laín S and Westwood NJ. Novel cambinol analogs as sirtuin inhibitors: Synthesis, biological evaluation and rationalization of activity. J Med Chem 52, 2673, 2009. Newcomers Lars-Gunnar Larsson Tracking Down Fresh Footprints of the Myc Tumor Protein ers. Our expectations have been entirely I graduated as a B. Sc. in Microbiology at fulfilled since we moved to MTC in 2007. Uppsala University 1982. However, since From our side, we have contributed to further then, I dedicated myself to Tumor Biology – strengthen Tumor Biology research at MTC, in my view a most fascinating field of research in particular we concentrated on the analyembracing the most fundamental questions of sis of different aspects of oncogene/tumor how cell growth and development is regulated. suppressor gene function and regulation. I I got my PhD in Uppsala 1989 studying presume my long experience in teaching is oncogenes and their role in differentiation. also an asset to the department. During this period it became increasingly clear that Myc, one the oncogenes I studied, was a key player in tumor development. I Visualize protein interactions therefore decided to specialize in Myc and In my career as an independent researcher I made a postdoc 1991–94 in the laboratory of have focused mainly on post-translational Bernhard Lüscher in Hannover, Germany. regulation of the oncoprotein/transcription During this period I published one of the factor Myc in response to signaling and tried first papers on the Mad family of Myc antagoto elucidate the most important functions nists1. By this time I had of Myc. One of our major achievements was the become truly “addicted” demonstration that phosto Myc, and returnphorylation of Myc reguing to Sweden in 1994 I lates turnover by the ubiqstarted my own research uitin/proteasome pathway group at Rudbeck lab and we subsequently idenin Uppsala continuing tified the first E3 ubiquitin Myc research. 1998 I got Myc/Ras oncogene cooperativity in ligase, Skp2, targeting Myc a senior lecturer position transformation. Apoptosis and cellular for degradation2. Surprisat the Swedish University senescence are two main barriers of oncogenic transformation of cells. Lars- ingly, we found that Skp2 of Agricultural Sciences son’s recent research suggests that (SLU) in Uppsala and simultaneously promotes Myc and Ras complement each other in 2001 I was appointed Myc-driven transcripby opposing each other’s anti-tumorigenic activities by repressing senesProfessor of Molecular tion, thereby supporting cence and apoptosis, respectively, Genetics. I eventually the so-called “Kamikazeresulting in tumor development. decided to move on and model” of transcription. got recruited to MTC in 2007. Another important finding was that Myc The reason I chose MTC was its strong directs transcription from all three RNA position in Tumor Biology, and in particular polymerases3. We also contributed to the its long-standing and continuous interest in developments of new methods to visualize Myc and other transcription factors of releprotein interactions in living or fixed cells. vance for Tumor Biology such as p53. Other After moving to MTC we partially changed reasons were my already ongoing collaboradirection and discovered that an important tions with groups at MTC and other departfunction of Myc is the regulation of cellular ments at the Karolinska Institutet (KI) and my senescence4,5. Inspired by many other groups increasing interest in translational research, at MTC we have also put more emphasis where MTC is strong. My expectations were on translational research and are actively to gain increased interactions, discussions developing small molecules targeting Myc. and collaborations with researchers sharFurther, we started research on Myc-driven ing my interest in Tumor Biology. Further, I tumor development in animal models. We was looking forward that my research group will develop these new aspects in the future would become part of an exciting and interacin collaboration with other groups at MTC, tive scientific environment, exchanging ideas KI and in the world. I have also started to be as well as technical know-how through semiengaged more actively in teaching at basic and nars and daily contacts with other researchadvanced courses at MTC and KI and hope to be able to contribute more to teaching activities in the future. Key publications 1. Larsson L-G, Pettersson M, Öberg F, Nilsson K and Lüscher B. Expression of mad, mxi1, max and c-myc during induced differentiation of hematopoietic cells: opposite regulation of mad and c-myc. Oncogene 9, 1247, 1994. 2. von der Lehr N, Johansson S, Wu S, Bahram F, Castell A, Cetinkaya C, Hydbring P, Weidung I, Nakayama K, Nakayama KI, Söderberg O, Kerppola K and Larsson L-G. The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-myc-regulated transcription. Mol Cell 11, 1189, 2003. 3. Arabi A, Wu S, Ridderstråle K, Bierhoff H, Shiue C, Fatyol K, Fahlén S, Hydbring P, Söderberg O, Grummt I, Larsson L-G and Wright APH. c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat Cell Biol 7, 303, 2005. 4. Hydbring P, Bahram F, Su Y, Tronnersjö S, Högstrand K, von der Lehr N, Lilischkis R, Hein, Wu S, Vervoorts J, Henriksson M, Grandien A, Lüscher B and Larsson L-G. Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation. Proc Natl Acad Sci USA 107, 58, 2010. 5. Campaner S, Doni M, Hydbring P, Verrecchia A, Bianchi L, Sardella D, Schleker T, Perna D, Tronnersjö S, Murga M, Fernandez-Capetillo O, Barbacid M, Larsson L-G and Amati B. Cdk2 suppresses cellular senescence induced by the myc oncogene. Nat Cell Biol 12, 54, 2010. Department of Microbiology, Tumor and Cell Biology – MTC 11 Newcomers Pontus Aspenström The Secret Garden of Rho GTPases with the clear objective to become a botanist. I wanted to spend my life knowing all about flowers; it was not on the agenda to lock myself up within the confinement of a laboratory. Strangely enough, this was exactly what I did as a PhD student when I found myself studying microfilament proteins, such as actin. The concept that microfilaments acted as the primum movens for cell migration was gaining general acknowledgement during the late eighties. However, the signaling pathways that regulate the dynamic reorganization of the actin filament system were essentially unknown. I started my academic studies Wiskott-Aldrich syndrome protein The identification of the Ras-like small GTPases Rac and Rho as the key elements in the signaling pathways that link transmembrane receptors and the actin filament system resulted in a true shift of paradigm. This seminal discovery by Alan Hall and co-workers triggered my interest in Rho GTPases and I decided for a post-doctoral visit to his laboratory at University College London. There, I became engaged in the hunt for binding partners for the Rho member Cdc42. During this period, we identified the Wiskott-Aldrich syndrome protein (WASP)1, a key component in the regulation of actin polymerization and Rho GTPases control the integrity and reorganization of the actin filament system. Expression of the Rho member RhoD (green) in a porcine aortic endothelial cell triggers the formation of actin bundles (red), as well as, finger-like protrusions, so called filopodia, at the cell periphery. 12 Department of Microbiology, Tumor and Cell Biology – MTC PAR-6, a component of the polarity complex involved in the regulation of directed cell migration as well as for the establishment of epithelial cell polarity2. I was also involved in the identification of F-BAR domain-containing proteins3. The function of the F-BAR proteins is to coordinate cytoskeletal organization and membrane dynamics as they can bind and induce curvature to lipid bilayers. A new family of GTPases After returning to Sweden, I became a group leader at the Ludwig Institute for Cancer Research in Uppsala and my group continued to study the link between Rho GTPases and cytoskeletal organization4. In the search for novel Rho GTPases, a new family of GTPases was identified, which we named Miro (mitochondrial Rho)5. The Miro GTPases have emerged as key components in the regulation of mitochondrial movement along microtubules. Deregulated mitochondrial transport and morphology is linked to severe neuropathogical diseases, such as type 2A Charcot-Marie-Tooth disease and dominant optic atrophy. After twelve years in Uppsala, the time was ripe to move on and I settled with my group at MTC during 2008. My main reason for this decision was the feeling that this department could provide a dynamic and attractive work environment. Our work is to a large extent based on microscopy and the presence of the KI visualization facility (KIVIF) at MTC will be an asset. We intend to continue to study Miro, as well as the less known members of the Rho GTPases, to identify novel binding partners and to study how these networks of interactions can orchestrate cell functions. The microenvironment in tumors differs substantially from the surrounding tissue with regard to cell stiffness. Therefore, we want to study how extracellular force and rigidity affect vital processes such as cell contraction, migration and cell cycle progression. The diverse environments of the Karolinska Institute and the proximity to the Karolinska University hospital will be a benefit since the emerging view strongly indicates that Miro and Rho GTPases, as well as several of their binding partners, are dysfunctional or deregulated in cancer and in neuronal pathologies. It is my hope to increase the connections to clinical expertise in order to define to what extent the main targets for our research interests could also serve as targets for diagnostics and medication in human disease. Key publications 1. Aspenström P, Lindberg U and Hall A. Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich Syndrome. Curr Biol 6, 70, 1996. 2. Johansson A-S, Driessens M and Aspenström P. The mammalian homologue for the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for Cdc42. J Cell Sci 113, 3267, 2000. 3. Aspenström P. A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton. Curr Biol 7, 479, 1997. 4. Aspenström P, Fransson Å and Saras J. The Rho GTPases have diverse effects on the organization of the actin filament system. Biochem J 377, 327, 2004. 5. Fransson Å, Ruusala A and Aspen ström P. Atypical Rho GTPases have roles in mitochondrial homeostasis and apoptosis. J Biol Chem 278, 6495, 2003. Department of Microbiology, Tumor and Cell Biology – MTC 13 Awards, Major Grants and Appointments 2007 n Foreign Adjunct Professors Alexander von Gabain, Intercell AG, Vienna Biocenter, Austria; Margaret Liu, Carolina Vaccine Institute, University of North Carolina, USA; Birgit Lane, University of Dundee, Scotland UK and Centre for Molecular Medicine, Singapore, and Sir David Lane, University of Dundee, Scotland UK and Institute of Molecular and Cell Biology, Singapore, were appointed Foreign Adjunct Professors at MTC. n Adjunct Professors Annelie Brauner, Anneka Ehrnst, Gunilla Källenius and Åke Lundkvist were appointed Adjunct Professors at MTC. n New Professor Agneta Richter-Dahlfors was appointed Professor in Cellular Microbiology. n From MTC to the Government Former MTC group leader and Associate Professor Ewa Björling was appointed Minister of Foreign Trade in the Swedish Government by Prime Minister Fredrik Reinfeldt. n Peoples Republic of China National Friendship Award Ingemar Ernberg received the People’s Republic of China National Friendship Award from the president of the People’s Republic of China, Hu Jintao, and the premier of the People’s Republic of China, Wenjao Bao, in Beijing, China. The Award is the most important Chinese prize to foreign experts. n Peoples Republic of China Award for International Cooperation in Science and Technology Ingemar Ernberg received the People’s Republic of China Award for International Cooperation in Science and Technology by the Ministry of Science and Technology (MOST) in Beijing. n Best Scientific Achievement of the year Annelie Brauner was awarded Best Scientific Achievement of the year at the Karolinska University Hospital, Solna. 14 Department of Microbiology, Tumor and Cell Biology – MTC n Sven Gard Scholarship Hanna Sjölin received the Sven Gard Scholar ship for the best Virology Thesis in 2006. n New Associate Professors (Docents) Maria Isaguliants and Tobias Allander were appointed associate professor at KI. n Honorary Member of the Slovak Pediatric Society Annelie Brauner was elected Honorary Member of the Slovak Pediatric Society, a lifelong membership. n Knut and Alice Wallenberg Foundation Klas Kärre and co-applicants received a grant for the purchase of state of the art FACS equipment. n Ministry of Education of China n MTCers of 2007 The organizers of the MTC pub: “The Muppet Crew”, Danika Shepsis, Emma Hutric and Alberto Cagigi. n MTC Scientist of 2007 Mats Wahlgren. n MTC Paper of 2007 From the Yihai Cao group: “Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis” Lars Johan Nissen, Renhai Cao, Eva-Maria Hedlund, Zongwei Wang, Xing Zhao, Daniel Wetterskog, Keiko Funa, Ebba Bråkenhielm and Yihai Cao. J Clin Invest 117, 2766, 2007. n MTC Pedagogical Prize of 2007 Roland Möllby. Hao Mo received the Prize for Outstanding Chinese PhD Students, which comes with an award from the Chinese government. n Positions from the Swedish Research Council Pontus Aspenström received a 6-year Senior Researcher position for molecular studies of cell motility. Laura Plant and Markus Sköld received 4-year Research Assistant positions. n Position from the Swedish Childhood Cancer F oundation Marina Vita received a 2-year postdoc position. n Position from the Swedish Cancer S ociety Marie Arsenian Henriksson received the Senior Investigator Award from the Swedish Cancer Society. n MTC Annual Talks Poster prizes 2007 Keira Melican, Emilie Flaberg and Jakob Lovén. Ingemar Ernberg, recipient of the Peoples Republic of China National Friendship Award. 2008 n New Professors Rolf Ohlsson was appointed a strategic professorship in Genomic Integrity. Birgitta H enriques Normark was appointed Professor in Medical Microbial Pathogenesis. n KI University Board Klas Kärre was elected member of the University Board of the Karolinska Institutet for the coming three years. n KI Pedagogical Prize Annelie Brauner and Tomas Cronholm (MBB) received the KI Pedagogical Prize. Annelie Brauner received the prize for her outstanding achievements in education. n Science Business Innovation Board Hans Wigzell received The Bridge Award “for an individual who has done the most to promote policies for entrepreneurship in university or public research institutions”. n Chairman of the KI Cultural Board Ingemar Enberg was elected chairman of the KI Cultural Board, replacing professor Jan Lindsten. n Herbert J. Block Memorial Lectureship Award Georg Klein received the Herbert J. Block Memorial Lectureship Award, Ohio State University, USA. n Bill and Melinda Gates Foundation Gunilla Karlsson Hedestam and Bruce Beutler at the Scripps Research Institute received a 3-year grant to study “Immunoadjuvant effects influencing induction of anti-viral B cell responses”. n Torsten och Ragnar Söderberg Foundation Sven Pettersson and former MTC PhD student Fredrik Bäckhed received a grant for the project “Gut flora, peroxisome proliferating activating receptor (PPARg), adipocytes, macrophages and mechanisms underlying obesity”. Yihai Cao received a grant for the project “Angiogenesis and lymphangiogenesis in cancer and metastasis”. Galina Selivanova recieved a grant for the project “Targeting p53 to combat cancer: From laboratory bench to patient”. Poster competition winners at the MTC Annual Talks 2007 from left to right, PhD students Keira Melican, Emilie Flaberg and Jakob Lovén. n Positions from the Swedish Research Council Martin Rottenberg received a 6-year Senior Researcher position for studies of congenital immunity. Birgitta Henriques-Normark received a 50 percent 6-year Senior Researcher position in clinical bacteriology. Bence Rethi and Björn Önfelt received 4-year Research Assistant positions. Hanna Sjölin received a 3-year postdoc fellowship. Katarina Reis received a 2-year postdoc position. n KI Senior Researcher The Board of Research at Karolinska Institutet granted Katrin Pütsep funds for employment as a Senior Researcher for two years. n KI Ethics Council Marie Arsenian Henriksson was appointed as member of the KI Ethics Council. n Jonas Söderqvists Scholarship Åsa Hidmark received the 2008 year scholar ship from Jonas Söderqvists Scholarship Foundation. n The Swedish Foundation for Strategic Research Björn Önfelt was awarded a grant within the program “Future Research Leaders” to work at MTC and the Department of Applied Physics, The Royal Institute of Technology (KTH). n Wenner-Gren Foundations fellowship Hanna Sjölin has been awarded a WennerGren Fellowship for 3 years to study in the US with the possibility for an additional two year Research Assistant position on returning to Sweden. n MTC “15 year Celebration” Poster prizes Yuan Xue, Sandra Nilsson and Petra Luthje. n MTCers of 2008 The Economy Unit: Lada Larsson, Carina Lingonbacke and Eva Wichert. n MTC Junior Scientist of 2008 Anna Nilsson. n MTC Paper of 2008 From the Petter Höglund group: “The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells.” Petter Brodin, Tadepally Lakshimikanth, Susanne Johansson, Klas Kärre and Petter Höglund. Blood 113, 2434, 2009. n MTC Photographer of 2008 Lennart Nilsson. n MTC Artist of 2008 Ewert Linder. n MTC Life Science Achievement Award Hans Wigzell. n The Swedish Childhood Cancer Foundation Helén Nilsson received a 2-year postdoc position. Department of Microbiology, Tumor and Cell Biology – MTC 15 2009 n KI Distinguished Professor Awards Yihai Cao, Klas Kärre, Staffan Normark and Mats Wahlgren received this special grants for KI professors who carry out research of the highest quality at the international level. n Torsten och Ragnar Söderberg Foundation Mats Wahlgren received the 5-year Söderberg Research Professorship in Medicine awarded by The Royal Swedish Academy for Sciences. n The Royal Academy for Engineering Science (IVA) Hans Wigzell was awarded the Gold Medal for his entrepreneurial work within the field of Biotechnology. n EU grant Martin Rottenberg received a 3-year grant to coordinate an EU consortium, including nine countries, to study “Mycobacterium-host interactions”. n Position from Marianne och Marcus Wallenbergs Foundation Petter Höglund received a 3-year position. n International AIDS Vaccine Initiative Gunilla Karlsson Hedestam received a 5-year grant to investigate B cell responses to the HIV-1 envelope glycoproteins with a focus on induction of broadly neutralizing antibodies. n The Heart and Lung Foundation and King n The Royal Swedish Academy of Sciences Klas Kärre was elected member of the Royal Swedish Academy of Sciences. Oscar II’s Jubilee Foundation Markus Maeurer, Martin Rottenberg and Markus Sköld were awarded a grant for research on a recombinant BCG vaccine expressing novel antigens. n Academic Medal n Position from the Swedish Research Georg Klein was awarded the medal of Biomedicum from Helsinki, Finland. Council Gunilla Karlsson Hedestam was awarded a 6-year Senior Research position for development of novel innovative vaccines. n Research Strategy Committee (FSK) Galina Selivanova and Sten Nilsson were appoined coordinators for a new translational research center entitled: “Preclinical and clinical development of novel targeted anti-cancer therapies (ACT!).” They received the grant for the initial period 2010-2012 by the Research Strategy Committee, which is a collaborative committee between KI and Stockholm County Council (SLL). n Position from the Swedish Cancer Society Petter Höglund received a 50 percent 6-year research position from the Swedish Cancer Society. n Bill and Melinda Gates Foundation Markus Maeurer received a 2-year grant to study “Pattern recognition in immune responses directed against Mycobacterium tuberculosis (Mtb) using high content peptide microarray chips”. n New Associate Professors (Docents) Annika Karlsson and Elena Kasuba were appointed associate professor at KI. n The Swedish Society for Medical Research (SSMF) Mattias Forsell received a postdoctoral scholarship. n MTCer of of 2009 John Sennett. n MTC Scientists of of 2009 Galina Selivanova and Yihao Cao. n MTC Paper of of 2009 From the Rolf Ohlsson group: ”Nonallelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development”. Kuljeet Singh Sandhu, Chengxi Shi, Mikael Sjölinder, Zhihu Zhao, Anita Göndör, Liang Liu, Vijay K Tiwari, Sylvain Guibert, Lina Emilsson, Marta P Imreh and Rolf Ohlsson. Gen Dev, 23, 2598, 2009. n MTC Pedagogical Prize of 2009 Anneka Ehrnst. Markus Maeurer briefing Bill Gates on drug resistance in tuberculosis. Photo: Ewert Linder 16 Department of Microbiology, Tumor and Cell Biology – MTC Impressions from the MTC 15 years anniversary Speakers at the MTC Jubilee Symposium at Nobel forum June 4-5, 2008. Georg Klein explaining the main principles of tumor biology to an interested audience of technical and administrative staff in June 2008. KI President Harriet Wallberg-Henriksson opens the Jubilee Symposium on June 4, 2008. Hannah Akuffo, Francesca Chiodi and Peter Krammer mingling during a coffee break between the lectures on the Jubilee Symposium focusing on TB and HIV in November 2008. Department of Microbiology, Tumor and Cell Biology – MTC 17 Scientists in Focus Birgitta Henriques Normark From the Clinics to Basic Science I received my medical degree from the Karolinska Institutet (KI) in 1983 and then worked as a clinician for several years at different hospitals in the Stockholm area. I developed my interest in respiratory tract infections when I worked as an ear-noseand throat doctor/surgeon for a long time at Huddinge hospital. I first thought I would become a clinical fellow but when, before I did my internship, I was involved in research on the bacterium Clostridium difficile together with professor Monica Thelestam at the Department of Bacteriology. I enjoyed it so much that I decided that I would like to continue with research on microbes. After internship at Huddinge and Södertälje hospitals I started as a physician at The National Bacteriological Laboratory (SBL) that later became The Swedish Institute for Infectious Disease Control (SMI). I defended my thesis in the year 2000, became docent in 2004 and professor at KI in 2008. Translational approach My research focuses on the major pathogen Streptococcus pneumoniae (or pneumococci) and has a translational approach going from the patient to the small molecule. Pneumococci are a common cause of morbidity and mortality world-wide, primarily among children and the elderly, but also a common colonizer of the nasopharynx of pre-school children. To understand why pneumococcal Electron micrograph of pneumococcal pili. 18 Department of Microbiology, Tumor and Cell Biology – MTC infection can result either in healthy carriage, local or invasive disease we first built up a molecular epidemiological platform allowing us not only to serotype but also to group pneumococcal isolates into genetically related clonal types. We identified pneumococcal clones with different likelihood of causing carriage and invasive disease respectively. We also began to understand the correlation between clonal types and the severity of disease and other clinical aspects such as disease type, mortality and to what extent underlying diseases in the patients are required for certain clonal types to cause disease. Isolates belonging to defined clonal types were characterized in mice model infection and a genome wide analysis was performed by microarray analyses using a whole genome array that we set up, as well as whole genome sequencing. We identified pathogenicity islets being of importance for pneumococcal epidemicity as well as for disease outcome. A major finding was that pneumococci carry pilus like structures on their surface. a special emphasis on cell wall recognition by NOD proteins and the potential immunomodulatory role played by peptidoglycan receptors. Our research program also embraces other pathogens such as Group A streptococci, enterococci and Chlamydia. Promotes global spread 5 selected references The biogenesis, regulation, epidemiology, and structure as well as the role of the pilus and its components in disease are now being actively pursued. Our recent finding that piliation promotes global spread of antibiotic resistant pneumocococcal clones is particularly important and suggests that pilus proteins may be used as protective antigens in vaccines against the spread of resistant pneumococci. Host defense factors and innate immune responses involved in pneumococcal pathogenesis are actively being pursued in vivo and in vitro. Thus, we have defined the role of Toll like receptor signaling in host defense, characterized the role of neutrophil extracellular traps (NETs) and the interaction with dendritic cells in pneumococcal infection. The role of various host components in clearing the organism at local sites, and in the blood stream, is being pursued as well with 1. Beiter K et al., An endonuclease allows Streptococcus pneumoniae to escape Neutrophil Extracellular Traps. Curr Biol 16, 1, 2006. 2. Barocchi M et al., A Pneumococcal pilus influences virulence and host inflammatory responses. Proc. Natl Acad Sci USA 103, 2857, 2006. 3. Muschiol S et al., A small molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis. Proc Natl Acad Sci USA 103, 14566, 2006. 4. Sjöström K et al., Clonal success of piliated penicillin non-susceptible pneumococci. Proc Natl Acad Sci USA 104, 12907, 2007. 5. Henriques-Normark B et al., The rise and fall of bacterial clones: with a focus on Streptococcus pneumoniae. Nat Microbiol Rev 6, 827, 2008. Scientists in Focus Hannah Akuffo Towards an Adjunct Professorship in Parasitology I was educated in Ghana and England and have been keen to explore different areas within my life-long education. Bachelors’ degree in Biochemistry and Food Science in Ghana made me curious about immunology. Master of Science in Immunology in London focusing on BCG infection in mice, created an interest in a mouse model of leprosy. PhD and postdoctoral studies in London on the mechanisms of resistance to Mycobacterium lepraemurium infection in mice as a model for leprosy, led to a decision to study human leprosy. A position as senior scientist in Ethiopia taught me a lot about this disease, but I was also introduced to leishmaniasis, which like leprosy presents a spectrum of clinical manifestations. Coming to Sweden (for love) in 1986 and working at the Karolinska Institutet, but now a dedicated leishmaniac, my work concentrated on finding out why Swedish healthy individuals, unexposed to Leishmania, can still have a response to Leishmania parasites. The answer pointed to natural killer (NK) cells, taking my research interest towards understanding the interaction between NK cells and Leishmania parasites and the role of NK cells in the development and outcome of Leishmania infection. Working with human leishmaniasis has resulted in collaborations in Ethiopia, Iran, Nicaragua, Somalia, Sudan, USA and Uzbekistan with a coupled interest Ulcer formation during Leishmania major induced cutaneous leishmaniasis is associated with the expression of TRAIL and Fas in the epidermis. Photo by Liv Eidsmo. in research training of students from many of these countries. The irony for me and my heritage is that leishmaniasis is one of the few parasitic diseases that was not found in Ghana in the 1990s, which drew me to explore another disease which is manifested as a clinical spectrum, namely river blindness caused by the worm Onchocerca volvulus. These cellular immunology studies were performed in the Volta region of Ghana, in the World Health Organization (WHO) dedicated clinical trials center in Hohoe. The possibility to engage my students from Guatemala and Sweden in these activities in Ghana was very enriching. In 2003, an outbreak of skin ulcers in the Volta region of Ghana was found to be caused by leishmaniasis! Most people would advice that to get on in science one has to focus. I, however, believe that the knowledge gained from immunological studies on Mycobacteria, Leishmania and worms have culminated in our studies on de-worming and improving vaccine efficacy. Linking some experience from antileishmaniasis vaccine research in dogs, we set out to study the effect of de-worming on the response to BCG, the anti-tuberculosis vaccine which is highly effective in high income countries, but less effective in low income countries. Our studies in humans showed that de-worming indeed does improve BCG response and studies in mice showed that worms certainly compromised the efficacy of BCG vaccine against Mycobacterium tuberculosis infection. I had the good fortune, at the end of 1998, to be employed by The Department of Research Cooperation, SAREC, at the Swedish International Development Agency (Sida). This led me to obtain an increased understanding of research capacity building. In 2006 I formally became an Adjunct Professor in Parasitology. I am in a truly privileged situation. I have been able to remain in the academia doing research on diseases of importance for low income countries and at the same time I am able to work with Sida to contribute to enhancing the conditions for research and research capacity in these countries. This work also allows me to be involved in changes at the institutional and national levels to foster research efforts by individuals in low income countries. The experience from my work at MTC feeds directly into my work at Sida and vice versa. 5 selected references 1. Eidsmo L et al., The contribution of the Fas/FasL apoptotic pathway in ulcer formation during Leishmania major-induced cutaneous Leishmaniasis. Am J Pathol 166, 1099, 2005. 2. Nylén S et al., Surrogate markers of immunity to Leishmania major in leishmanin skin test negative individuals from an endemic area re-visited. Vaccine 24, 6944, 2006. 3. Lieke T et al., Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation. Clin Exp Immunol 153, 221, 2008. 4. Elias D et al., Poor immunogenicity of BCG in helminth infected population is associated with increased in vitro TGF-beta production. Vaccine. 26, 3897, 2008. 5. Nylén S and Akuffo H. Tracing immunity to human leishmaniasis. Future Microbiol 4, 241, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 19 Hans G Boman in Memoriam (1924–2008) Professor Hans G Boman was an exceptionally creative scientist whose ground-breaking discoveries have changed our understanding of the firstline of immune defence. He initiated the flourishing field of innate immunity and antimicrobial peptides. As professor emeritus, he moved from Stockholm University to join MTC and Karolinska Institutet (KI) in 1997. During his time at MTC, Hans G Boman was awarded some of the most important Nordic prizes, the Nordic Fernström Prize (2000), the Söderberg Prize (2002) and the Olof Rudbeck Prize (2005). He was honorary Doctor in Medicine at Umeå University and a member of the Royal Academy of Sciencies in Sweden. Hans is deeply missed by his colleagues and friends (Stromminger, 2009; Pütsep and Faye, 2009). peptide production is developmentally regulated rather than regulated by environmental challenge (Pütsep et al., 2000). One third of the world population is colonized by the gastric bacterium Helicobacter Hans G Boman’s research at MTC pylori, but most infections are asymptomatic. At MTC Hans formed, together with his Together with his colleagues at MTC, Hans wife Anita, what became his last research discovered that this commensal bacterium group, in which he focused on antimicropossess antibacterial activity, but is selfbial peptide expression and its regulation in resistant. Hans, and others, have suggested animals and man. As animal model systems, that H. pylori may have beneficial effects on Hans used frogs and mice. Frogs produce its host, as this antimicrobial peptide may high amounts of antimicrobial peptides in contribute to the defence against gastrointheir skin secretions. In a series of elegant testinal pathogens (Pütsep et experiments in collabo“Hans G Boman’s al., 1999). ration with colleagues in intellectual insight Working at a medical Rome, Hans could demonreached far beyond university, Hans wished to strate that glucocorticoids, the scientific world.” identify diseases with abera class of steroid hormones, rant expression of antimiinhibit antimicrobial peptide crobial peptides. Indeed, in a Lancet publiexpression in frog skin resulting in higher cation from 2002, Hans’ research group, in bacterial load (Miele et al., 1998; Simmaco et collaboration with haematologists at Karoal., 1998). Mice, in turn, protect the epithelinska University Hospital, demonstrated lial integrity of the small intestine largely deficiency in the production of the antimithrough high production of antimicrobial crobial peptide hCAP18/LL-37 in neutrophils peptides. Unexpectedly, germ-free animals of subjects with severe congenital neutropeand conventional mice showed essentially nia (Kostmann disease) (Pütsep et al., 2002). very small differences in peptide production. Kostmann subjects have an abnormally low Thus mice are already at birth prepared for a number of neutrophils, the most important co-existence with microbes as antimicrobial 20 Department of Microbiology, Tumor and Cell Biology – MTC type of white blood cell, in their blood. The loss of LL-37 production in neutrophils is most likely one reason for the subjects still being infection prone, in spite of treatment with human granulocyte colonystimulating factor, which elevates neutrophil levels. Since that publication a number of other groups have demonstrated the importance of antimicrobial peptides in various diseases, such as inflammatory bowels diseases, urinary tract infections and skin disorders. Foundation of a novel research field Hans entered his scientific career with the perspective of a biochemist. He received his PhD in Biochemistry in 1958 under the guidance of Arne Tiselius at Uppsala University, followed by post-doctoral studies on protein biosynthesis in bacteria in the excellent and dynamic research environment of Fritz Lipmann, a former Nobel Prize laureate, at the Rockefeller Institute in New York. He returned to Uppsala University in 1960, where he established his research group at the Department of Biochemistry with focus on the biosynthesis of tRNAs. In 1966, Hans was appointed the first professorship in Microbiology ever in Sweden at Umeå University. Initially, he focused on mechanisms of bacterial resistance against penicillins. However, he was soon fascinated by the discrepancy between the mounting time for an efficient adaptive immune response and the fast growth of bacteria. There had to be other efficient mechanisms for protection! Since insects lack the key factors of adaptive immunity, B cells and T cells, Hans turned to insects as model systems. In 1972 Hans demonstrated, in collaboration with Bertil Rasmusson, the existence of an inducible defence system in Drosophila (Boman et al., 1976). When Hans later moved to Stockholm University he devoted his research to unrav- elling the characteristics and mechanisms of this inducible defence system. It took Hans almost a decade and a change to the silk moth Hyalophyra cecropia as a model system to demonstrate that peptides with antimicrobial activity mediated the previously described inducible defence (Steiner et al., 1981). Other research groups entered the field and soon it became evident that immune defence mediated by antimicrobial peptides is an ancient and evolutionary conserved principle throughout all kingdoms of life. In collaboration with Viktor Mutt at KI, Hans characterized the antimicrobial peptides PR-39 and NK-lysin. The cloning of PR-39 by Hans and colleagues lead to the discovery of a major human antimicrobial peptide, LL-37 (Agerberth et al., 1995). New scientific avenues Throughout his scientific life, Hans strived to find new scientific avenues by asking the most simple, but unanswered, questions. Hans communicated this philosophy and his strategy for performing good research in an essay – How do you do Life Science? – which is available at the KI-website (Hans G Boman, KI website). Hans was a modern and free-thinking supervisor who took great interest and pleasure in discussions with students. He was an appreciated lecturer and when he moved to KI he engaged in graduate student courses, teaching theoretical research methodology, References l l l l Boman H, Nilsson GI and Rasmusson B. Inducible antibacterial defence system in Drosophila. Nature 237, 232, 1976. Steiner H, Hultmark D, Engström A, Bennich H and Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246, 1981. Agerberth B, Gunne H, Odeberg J, Kogner P, Boman HG and Gudmundsson GH. FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci USA 92, 195, 1995. Miele R, Ponti D, Boman HG, Barra D and Simmaco M. Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-κB and NF-IL6 binding sites in its promoter. FEBS Lett 431, 23, 1998. The antimicrobial peptide Cecropin P1 was previously considered to be synthesized by the pig intestine. Hans and his collegues could demonstrate that contaminating nematodes were the source of Cecropin P1 (Andersson et al., 2003). The large parasitic nematode worm of the genus Ascaris infects human (Ascaris suum) and pig (Ascaris lunbricoides) intestines. Mass spectrometry analysis of A) HPLC fraction with antimicrobial activity from Ascaris lumbricoides B) synthetic antimicrobial peptide Cecropin P1. Hans G Boman welcomed state-of-the-art techniques and with financial support from the Wallenberg foundation, he brought a MALDI-TOF spectrometer to MTC, which is still in use. The art of science as decision making. Being a scientist driven by curiosity, he was a frequent visitor of seminars on virtually any topic, including the MTC student seminars. In 2003, when Hans’ physical health began to create hindrances, he declined the position as a group leader. He was however, still actively participating in the scientific discussions and he concluded his view and perspectives on peptide-mediated defense in a review (Boman, 2003) and on science and academia in an autobiograpy (Boman, 2002). Hans inspired many colleagues at MTC and elsewhere at KI to search deeper into l l l l Simmaco M, Mangoni ML, Boman A, Barra D and Boman HG. Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora. Scand J Immunol 48, 357, 1998. Pütsep K, Brändén CI, Boman HG and Normark S. Antibacterial peptide from Helicobacter pylori. Nature 398, 671, 1999. Pütsep K, Axelsson LG, Boman A, Midtvedt T, Normark S, Boman HG and Andersson M. Germ-free and colonized mice generate the same products from enteric prodefensins. J Biol Chem 275, 40478, 2000. Pütsep K, Carlsson G, Boman HG and Andersson M. Deficiency of antibacterial peptides in patients with morbus Kostmann: an observation study. Lancet 360, 1144, 2002. the field of innate immunity. In addition to being an enthusiastic scientist, he surprised his colleagues with an arts exhibition of his collage paintings at KI in 2006. Hans G Boman’s intellectual insight reached far beyond the scientific world. It was genuinely enjoyable to discuss with him any subject, may it be movies, politics, literature, arts or music. Every summer, Hans together with his wife Anita went sailing for at least six weeks. Here, Hans could charge his batteries and find new perspectives on unsolved questions in biology. Katrin Pütsep l l l l l Boman HG. En egensinnig biologs liv, skildrat av honom själv. In free translation: ’The life of a headstrong biologist, told by himself.’ Autobiography 2002. Boman HG. Antibacterial peptides: basic facts and emerging concepts. J Intern Med 254, 197, 2003. Strominger JL. Pillars of Immunology. Animal antimicrobial peptides: ancient players in innate immunity. J Immunol 182, 6633, 2009. Pütsep K and Faye I. Hans G Boman (1924-2008): pioneer in peptide-mediated innate immune defence. Scand J Immunol 70, 317, 2009. How do you do Life Science? http://ki.se Department of Microbiology, Tumor and Cell Biology – MTC 21 22 Department of Microbiology, Tumor and Cell Biology – MTC High Impact Publications 2007–2009 Publications with a Journal Impact Factor above 8. 2007 Blood 2007, 109:643–52. Differentiation of CD1a- and CD1a+ monocyte-derived den– dritic cells is biased by lipid environment and PPARg. Gogolak P, Rethi B, Szatmari I, Lanyi A, Dezso B, Nagy L and Rajnavolgyi E. Neutrophil extracellular traps: casting the NET over pathogenesis. Wartha F, Beiter K, Normark S and Henriques-Normark B. Curr Opin Microbiol. 2007, 10:52–6. Proc Natl Acad Sci USA. 2007, 104:3919–3924. Filamin B deficiency in mice results in skeletal malformations and impaired microvascular development. Zhou X, Tian F, Sandzen J, Cao R, Flaberg E, Szekely L, Cao Y, Ohlsson C, Bergo MO, Boren J and Akyurek LM. J Clin Invest. 2007, 117:2362–2368. Angiogenesis modulates adipogenesis and obesity. Cao Y. A molecular link between malaria and Epstein-Barr virus reactivation. Chêne A, Donati D, GuerreiroCacais AO, Levitsky V, Chen Q, Falk KI, Orem J, Kironde F, Wahlgren M and Bejarano MT. PLoS Pathog. 2007, 3:e80. Proc Natl Acad Sci USA. 2007, 104:12140–5. Combinatorial protein therapy of angiogenic and arteriogenic factors remarkably improves collaterogenesis and cardiac function in pigs. Lu H, Xu X, Zhang M, Cao R, Bråkenhielm E, Li C, Lin H, Yao G, Sun H, Qi L,Tang M, Dai H, Zhang Y, Su R, Bi Y, Zhang Y and Cao Y. Proc Natl Acad Sci USA. 2007, 104:12907–12. Clonal success of piliated penicillin nonsusceptible pneumococci. Sjöström K, Blomberg C, Fernebro J, Dagerhamn J, Morfeldt E, Barocchi MA, Browall S, Moschioni M, Andersson M, Henriques F, Albiger B, Rappuoli R, Normark S and Henriques-Normark B. Nat Mater. 2007, 6:673–9. Electronic control of Ca(2+) signaling in neuronal cells using an organic electronic ion pump. Isaksson J, Kjäll P, Nilsson D, Robinson N, Berggren M and Richter-Dahlfors A. Adv Cancer Res. 2007, 98:1–16. Why do we not all die of cancer at an early age? Klein G, Imreh S and Zabarovsky ER. Gastroenterology. 2007, 133:808–17. Neuro peptide s receptor 1 gene polymorphism is associated with susceptibility to inflammatory bowel disease. D’Amato M, Bruce S, Bresso F, Zucchelli M, Ezer S, Pulkkinen V, Lindgren C, Astegiano M, Rizzetto M, Gionchetti P, Riegler G, Sostegni R, Daperno M, D’Alfonso S, Momigliano-Richiardi P, Torkvist L, Puolakkainen P, Lappalainen M, Paavola-Sakki P, Halme L, Farkkila M, Turunen U, Kontula K, Lofberg R, Pettersson S and Kere J. Proc Natl Acad Sci USA. 2007, 104:15364–9. IL-20 is an arteriogenic cytokine that remodels collateral networks and improves functions of ischemic hind limbs. Tritsaris K, Myren M, Ditlev SB, Hübschmann MV, van der Blom I, Hansen AJ, Olsen UB, Cao R, Zhang J, Jia T, Wahlberg E, Dissing S and Cao Y. Proc Natl Acad Sci USA. 2007, 104:15835–40. PfEMP1-DBL1a amino acid motifs in severe disease states of Plasmodium falciparum malaria. Normark J, Nilsson D, Ribacke U, Winter G, Moll K, Wheelock CE, Bayarugaba J, Kironde F, Egwang TG, Chen Q, Andersson B and Wahlgren M. Natural killer cells trigger differentiation of monocytes into dendritic cells. Zhang AL, Colmenero P, Purath U, Teixeira de Matos C, Hueber W, Klareskog L, Tarner IH, Engleman EG and Söderström K. Blood. 2007, 110:2484–93. J Clin Invest. 2007, 117:2766–2777. Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis. Nissen L J, Cao R, Hedlund EM, Wang Z, Zhao X, Wetterskog D, Funa K, Bråkenhielm E and Cao Y. Gastroenterology. 2007, 133:1499–509. DMBT1 confers mucosal protection in vivo and a deletion variant is associated with crohn’s disease. Renner M, Bergmann G, Krebs I, End C, Lyer S, Hilberg F, Helmke B, Gassler N, Autschbach F, Bikker F, Strobel-Freidekind O, GronertSum S, Benner A, Blaich S, Wittig R, Hudler M, Ligtenberg AJ, Madsen J, Holmskov U, Annese V, Latiano A, Schirmacher P, Amerongen AV, D’Amato M, Kioschis P, Hafner M, Poustka A and Mollenhauer J. The world according to MYC. Conference on MYC and the transcriptional control of proliferation and oncogenesis. Lüscher B and Larsson LG. EMBO Rep. 2007, 8:1110–4. Blood. 2007, 111:2693–703. Leukotriene B4 activates T cells which inhibit B cell proliferation in EBV infected cord blood derived mononuclear cell cultures. Liu A, Claesson HE, Mahshid Y, Klein G and Klein E. 2008 J Exp Med. 2008, 205:183–93. Developmental switch of intestinal antimicrobial peptide expression. Ménard S, Förster V, Lotz M, Gütle D, Duerr CU, Gallo RL, HenriquesNormark B, Pütsep K, Andersson M, Glocker EO and Hornef MW. Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies. Giefing C, Meinke AL, Hanner M, Henics T, Minh DB, Gelbmann D, Lundberg U, Senn BM, Schunn M, Habel A, HenriquesNormark B, Örtqvist Å, Kalin M, von Gabain A and Nagy E. J Exp Med. 2008, 205:117–31. Expression of granule-associated proteins in neutrophils from patients with severe congenital neutropenia. Andersson M, Karlsson J, Carlsson G and Pütsep K. Blood. 2007, 110:2772–3. Nature. 2007, 449:663. Science & Politics: when ministers are well primed. Wigzell H. Department of Microbiology, Tumor and Cell Biology – MTC 23 Citation indicators Two common bibliometric indicators for MTC publications 2005-2008. Both indicators compare citation counts for MTC articles to citation counts for world articles from the same year and in the same research field. A field normalized citation score average of 1,5 (2005– 2008) means that MTC publications are cited 50% above the world average. A Top 5% value of 8% (the average of MTC for 2005-2008) means that 8% of MTC publications are among the worlds 5% most highly cited. Publications from 2009 are still too new to be evaluated with citation counts. More information about bibliometric indicators used at Karolinska Institutet is available at http://ki.se/bibliometrics. Certain data included herein are derived from the Web of Science® prepared by THOMSON REUTERS®, Inc. (Thomson®), Philadelphia, Pennsylvania, USA: ©Copyright THOMSON REUTERS® 2010. All rights reserved. J Exp Med. 2008, 205:63–77. An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses. Harari A, Bart PA, Stöhr W, Tapia G, Garcia M, MedjitnaRais E, Burnet S, Cellerai C, Erlwein O, Barber T, Moog C, Liljestrom P, Wagner R, Wolf H, Kraehenbuhl JP, Esteban M, Heeney J, Frachette MJ, Tartaglia J, McCormack S, Babiker A, Weber J and Pantaleo G. Gut. 2008, 57:764–71. Secreted enteric antimicrobial activity localizes to the mucus surface layer. Meyer-Hoffert U, Hornef MW, Henriques-Normark B, Axelsson LG, Midtvedt T, Pütsep K and Andersson M. The challenges of eliciting neutralizing antibodies to HIV-1 and to influenza virus. Karlsson Hedestam GB, Fouchier RA, Phogat S, Burton DR, Sodroski J and Wyatt RT. Nat Rev Microbiol. 2008, 6:143–55. 2,2 Field Normalized Citation Score rococcus faecalis from newborn babies regulate endogenous PPARg activity and IL-10 levels in colonic epithelial cells. Are A, Aronsson L, Wang S, Greicius G, Lee YK, Gustafsson JA, Pettersson S and Arulampalam V. Nat Cell Biol. 2008, 10:211–9. Membrane nano- tubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Köhler K, Oddos S, Eissmann P, Brodsky FM, Hopkins C, Onfelt B, Sattentau Q and Davis DM. 24 Department of Microbiology, Tumor and Cell Biology – MTC Top 5% 11% 2,0 10% 1,8 9% 1,6 8% 1,4 7% 1,2 6% 1,0 5% 0,8 4% 0,6 3% 0,4 2% 0,2 1% 0% 0,0 2005 2006 Genome Res. 2008, 18:370–9. Segmental duplications and evolutionary plasticity at tumor chromosome break- prone regions. DaraiRamqvist E, Sandlund A, Müller S, Klein G, Imreh S and Kost-Alimova M. J Clin Invest. 2008, 118:913–23. VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats. Li Y, Zhang F, Nagai N, Tang Z, Zhang S, Scotney P, Lennartsson J, Zhu C, Qu Y, Fang C, Hua J, Matsuo O, Fong GH, Ding H, Cao Y, Becker KG, Nash A, Heldin CH and Li X. Proc Natl Acad Sci USA. 2008, 105:4358–63. Helicobacter pylori evolution during progression from chronic atrophic gastritis to gastric cancer and its impact on gastric stem cells. Giannakis M, Chen SL, Karam SM, Engstrand L and Gordon JI. Proc Natl Acad Sci USA. 2008, 105:5489–94. EBV- Proc Natl Acad Sci USA. 2008, 105:1943–8. Ente- World Average encoded EBNA-6 binds and targets MRS18–2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes. Kashuba E, Yurchenko M, Yenamandra SP, Snopok B, Isaguliants M, Szekely L and Klein G. Nat Immunol. 2008, 9, 477–80. Natural killer cell recognition of missing self. Kärre K. Twin ushers guide pili across the bacterial outer membrane. Daniels R and Normark S. Cell. 2008, 133:574–6. 2007 2008 Blood. 2008, 112:1461–71. Neutrophil secretion products pave the way for inflammatory monocytes. Soehnlein O, Zernecke A, Eriksson EE, Rothfuchs AG, Pham CT, Herwald H, Bidzhekov K, Rottenberg ME, Weber C and Lindbom L. Priming of T cells to Fas-mediated proliferative signals by interleukin-7. Rethi B, Vivar N, Sammicheli S, Fluur C, Ruffin N, Atlas A, Rajnavolgyi E and Chiodi F. Blood. 2008, 112:1195–204. Adv Cancer Res. 2008, 100:113–131. Molecular mechanisms and therapeutic development of angiogenesis inhibitors. Cao Y. Proc Natl Acad Sci USA. 2008, 105:10167–72. FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue. Xue Y, Cao R, Nilsson D, Chen S, Westergren R, Hedlund EM, Martijn C, Rondahl L, Krauli P, Walum E, Enerbäck S and Cao Y. Proc Natl Acad Sci USA. 2008, 105:13203–5. A review of Judah Folkman’s remarkable achievements in biomedicine. Cao Y and Langer R. Proc Natl Acad Sci USA. 2008, 105:15028–33. Inflammation and autoimmunity caused by a SHP1 mutation depend on IL-1, MyD88, and a microbial trigger. Croker BA, Lawson BR, Berger M, Eidenschenk C, Blasius AL, Moresco EM, Sovath S, Cengia L, Shultz LD, Theofilopoulos AN, Pettersson S and Beutler BA. PLoS Pathog. 2008, 4:e1000171. B cell recognition on the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4. Forsell MN, Dey B, Mörner A, Svehla K, O’dell S, Högerkorp CM, Voss G, Thorstensson R, Shaw GM, Mascola JR, Karlsson Hedestam GB, Wyatt RT. Högerkorp CM, Voss G, Thorstensson R, Shaw GM, Mascola JR, Karlsson Hedestam GB and Wyatt RT. 2009 Cell Metab. 2009, 9:99–109. Hypoxia-independ- ent angiogenesis in adipose tissues during cold acclimation. Xue Y, Petrovic N, Cao R, Larsson O, Lim S, Chen S, Feldmann HM, Liang Z, Zhu Z, Nedergaard J, Cannon B and Cao Y. Proc Natl Acad Sci USA. 2009, 106:7939–44. Timing of HAART defines the integrity of memory B cells and the longevity of humoral responses in HIV-1 vertically-infected children. Pensieroso S, Cagigi A, Palma P, Nilsson A, Capponi C, Freda E, Bernardi S, Thorstensson R, Chiodi F and Rossi P. Proc Natl Acad Sci USA. 2009, 106:859-63 Towards a genetics of cancer resistance. Klein G. Mycobacterial glycoconjugates as vaccine candidates against tuberculosis. Källenius G, Pawlowski A, Hamasur B and Svenson SB. Trends Microbiol. 2008, 16:456–62. Nat Rev Microbiol. 2008, 6:827–37. The rise and fall of bacterial clones: Streptococcus pneumoniae. Henriques-Normark B, Blomberg C, Dagerhamn J, Bättig P and Normark S. Circ Res. 2008, 103:1092–9. Vascular endothelial growth factor-A and platelet-derived growth factor-B combination gene therapy prolongs angiogenic effects via recruitment of interstitial mononuclear cells and paracrine effects rather than improved pericyte coverage of angiogenic vessels. Korpisalo P, Karvinen H, Rissanen TT, Kilpijoki J, Marjomäki V, Baluk P, McDonald DM, Cao Y, Eriksson U, Alitalo K and Ylä-Herttuala S. Cytokine Growth Factor Rev. 2008, 19:395–404. Interferon and cytokine responses to Crimean Congo hemorrhagic fever virus; an emerging and neglected viral zonoosis. Weber F and Mirazimi A. Proc Natl Acad Sci USA. 2008, 105:18513–8. Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancerassociated systemic syndrome. Xue Y, Religa P, Cao R, Hansen AJ, Lucchini F, Jones B, Wu Y, Zhu Z, Pytowski B, Liang Y, Zhong W, Vezzoni P, Rozell B and Cao Y. Blood. 2008, 112:4401–10. Altered expression of the receptor-ligand pair CXCR5/CXCL13 in B-cells during chronic HIV-1 infection. Cagigi A, Mowafi F, Phuong Dang LV, TennerRacz K, Atlas A, Grutzmeier S, Racz P, Chiodi F and Nilsson A. Great times for small molecules: c-AMP, a second messenger candidate in Bacteria and Archaea. Römling U. Sci Signal. 2008, 1:pe39. Positive and negative modulation of angiogenesis by VEGFR1 ligands. Cao Y. Sci Signal. 2009, 2:re1. Blood. 2009, 113:2434–41. The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Brodin P, Lakshmikanth T, Johansson S, Kärre K and Höglund P. Nat Rev Genet. 2009, 10:269–76. Replication timing and epigenetic reprogramming of gene expression: a two-way relationship? Göndör A and Ohlsson R. Proc Natl Acad Sci USA. 2009, 106:6152–7. VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis. Zhang F, Tang Z, Hou X, Lennartsson J, Li Y, Koch AW, Scotney P, Lee C, Arjunan P, Dong L, Kumar A, Rissanen TT, Wang B, Nagai N, Fons P, Fariss R, Zhang Y, Wawrousek E, Tansey G, Raber J, Fong GH, Ding H, Greenberg DA, Becker KG, Herbert JM, Nash A, Yla-Herttuala S, Cao Y, Watts RJ and Li X. NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo. Lakshmikanth T, Burke S, Ali TH, Kimpfler S, Ursini F, Ruggeri L, Capanni M, Umansky V, Paschen A, Sucker A, Pende D, Groh V, Biassoni R, Höglund P, Kato M, Shibuya K, Schadendorf D, Anichini A, Ferrone S, Velardi A, Kärre K, Shibuya A, Carbone E and Colucci F. Cancer Cell. 2009, 15:171–83. MDM2-Dependent downregulation of p21 and hnRNP K provides a switch between apoptosis and growth arrest induced by pharmacologically activated p53. Enge M, Bao W, Hedström E, Jackson SP, Moumen A and Selivanova G. Proc Natl Acad Sci USA. 2009, 106:11966–71. The proapoptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease. Nagy N, Matskova L, Kis LL, Hellman U, Klein G and Klein E. J Clin Invest. 2009, 119:1251–63. Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis. Grinkevich VV, Nikulenkov F, Shi Y, Enge M, Bao W, Maljukova A, Gluch A, Kel A, Sangfelt O and Selivanova G. Cancer Cell. 2009, 15:441–53. Proc Natl Acad Sci USA. 2009, 106:14478–83. Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Cheung AK, Lung HL, Ko JM, Cheng Y, Stanbridge EJ, Zabarovsky ER, Nicholls JM, Chua D, Tsao SW, Guan XY and Lung ML. PLoS Pathog. 2009, 5:e1000584. Getting to grips with strangles: an effective multi-component recombinant vaccine for the protection of horses from Streptococcus equi infection. Guss B, Flock M, Frykberg L, Waller AS, Robinson C, Smith KC and Flock JI. Nature. 2009, 461:212–7. Chromosome crosstalk in three dimensions. Göndör A and Ohlsson R. Department of Microbiology, Tumor and Cell Biology – MTC 25 Proc Natl Acad Sci USA. 2009, 106:17505–10. Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature. Hedlund EM, Hosaka K, Zhong Z, Cao R and Cao Y. Semin Cancer Biol. 2009, 19:338–43. Improvement of antiangiogenic cancer therapy by understanding the mechanisms of angiogenic factor interplay and drug resistance. Cao Y, Zhong W and Sun Y. Semin Cancer Biol. 2009, 19:277–8. Angiogen- esis in malignancy. Cao Y. Cell. 2009, 139:679–92. Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression. Genander M, Halford MM, Xu NJ, Eriksson M, Yu Z, Qiu Z, Martling A, Greicius G, Thakar S, Catchpole T, Chumley MJ, Zdunek S, Wang C, Holm T, Goff SP, Pettersson S, Pestell RG, Henkemeyer M and Frisén J. Proc Natl Acad Sci USA. 2009, 106:19866–71. MRPS18–2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell-like properties. Kashuba E, Pavan Yenamandra S, Deoram Darekar S, Yurchenko M, Kashuba V, Klein G and Szekely L. Proc Natl Acad Sci USA. 2009 106:18408–13. Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arteriallymphatic conduits in zebrafish and glass catfish. Dahl Ejby Jensen L, Cao R, Hedlund EM, Söll I, Lundberg JO, Hauptmann G, Steffensen JF and Cao Y. Genes Dev. 2009, 23:2598–603. Nonallelic trans- vection of multiple imprinted loci is organized by the H19 imprinting control region during germline development. Sandhu KS, Shi C, Sjölinder M, Zhao Z, Göndör A, Liu L, Tiwari VK, Guibert S, Emilsson L, Imreh MP and Ohlsson R. Proc Natl Acad Sci USA. 2009, 106:19485–90. Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model. Lee SL, Rouhi P, Dahl Jensen L, Zhang D, Ji H, Hauptmann G, Ingham P and Cao Y. Semin Cancer Biol. 2009, 19:411–20. Endemic Burkitt’s lymphoma as a polymicrobial disease: new insights on the interaction between Plasmodium falciparum and Epstein-Barr virus. Chêne A, Donati D, Orem J, Mbidde ER, Kironde F, Wahlgren M and Bejarano MT. Semin Cancer Biol. 2009 19:407–10. To the genesis of Burkitt lymphoma: regulation of apoptosis by EBNA-1 and SAP may determine the fate of Ig-myc translocation carrying B lymphocytes. Nagy N, Klein G and Klein E. Connections between MTC research groups and their main research areas (as identified with MeSH terms). The MeSH headings must occur over 10 times in MTC articles to be shown and the connection to each individual research group must be at least 2 articles strong to show up as a line. The MeSH Headings used in this analysis were from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. NLM represents that its data were formulated with a reasonable standard of care. Except for this representation, NLM makes no representation or warranties, expressed or implied. This includes, but is not limited to, any implied warranty of merchantability or fitness for a particular purpose, with respect to the NLM data, and NLM specifically disclaims any such warranties and representations. Certain data included herein are derived from the Web of Science ® prepared by THOMSON REUTERS ®, Inc. (Thomson®), Philadelphia, Pennsylvania, USA: ©Copyright THOMSON REUTERS® 2010. All rights reserved. 26 Department of Microbiology, Tumor and Cell Biology – MTC Schistosome worm sections “Andyfied” by Ewert Linder. Department of Microbiology, Tumor and Cell Biology – MTC 27 Research Reports Immunobiology Immunology Research MTC is one of the most active Departments at the Karolinska Institutet (KI) in the field of immunology. This is in no small part thanks to our excellent animal facility, which harbors a number of animal models for human disease and an extensive panel of genetically modified mice of interest to immunologists. Many mouse models to study immune responses to infectious agents as well as anti-tumor responses and autoimmunity are available at MTC and the number of mouse strains housed at the facility is continuously expanding. While the majority of the immunobiology projects at MTC rely on pre-clinical experimental systems, there is also a focus on translational research. Several investigators have a clinical background and/or collaborate with clinicians at Karolinska University Hospital or the Stockholm South General Hospital (Södersjukhuset), which ensures access to important expertise and unique patient samples. Also of note, the KI-wide research network “KI immunology and inflammation (KiiM)” was initiated from MTC and researchers from MTC play active roles in managing this network. The regular KiiM Gunnel Biberfeld Francesca Chiodi retreat is an increasingly popular event, which has attracted a number of distinguished international speakers over the years. Natural killer cell immunobiology A highly visible research area at MTC is the field of natural killer (NK) cell immunobiol- Petter Höglund Mikael Jondal Gunilla Karlsson Hedestam ogy. Several research groups study NK cells to understand their development and function, as well as the mechanisms underlying the process of NK cell education. The activities of NK cells are regulated by a number of activating and inhibitory receptors that can be manipulated to alter NK cell func- Project leaders: Louise Berg Maria Johansson Gerald McInerney Björn Önfelt Bence Rethi Markus Sköld Jonas Sundbäck Klas Kärre 28 Peter Liljeström Markus Maeurer Department of Microbiology, Tumor and Cell Biology – MTC Martin Rottenberg Hans Wigzell Research Reports Immunobiology Photo: Lasse Skog tion. Groups at MTC have shown that by blocking inhibitory receptors expressed by NK cell, enhanced rejection of leukemia cells or enhanced anti-viral activity against HIV-1 or herpes viruses infected cells may be achieved. NK cells in organ-specific autoimmunity are also studied, such as NK cells in the pancreas in a mouse model of human type I diabetes. Furthermore, new technological approaches for understanding how NK cells and T cells communicate with target cells to exert their function were recently established at MTC. Human immune responses Many additional basic immunology questions are also investigated at MTC, such as how immunological aging occurs and how this relates to the loss of thymic tissue. Efforts are also underway to extend the mouse model beyond its current use by engrafting human CD34+ hematopoietic stem cells to develop mice harboring human immune cells to study human immune responses to pathogens such as mycobacteria. A Mycobacterium tuberculosis (Mtb) aerosol infection model has also been set up, (HIV-1). The mechanisms through which T which mimics the natural route of infection and B cells are damaged during HIV-1 infecallowing the analysis of monocyte differentiation are studied and amongst other findings it tion and function during was shown that activationMtb infection. A further induced apoptosis increase “While the majority of the effort in the field of Mtb is with disease progression. immunobiology projects to identify target antigens The need to halt the at MTC rely on pre-clinical within the Mtb proteome global spread of HIV-1 calls experimental systems, for use in diagnostics for improved approaches to there is also a focus on or as vaccine antigens. induce protective immune translational research.” Additional bacteria and responses against this parasites that are studgenetically diverse virus. ied at MTC include the bacteria Salmonella, This goal is approached by several groups and Chlamydia, Listeria and Pneumococci and has resulted in the development of recomthe parasites Trypanozomes and Leishmania, binant viral vaccine vectors and engineered all of which have evolved means to interfere protein antigens, some of which have entered with the host immune response. clinical trials already. The need to stimulate responses to highly conserved and often subdominant viral epitopes puts special demands The mechanisms of HIV-1 infection on the design of vaccine immunogens and The interaction between viruses and the host on understanding how to focus T and B cell immune system is another main area within responses on relevant antigenic determinants. the field of immunology at MTC. An imporThis is an active area of investigation at MTC, tant viral pathogen that causes chronic infecwhich also covers the mechanism of action of tion and, in the absence of anti-retroviral thervaccine adjuvants. apy, renders the host immune system defective is human immunodeficiency virus type 1 Gunilla Karlsson Hedestam Department of Microbiology, Tumor and Cell Biology – MTC 29 Research Reports Immunobiology Translational studies of NK cell function in HIV and herpes virus infection We investigate natural killer (NK) cell function in patients with HIV infection and in healthy children in relation to early encounter of herpes virus infections. NK cells Louise Berg are innate lymphocytes regulated [email protected] by expression of a multitude of inhibitory and activating receptors, interacting with endogenous or virally encoded cell surface bound ligands. Although antiretroviral therapy greatly delays disease progression in HIV, there is still no cure for infection. We focus on investigating NK cell based treatment in HIV. Susanne Johansson has characterised the function and receptor expression of NK cells from viremic patients, patients under antiretroviral treatment and patients who efficiently control viremia without treatment. She has seen that blockade of NK cell inhibitory receptors increases NK cell recognition of HIV infected autologous T cells in vitro. We are presently studying antibody dependent cellular cytotoxicity (ADCC) in HIV, and aim to investigate whether inhibitory receptor blockade in combination with ADCC may be an effective treatment strategy in HIV. This project is run in collaboration with the Stockholm South General Hospital. NK cells have been extensively studied in herpes virus infection, mainly with cytomegalovirus (CMV) (Figure). In a collaboration with researchers at Stockholm University, we have investigated NK cells during childhood Immune evasion strategies of and seen that recepcytomegalovirus. tor expression develops during the first years of life. In addition, we found that children who encounter herpes virus infections early in life have a decreased monocyte induced NK cell function, as seen during asymptomatic chronic EBV and CMV infection. This may bear impact on the development of allergy, which is less frequent in children encountering early infections with herpes viruses. We conclude that environmental factors, such as chronic herpes virus infections, shape innate immune functions in early life. This project is run in collaboration with clinicians from the Department of Pediatrics, Sachs’ Children’s Hospital at the Stockholm South General Hospital. Prevention of mother-tochild transmission of HIV in Dar es Salaam, Tanzania My research group has collaborated with the Muhimbili University of Health and Allied Sciences in Dar es Salaam, Tanzania for more than 20 years in the Swedish International Gunnel Biberfeld Development Cooperation Agency [email protected] (Sida)-supported TANSWED HIV program. Two of the main projects in this program are: “Prevention of mother-to-child transmission of HIV” and “Evaluation of HIV vaccines”. About 400,000 children become HIV-infected each year in subSaharan Africa, the majority through mother-to-child transmission (MTCT) of HIV. Prophylactic antiretroviral (ARV) treatment is given to HIV-infected mothers and their infants before and around delivery to reduce the risk of early MTCT of HIV in developing countries. However, this prophylactic treatment does not prevent postnatal HIV transmission through breast milk. Replacement feeding from birth is not feasible, safe or affordable in most resource-limited settings. We have performed two studies in Dar es Salaam to evaluate prevention of breast milk transmission of HIV by giving extended ARV prophylaxis to HIV-infected mothers or to their infants during breast feeding for 6 months after delivery. ARV treatment continued for mothers who needed treatment for their own health. These studies showed that infant or maternal ARV prophylaxis for 6 months during breast feeding resulted in a similar low risk of acquisition of infant HIV infection between 6 weeks A mother and her child waitand 6 months (1%) and a similar low ing for examination in the research clinic in Dar es cumulative infant HIV infection rate at Salaam. 6 months of age (5%). Based on these and other similar studies in sub-Saharan Africa, the World Health Organization has issued new guidelines for prevention of postnatal infection in infants of HIV-infected mothers who do need treatment for their own health recommending ARV prophylaxis to either the mother or the infant during the whole duration of breast feeding. Selected publications l Selected publications l l l 30 Sundström Y, Nilsson C, Lilja G, Kärre K, Troye-Blomberg M and Berg L. The expression of human NK cell receptors in early life. Scand J Immunol 66, 335, 2007. Shagafian-Hedengren S, Sundström Y, Sohlberg E, Nilsson C, Linde A, Troye-Blomberg M, Sverremark-Ekström E and Berg L. Herpesvirus seropositivity in childhood associates with decreased monocyte-induced NK-cell IFN-g production. J Immunol, 182, 2511, 2009. Johansson S, Hinkula J, Hejdeman B, Johansson M, Wahren B, Wagtmann N R, Kärre K and Berg L. NK-cell activation by KIR binding antibody 1-7F9 and response to in vitro HIV infected autologous cells in viremic and controller HIV infected patients. Clin Immunol 134, 158, 2010. Department of Microbiology, Tumor and Cell Biology – MTC l l Kilewo C, Karlsson K, Massawe A, Lyamuya E, Swai A, Mhalu F, Biberfeld G and the Mitra Study Team. Prevention of mother-tochild transmission of HIV-1 through breast-feeding by treating infants prophylactically with lamivudine in Dar es Salaam, Tanzania: the Mitra Study. J Acquir Immune Defic Syndr 48, 315, 2008. Sandström E, Nilsson C, Hejdeman B, Bråve A, Bratt G, Robb M, Cox J, Vancott T, Marovich M, Stout R, Aboud S, Bakari M, Pallangyo K, Ljungberg K, Moss B, Earl P, Michael N, Birx D, Mhalu F, Wahren B, Biberfeld G and the HIV Immunogenicity Study 01/02 Team. Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara. J Infect Dis 198, 1482, 2008. Kilewo C, Karlsson K, Ngarina M, Massawe A, Lyamuya E, Swai A, Lipyoga R, Mhalu F, Biberfeld G and the Mitra Plus Study Team. Prevention of mother-to-child transmission of HIV-1 through breastfeeding by treating mothers with triple antiretroviral therapy in Dar es Salaam, Tanzania: the Mitra Plus study. J Acquir Immune Defic Syndr 52, 406, 2009. Research Reports Immunobiology Understanding the mechanism of damage induced by HIV-1 to T and B cells may lead to faster development of a HIV-1 vaccine Only a proportion of T cells which die during HIV-1 infection are directly infected by the virus; and memory B cells, whose number is reduced during Francesca Chiodi HIV-1 infection, are not a [email protected] direct target for HIV-1. My group aims to understand the mechanism(s) through which T and B cells are damaged during HIV-1 infection. To determine whether dysfunctional cell homing may play a role in impairments of B cell responses, the expression of chemokine receptors and their respective ligands were examined on B cells. We reported a decreased CXCR5 expression and an increased expression of CXC chemokine ligand 13 (CXCL13) on B cells from HIV-1 infected patients, in association with low CD4 T cell counts. Altered expression of the chemokine receptor-ligand pair, CXCR5/ CXCL13, may thus participate in the establishment of B cell dysfunctions during chronic HIV-1 infection. We reasoned that an early control of viral replication through anti-viral treatment could preserve the normal development of the memory B cell compartment and responses to routine childhood vaccines. Accordingly, we evaluated the effects of different medication schedules in 70 HIV-1 vertically-infected pediatric subjects on B cell functions. We showed that timing of medication initiation is the major factor predicting the longevity of B cell responses in vaccinated HIV-1-infected children. The Fas receptor, primarily associated with T and B cell depletion in lymphopenic settings, was previously shown to contribute to compensatory Tcell expansion. As IL-7 levels are elevated during HIV-1 infection in association with increased Fas expression, we analyzed whether IL-7 influences Fas-mediated proliferative signals in T cells. We showed that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. High IL-7 levels may simultaneously induce sensitivity to Fas-mediated apoptosis in non-activated T cells and increase Fas-induced co-stimulatory signals in T cells recognizing low-affinity antigens. Dissecting natural killer cell tolerance in models of transplantation and autoimmunity Natural killer (NK) cells mediate graft-versus-leukemia effects after stem cell transplantation and contribute to autoimmunity. NK cell functions are regulated by MHC class I molecules, which Petter Höglund also control development of NK [email protected] cells, a process we have termed “NK cell education”. A major objective of our work is to identify the molecular mechanisms for this education process using murine model systems. The inhibitory receptors, called Ly49 in mice, and their MHC ligands are both encoded in multigenic gene families and display polymorphisms and crossreactivities. By studying transgenic mice expressing individual MHC class I alleles in isolation, pairs and triplets using polychromatic flow cytometry protocols, we are able to dissect the system despite this complexity. We have found that some MHC class I alleles have a strong “educating” impact on the NK cell system, while some alleles have a weak impact. In addition, we have shown that NK cell education is regulated quantitatively in individual NK cells. Several novel insights into the process of NK cell education have been delinated, which are further studied in clinical collaborations involving patients undergoing stem cell transplantation against hematological malignancy. In a second objective, NK cells in organ-specific autoimmunity is studied. Non-obese diabetic (NOD) mice develop a type I diabetes similar to the human disase. We focus on NK cells in the target organ, the pancreas. Pancreatic NK cells show an altered phenotype compared to NK cells in the hematopoietic system and localize both in the islets of Langerhans and in the exocrine part of the organ. The fraction of NK cells inside the islets increased as disease progresses, implyAn islet of Langerhans with infiltrating ing a local role of NK cells in NK cells. Red = Insulin; blue = T cells and green = NK cells. diabetes pathogenesis. NK cell depletion studies suggest that NK cells indeed promote the development of diabetes, and our future work focusses on identifying the cellular and molecular regulation of this effect. Selected publications Selected publications l l l Cagigi A, Mowafi F, Phuong Dang LV, Tenner-Racz K, Atlas A, Grutzmeier S, Racz P, Chiodi F and Nilsson A. Altered expression of the receptor-ligand pair CXCR5/CXCL13 in B cells during chronic HIV-1 infection. Blood 112, 4401, 2008. Rethi B, Vivar N, Sammicheli S, Fluur C, Ruffin N, Atlas A, Rajnavolgyi E and Chiodi F. Priming of T cells to Fas-mediated proliferative signals by interleukin-7. Blood 15, 112, 2008. Pensieroso S, Cagigi A, Palma P, Nilsson A, Capponi C, Freda E, Bernardi S, Thorstensson R, Chiodi F and Rossi P. Timing of HAART defines the integrity of memory B cells and the longevity of humoral responses in HIV-1 vertically-infected children. Proc Natl Acad Sci USA 106, 7939, 2009. l l l Johansson S, Salmon-Divon M, Johansson MH, Pickman Y, Brodin P, Kärre K, Mehr R and Höglund P. Probing natural killer cell education by Ly49 receptor expression analysis and computational modelling in single MHC class I mice. PLoS One 4, e6046, 2009. Brodin P, Lakshmikanth T, Johansson S, Kärre K and Höglund P. The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Blood 113, 2434, 2009. Brauner H, Elemans M, Lemos S, Broberger C, Holmberg D, Flodström-Tullberg, M, Kärre K and Höglund P. Distinct Phenotype and function of natural killer cells in the pancreas of non obese diabetic mice. J Immunol 184, 2272, 2010. Department of Microbiology, Tumor and Cell Biology – MTC 31 Research Reports Immunobiology Natural killer cells – anti-tumor reactivity and self-tolerance during inhibitory receptor blockade Our work aims at developing and using mouse models where antileukemic reactivity can be improved by employing natural killer (NK) cells. NK cells are Maria Johansson lymphocytes in the innate [email protected] immune system and their activity is regulated by activating and inhibitory receptors. We study well characterized inhibitory receptors for self MHC class I on NK cells. By blocking these receptors, one can induce NK cell rejection of leukemia cells in mice. We found that this anti-tumor reactivity can be potentiated by exogenous IL-2. In addition, we investigated the risk of breaking NK cell tolerance to normal cells by such treatment, and found no signs of autoreactivity (Vahlne G et al., 2008). The presence of NK cell activating ligands on normal hematopoietic cells suggest that also such cells should be killed during inhibitory receptor blockade. We are now further investigating the basis for the robust self-tolerance during treatment. We are also developing mouse models for utilization of NK cells in hematopoietic stem cell (HSC) transplantation (see report by Blocking of inhibitory receptors on NK cells Klas Kärre). In closely induce killing of tumor cells related projects we are investigating if blockade of inhibitory NK cell receptors could induce beneficial reactivity against virus infected cells by mouse as well as by human NK cells (see report by Louise Berg). Sensitive and accurate assays are a cornerstone of experimental research. We are developing single cell assays for cellular effector functions. We have adapted to the mouse NK cell system, a method for measuring exocytosis of secretory lysosomes (degranulation) utilizing cell surface detection of the marker CD107a (Vahlne G et al., 2008). By the use of multicolor flow cytometry, this analysis can be combined with analysis of other functions such as cytokine production as well as with phenotypic analysis. The involution of the thymus gland Immunological aging occurs at many levels within the immune system but one decisive factor is the loss of thymic tissue (thymic involution) as thymus is the only site where Mikael Jondal new T cells can be formed. The decline in [email protected] the formation of T cells leads to an agerelated loss of function within the cellular immune system. Thymic involution is especially related to the formation of sex hormones, formed during puberty. In mice, we have found that both epithelial and lymphoid (thymocytes) cells in the thymus can synthesize corticosterone (CS), a glucocorticoid which can kill lymphoid cells by the initiation of a cellular suicide program called “apoptosis”. For this reason we are testing the hypothesis that an age-related CS synthesis in the thymus contribute to thymic involution. We have found that CS synthesis in pre-puberty mice occurs mainly in epithelial cells whereas at puberty, thymocytes start to synthesize CS. This synthesis can be triggered by the male sex hormone testosterone in mice that have no endogenous production of this hormone (castrated mice). In addition, the pharmacological inhibition of local CS synthesis in thymocytes in old mice resulted in a strong regeneration of thymic tissue (see Figure) resulting in an increased formation of thymocytes. We are presently investigating how the reversal of thymic involution impacts the diversity and function of T cells in the peripheral immune system. As the immune system declines with age, infectious disease is a common cause of death in the elderly. This is becoming an issue of growing medical importance as the mean life expectancy is rapidly increasing, from around 40 to 80 years during the last 200 years. We believe that regeneration of thymic tissue in the elderly may boost cellular immunity and thus contribute to a longer and healthier life in this population. The inhibition of local glucocorticoid synthesis in the thymus may be one way to achieve this. Selected publications l l l 32 Vahlne G, Becker S, Brodin P and Johansson M.H. IFN-g production and degranulation are differentially regulated in response to stimulation in murine NK cells. Scand J Immunol 67,1, 2008. Johansson, S, Salmon-Divon M, Johansson M H, Pickman Y, Brodin P, Kärre K, Mehr R and Höglund P. Probing natural killer cell education by Ly49 receptor expression analysis and computational modeling in single MHC class I mice. PLoS ONE 4, e6046, 2009. Johansson S.E, Hejdeman B, Hinkula J, Johansson M.H, Romagné F, Wahren B, Wagtmann N R, Kärre K and Berg L. NK cell activation by KIR-binding antibody 1-7F9 and response to HIV-infected autologous cells in viremic and controller HIV-infected patients. Clin Immunol 134, 158, 2010. Department of Microbiology, Tumor and Cell Biology – MTC The effect of inhibition of the local glucocorticoid synthesis in the thymus. In treated mice there is a pronounced increase in thymocyte numbers seen as an expansion of the cortical area of the gland. Selected publications l l l Qiao S, Chen L, Okret S and Jondal M. Age-related synthesis of glucocorticoids in thymocytes. Exp Cell Res 314, 3027, 2008. Chen L, Fredholm BB and Jondal M. Adenosine, through the A1 receptor, inhibits vesicular MHC class I cross-presentation by resting DC. Mol Immunol 45, 2247, 2008. Qiao S, Okret S and Jondal M. Thymocyte-synthesized glucocorticoids play a role in thymocyte homeostasis and are down-regulated by adrenocorticotropic hormone. Endocrinology 150, 4163, 2009. Research Reports Immunobiology Virus-cell interactions and induction of anti-viral B cell responses MHC class I molecules: recognition by NK cells and T cells, novel functions in nerve cells We use experimental systems to investigate how immune responses are induced following virus infection and immunization. The quality of adaptive Gunilla Karlsson Hedestam immune responses is determined [email protected] by early events following the initial encounter between the pathogen and the host. We believe that these processes can be manipulated at multiple levels to improve the design of human vaccines. We are particularly interested in elicitation of anti-viral B cell responses as neutralizing antibodies are the correlates of protection for most vaccines that provide protection against infection and disease. Several projects in the group concern antibody responses against the HIV-1 envelope glycoproteins (Env), for which we have created a range of recombinant variants that lack or do not lack defined structural determinants. We recently showed that these immunogens stimulate B cell responses that are markedly different, both in terms of the specificities and the functions of the elicited antibodies. By further developing these approaches we hope to focus vaccine-induced responses on relevant neutralizing antibody targets to enhance the protective effect. To facilitate these analyses, we have developed new methods to enumerate different classes of B cells in immunized and infected subjects. In a different project, we investigate the intrinsic adjuvant effect of viruses, primarily using Semliki Forest virus (SFV), but also other RNA viruses. In one approach, we use SFV for an in vivo screen in mice to search for non-redundant cellular pathways that are required for elicitation of B cell responses against virus-encoded antigens. We have identified a number of candidate genes that are now under further investigation. While many projects in the group are based on in vivo models, we also study direct interactions between viruses and host cells in vitro to understand anti-viral signaling events, stress responses and antigen presentation, all of which influence the eliciThe HIV-1 envelope core structure with tation of adaptive immune key amino acid residues highlighted. responses. Natural killer (NK) cells express a panel of germ line encoded activating and inhibitory receptors. Most inhibitory ligands Klas Kärre are expressed by normal [email protected] cells, e.g. MHC class I molecules. Some activating ligands are also expressed on normal cells, while others are expressed only in stressed, transformed or infected cells. NK cells make decisions by integrating the input via these receptors. The consequence when activation prevails is release of cytokines (e.g. IFN-γ) and triggering of cytotoxicity, allowing NK cells to act in innate responses to tumors, transplants and infectious agents. A main challenge is to understand critical receptor-ligand interactions during NK cell development and education as well as in different pathophysiological situations. This is explored in collaborations with other MTC scientists Petter Höglund, Maria Johansson, Björn Önfelt, Louise Berg and Jonas Sundbäck (see their respective project reports). In addition, we have collaborated with E Carbone and F Colucci to identify NK receptors involved in recognition of primary and metastatic melanoma. As to autoimmunity, we have shown that the NKG2A receptor is a key regulator of cytokine production by NK cells in the synovial fluid of rheumatoid arthritis patients. Through M Uhlin, we have collaborated with the “Centre for Allogeneic Bone Marrow Transplantation” (KI Huddinge), in attempts to optimize expansion of T cells of therapeutic interest. A transdisciplinary collaboration led by S Cullheim at Department of Neuroscience, KI, has revealed a novel role for MHC class I molecules in synaptic plasticity and regeneration of motor neurons after axon injury, exerting influence on the afferent input as well as on restoration of function at the motor end plate. Selected publications Selected publications l l l Forsell MNE, Dey B, Mörner A, Svehla K, O’dell S, Högerkorp C-M, Voss G, Thorstensson R, Shaw G, Mascola J, Karlsson Hedestam GB and Wyatt RT. B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4. PLoS Pathog 4:e1000171, 2008. Mörner A, I Douagi A, Forsell M N E, Sundling C, Dosenovic P, O’dell S, Dey B, Kwong P, Voss G, Thorstensson R, Mascola J, Wyatt R and Karlsson Hedestam GB. HIV-1 Env-trimer immunization of macaques and impact of priming with viral vector or stabilized core protein. Virol 83, 540, 2009. Dosenovic P, Chakrabarti B, Soldemo M, Douagi I, Forsell MNE, Li Y, Phogat A, Paulie S, Hoxie J, Wyatt R T and Karlsson Hedestam G B. Selective expansion of HIV-1 Env-specific B cell subsets recognizing distinct structural elements following immunization. Immunol 183, 3373, 2009. Lesion induced increase of motoneuron MHC class Ia, with predominant expression in regenerating axons and at re- innervated neuromuscular junctions (NMJ). Figure shows colocalization between H2-Db (middle panel) and presynaptic markers (left panel) in normal and reinnervated NMJs (from Thams et al., 2009). l l l de Matos CT, Berg L, Michaëlsson J, Felländer-Tsai L, Kärre K and Söderström K. Activating and inhibitory receptors on synovial fluid natural killer cells of arthritis patients: role of CD94/NKG2A in control of cytokine secretion. Immunology 122, 291, 2007. Lakshmikanth T, Burke S, Ali TH, Kimpfler S, Ursini F, Ruggeri L, Capanni M, Umansky V, Paschen A, Sucker A, Pende D, Groh V, Biassoni R, Höglund P, Kato M, Shibuya K, Schadendorf D, Anichini A, Ferrone S, Velardi A, Kärre K, Shibuya A, Carbone E and Colucci F. NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo. J Clin Invest 119, 1251, 2009. Thams S, Brodin P, Plantman S, Saxelin R, Kärre K and Cullheim S. Classical major histocompatibility complex class I molecules in motoneurons: new actors at the neuromuscular junction. J Neurosci 29,13503, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 33 Research Reports Immunobiology Vaccines and Immunity We are developing new vaccines against infectious disease and cancer. In this context, we study innate and acquired immune responses to various types of vaccines. We also build Peter Liljeström vehicles for use in vaccination and one of our [email protected] most central vaccine technology is in-house developed alphavirus replicon system based on Semliki Forest virus (SFV), our working horse for many years. Using SFV as model we have studied how viruses interact with the host cell and how they trigger innate immune responses. Using this knowledge we are in a position to know how vaccines based on the SFV platform work and what kind of immune responses are obtained. We have also studied how additional innate receptor ligands can be incorporated in the SFV platform for driving the immune response in certain directions as for example to enhance the humoral (antibody) response. We have compared a number of vaccine technologies in an Display of the 4E10 peptide effort to expand our understanding on epitope of the HIV-1 gp41 envehow different vaccines induce protec- lope protein (picture: Daniel Johansson). tive immune responses. We have also combined different vaccine technologies in so called prime-boost regimens and found that such approaches modulate the responses in various ways. Our work has also included in-depth analysis of T cell responses in terms of quality and longevity with the goal of defining immune correlates for protection. We have found that there are significant differences between viral vector vaccines. In the area of HIV/AIDS, we have participated in the development of novel vaccine candidates, some of which have already entered several clinical trials. Indeed, a DNA prime – NYVAC (poxvirus) boost vaccination regimen proved to be very promising and is now scheduled to move into larger clinical testing. In our future work, we will study the immune responses to a variety of vaccine candidates and characterize in detail the responses in terms of quality, memory and recall capacity. In particular, we want to define how the initial innate responses in each case orchestrate the acquired immune responses. Visualizing protective immune responses Cellular CD4+ and CD8+ T cell responses are protective against tuberculosis and contribute to long-term containment of Mycobacterium tuberculosis (Mtb) infection. Definition of Mtb specific CD4+ and Mark Maeurer CD8+ T cell response would be desirable [email protected] to gauge protective immune responses in individuals i) vaccinated with BCG and/or ii) exposed to Mtb, but who are protected (latent TB). Screening of the 4500 proteins from Mtb with T cells from clinically well defined individuals (active BCG vaccination, latent TB = protected, active TB) is not feasible, yet screening for serum reactivity (IgG and IgA) is possible. We predicted that serum IgG and IgA responses are indicative of T cell responses. B-cell and CD4+ T cell responses are closely related: Screening of high-content Mtb chips with serum from clinically very well defined populations enabled us to select promising target antigens to gauge for clinically and biologically relevant biomarkers associated with Mtb infection and to identify rational vaccine targets. Humoral Mtb targets were screened for CD8+ and CD4+ T cell targets using peptide arrays and soluble recombinant MHC class II molecules. A particular feature of long-lasting memory CD8+ T cell responses turned out to be the expression of the homodimer CD8aa – and the dependency on IL-7-signaling. IL-7-signaling can be significantly impaired in long-lasting chronic infections and be altered by tissue-specific expression of IL-7 isoforms (alternative splicing). Of great interest, IL-7 and an alternatively spliced isoform showed effects on differentiation of human neuronal stem cells. We are currently testing promising Mtb vaccine candidates and evaluate IL-7 isoforms to augment long-lived cellular protective immune responses. Selected publications l l l 34 Näslund TI, Uyttenhove C, Nordström EK, Colau D, Warnier G, Jondal M, Van den Eynde BJ and Liljeström P. Comparative prime-boost vaccinations using Semliki Forest virus, adenovirus and ALVAC vectors demonstrate differences in the generation of a protective central memory CTL response against the P815 tumor. J Immunol 178, 6761, 2007. Harari A, Bart PA, Stöhr W, Tapia G, Garcia M, Medjitna-Rais E, Burnet S, Cellerai C, Erlwein O, Barber T, Moog C, Liljeström P, Wagner R, Wolf H, Kraehenbuhl JP, Esteban M, Heeney J, Frachette MJ, Tartaglia J, McCormack S, Babiker A, Weber J and Pantaleo G. An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional and long-lasting T cell responses. J Exp Med 205, 63, 2008. Mooij P, Balla-Jhagjhoorsingh SS, Beenhakker N, van Haaften P, Baak I, Nieuwenhuis IG, Heidari S, Wolf H, Frachette MJ, Bieler K, Sheppard N, Harari A, Bart PA, Liljeström P, Wagner R, Pantaleo G and Heeney JL. Comparison of human and rhesus macaque T cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens. J Virol 83, 5881, 2009. Department of Microbiology, Tumor and Cell Biology – MTC Humoral recognition pattern analysis using a peptide microarray chip. Pattern obtained in serum from individuals who received a 5 component pertussis vaccine (left, top panel) vs placebo (left, bottom) defined by epitope recognition intensities. Differences are visualized to the right: recognition hotspots induced by vaccination. Selected publications l l l Weichold FF, Mueller S, Kortsik C, Hitzler WE, Wulf M, Hone DM, Sadoff JC and Maeurer M. Impact of MHC class I alleles on the M. tuberculosis antigen-specific CD8+ T cell response in patients with pulmonary tuberculosis. Genes Immun 8, 334, 2007. Gaseitsiwe S, Valentine D, Ahmed R Mahdavifar S, Magalhaes I Zerweck J, Schutkowski M, Gautherot E, Montero F, Ehrnst A, Reilly M and Maeurer M. MHC class II molecule- peptide complex analysis on a microarray chip. Clin Vaccine Immunol. 16, 567, 2009. Moors M, Vudattu N K, Abel J, Kramer U, Rane L, Ulfig N, Seccatelli S, Fritsche E and Maeurer M. Interleukin-7 and Interleukin-7 splice variants differentiate human neural progenitor cells. Genes Immun 11, 11, 2010. Research Reports Immunobiology Regulation of gene expression during viral infection All viruses, regardless of their host tropism, genome structure or any other properties, must use components of the host cell gene expression machinery in order to propagate. This reliance has led Gerald McInerney to the development of an evolutionary [email protected] arms race between viruses and hosts. Viral strategies have evolved to ensure access to this machinery while host cells have developed means to recognize viruses as foreign entities and to restrict their expression. In turn, viruses have developed methods to counteract these restrictions and to reduce the cells’ ability to communicate with its neighboring cells. Our work concerns the mechanisms of regulation of gene expression during the early stages of viral infections. Using Semliki Forest virus (SFV) as a model, we are studying viral interactions with diverse cellular pathways and systems in order to understand the biology of the virus-infected cell. SFV induces the transient formation of stress granules in the cytoplasm of infected cells (McInerney et al., 2005). These granules are sites of mRNA triage which may play a role in the shut off of host cell mRNA translation. We are currently investigating the mechanisms by which these granules are assembled and disassembled and the nature of their contribution to SFV cytotoxicity. SFV infection induces the activation and nuclear localization of type I interferon transcription factors IRF-3 and NFκB (see figure). One of the SFV non-structural proteins, nsp2, also localizes to the nucleus of infected cells and acts to inhibit the expression of type I interferon (Breakwell, et al., 2007). Our recent work is aimed at understanding how this viral protein inhibits host cell innate immune signaling. In addition to these projects, we work closely with other members of the Gunilla Karlsson Hedestam group towards our common goal of a deeper understanding the cell biology of virus infection and the generation of virus-specific immune responses. Semliki Forest virus dependent activation of interferon regulatory factor 3: IRF3 staining (red) shows that the protein is activated by translocation to the nucleus only in the SFV-EGFP infected cells (green). Altered T cell homeostasis in HIV-1 infection T lymphocytes show high sensitivity to spontaneous and death receptor-induced apoptosis following HIV-1 infection. The death receptor molecule Fas may play an important role in T cell depletion as Fas expression on T cells, the levels of FasL and sensitivity to activation-induced apoptosis are all increased in parallel with disease progression in HIV-1 Bence Rethi [email protected] infected patients. In our studies we analyzed how the anti-apoptotic and T cell stimulatory cytokine IL-7 modulates the function of Fas on T cells. IL-7 is particularly interesting in HIV infection as the concentration of this cytokine is increasing in parallel with CD4+ T cell depletion, possibly as a feedback mechanism that counteracts further T cell loss. We demonstrated that IL-7 upregulates Fas expression on naïve and memory T cells and induces a cytoskeleton-dependent Fas polarization on the cell surface. T cells, when treated with IL-7, undergo apoptosis upon Fas cross-linking. IL-7 concentrations in the serum of HIV-1 infected patients correlated with levels of Fas expression on T cells and the sensitivity to Fas-mediated apoptosis. Fas, on the other hand, can stimulate proliferation when triggered on the surface of sub-optimally activated T cells. We demonstrated that T cells of HIV-infected individuals are characterized by high sensitivity to Fas-mediated costimulatory signals. Our results suggest a scenario where high IL-7 doses increase Fas expression and induce Fas membrane polariza- Regulation of Fas-mediated T cell apoptosis by IL-7 in lymphopenic conditions. tion, an expression pattern reminiscent of chronically activated T cells. Triggering of Fas molecules may lead to increased apoptosis of non-activated T cells whereas Fas signals, when induced in weakly activated T cells, may rather contribute to proliferation. T cell modulation by IL-7 gained further importance in light of recent advances in IL-7 therapy applied to lymphopenic patients. We continue to study how IL-7 modulates T cells functions with special emphasis on T cell-B cell communication. Selected publications Selected publications l l l Douagi I, McInerney GM, Hidmark AS, Miriallis V, Johansen K, Svensson L and Karlsson Hedestam GB. Role of interferon regulatory factor 3 in type I interferon responses in rotavirus-infected dendritic cells and fibroblasts. J Virol 81, 2758, 2007. Breakwell L, Dosenovic P, Karlsson Hedestam GB, D’Amato M, Liljeström P, Fazakerley J and McInerney GM. Semliki Forest virus nonstructural protein 2 is involved in suppression of the type I interferon response. J Virol 81, 8677, 2007. McInerney G and Karlsson Hedestam G. Direct cleavage, proteasomal degradation and sequestration: Three mechanisms of viral subversion of the type I interferon response. J Innate Immun 1, 599, 2009. l l l Fluur C, De Milito A, Fry TJ, Vivar N, Eidsmo L, Atlas A, Federici C, Matarrese P, Logozzi M, Rajnavölgyi E, Mackall CL, Fais S, Chiodi F and Rethi B. Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection. J Immunol 178, 5340, 2007. Fluur C, Rethi B, Thang PH, Vivar N, Mowafi F, Lopalco L, Foppa CU, Karlsson A, Tambussi G and Chiodi F. Relationship between serum IL-7 concentrations and lymphopenia upon different levels of HIV immune control. AIDS 21, 1048, 2007. Rethi B, Vivar N, Sammicheli S and Chiodi F. Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work? AIDS 23, 745, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 35 Research Reports Immunobiology Studies on the control of bacterial and parasitic infections The global threats to public health posed by the human pathogen Mycobacterium tuberculosis (Mtb) the causative agent of pulmonary tuberculosis (TB) require our immeMarkus Sköld diate attention. Our goal is to better [email protected] understand protective immunity during pulmonary TB focusing on the in vivo role of monocyte-derived cells. Monocytes are formed in the bone marrow and are constantly released into circulation during steady-state and inflammatory conditions. Using a newly developed monocyte adoptive transfer model, we have shown that many of the macrophage and dendritic cell (DC) subsets that appear in Mtb infected lung tissue and pulmonary lymph nodes (PLN) are monocyte-derived. Macrophages and DC are well suited to influence multiple aspects of host immunity during pulmonary TB. Our data show that activated monocyte-derived macrophages recruited to Mtb infected lungs induce expression of enzymes important for their bactericidal activity. In contrast, monocytes recruited to infected PLN draining the lung tissue acquire a mature DC phenotype to help initiate the adaptive immune response by priming naïve T lymphocytes. To study the host immune response during pulmonary TB we have, together with our colleagues at the Swedish Institute for Infectious Disease Control, established a murine low dose (20-200 colony forming units) nose-only Mtb aerosol infection model in a Biosafety Level-3 animal facility. This system mimics the natural Rod-shaped acid-fast bacilli (stained red) route of infection and detectable in lung tissue sections from infected mice infected with M. tuberculosis. allows us to determine how monocyte differentiation and function correlates with Mtb susceptibility. Better understanding of susceptibility to Mtb in the mouse model may provide valuable insights into susceptibility in humans. Because Mtb is the largest killer of people co-infected with human immunodeficiency virus-1 (HIV-1), we are currently extending our studies on monocyte function and the pathogen-specific T cell response in vivo to a unique murine model of Mtb/HIV co-infection. Selected publications Selected publications mechanisms. l l l 36 Monocyte differentiation and function during pulmonary tuberculosis We study the role and regulation of IFN-γ secretion and responses during intracellular chlamydial and mycobacterial infections, focusing on the role of “suppressor of cytokine signaling 1”, Martin Rottenberg a protein that inhibits IFN-γ signaling Hans Wigzell (Figure). We found that bacterial innate [email protected] receptor agonists induce SOCS1 which hampers bacterial growth control in macrophages and bacterial antigen presentation. SOCS1 inhibited not only IFN-γ-mediated responses but also the secretion of IFN-γ itself. In vivo, SOCS1 hampered Mycobacterium tuberculosis and Chlamydia growth control, but protected the host from severe pulmonary inflammation. We propose that targeting SOCS1 in defined cellular populations will contribute to protection against these infections. Immunodeficient mice inoculated with CD34+ human hematopoietic stem cells generate human myeloid and lymphoid cell populations de novo. We have used such a humanized mouse model to study human immune responses to mycobacteria. We found that human T cells differentiate into effector memory cells, migrate into the lung and secrete human IFN-γ in humanized mice infected with mycobacteria BCG. Human T cells were able to protect mice infected with BCG. The generation of human adaptive immune responses and the role of different T cell populations in the control of mycobacterial infections in humanized mice is currently being studied. There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with the protozoan Trypanosoma brucei. Trypanosomes do not synthesize their own purines. Cordycepin was selected from a parasite viability screening of a library of nucleoside analogues. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice even after parasites had penetrated into the brain. Cordycepin is cheap, non-toxic, and has been tested in clinical trials in leukemic patients. Our studies support the clinical testing of treatment with cordycepin and deoxycoformycin against SOCS1 is induced by bacterial innate recephuman African tor ligands and hampers secretion of IFN-γ, and trypanosomiasis. thereby the induction of IFN-γ-dependent bacterial Yang T, Stark P, Janik K, Wigzell H and Rottenberg ME. SOCS-1 protects against Chlamydia pneumoniae-induced lethal inflammation but hampers effective bacterial clearance. J Immunol 180, 4040, 2008. Vodnala SK, Ferella M, Lundén-Miguel H, Betha E, van Reet N, Amin DN, Oberg B, Andersson B, Kristensson K, Wigzell H and Rottenberg ME. Preclinical assessment of the treatment of secondstage african trypanosomiasis with cordycepin and deoxycoformycin. PLoS Negl Trop Dis 3:e 495, 2009. Claes F, Vodnala SK, van Reet N, Boucher N, Lunden-Miguel H, Baltz T, Goddeeris BM, Buscher P and Rottenberg ME. Bioluminescent imaging of Trypanosoma brucei shows preferential testis dissemination which may hamper drug efficacy in sleeping sickness. PLoS Negl Trop Dis 3,e 486, 2009. Department of Microbiology, Tumor and Cell Biology – MTC l l l Sköld M and Behar SM. Tuberculosis triggers a tissue dependent program of differentiation and acquisition of effector functions by circulating monocytes. J Immunol 181, 6349, 2008. Kiener HP, Watts GFM, Cui Y, Wright J, Thornhill TS, Sköld M, Behar SM, Niederreiter B, Lu J, Cernadas MM, Coyle AJ, Sims G, Smolen J, Warman ML, Brenner MB and Lee DM. Synovial fibroblasts self-direct multicellular lining architecture and synthetic function in three-dimensional organ culture. Arthritis Rheum 62, 742, 2010. Sada-Ovalle I, Sköld M, Tian T, Besra GS and Behar SM, a-Galactosylceramide as a therapeutic agent for pulmonary Mycobacterium tuberculosis infection. Am J Respir Crit Care Med, In press, 2010. Research Reports Immunobiology Regulation of anti-bacterial responses of macrophages by the receptor TRAPC We have cloned and are studying the function of a novel triggering receptor expressed on macrophages, dendritic cells and B cells, i.e. antigen presenting cells, which Jonas Sundbäck we have named TRAPC. TRAPC [email protected] belongs to the TREM family of molecules, which contains family members involved in activating macrophages during bacterial infections, are expressed on osteoclasts and involved in bone resorption, and are involved in modulating the function of microglial cells. The TRAPC receptor associates with the adaptor signaling molecule DAP12, and its expression is upregulated by bacterially derived LPS. Cross-linking of TRAPC on macrophages and dendritic cell activates the cells to produce nitric oxide, and protects macrophages from bacterial infection, suggesting a role for the receptor in early defense from bacteria. It has been shown that TLR signaling can be inhibited by DAP12 signaling. The TRAPC receptor can balance signals from TLR receptors in order to fine-tune the receptor signaling in presence or absence of a bacterial infection. One way to increase the understanding of the function of the receptor is to identifying the ligand for TRAPC. We have constructed a TRAPC-Fc fusion protein to use in the search of the ligand. This fusion protein bound a potential ligand on macrophage cell lines, and will continue by immunoprecipitating and protein sequencing to identify the ligand. Given the ability of TRAPC to balance LPS mediated activation of macrophages in an experimental setting of bacterial infection, it would be interesting to further study the role of the receptor using in vivo models, and to extend the studies to other cells where it is expressed. Genomic localization of TRAPC on mouse chromosome 17. Visualizing immune cell interactions The primary focus of our research is to understand the processes in which natural killer (NK) cells and T cells communicate with and eliminate cells that are potentially harmful to the body. The heterogeneity regarding Björn Önfelt the receptor expression patterns and effector [email protected] functions of the target cell populations makes these phenomena utterly complex. Most immunological setups in this field focus on readouts based on the average of the cell population. Thus far we have pursued the goal of developing new assays that can be used in academic research as well as the pharmaceutical and health care industry. We focus on approaches that provide the means to study the behavior of single cells and small subpopulations of cells. In the future this will enable us to effectively study how individual NK cells respond to different conditions. Cell mediated immunity by NK cells is achieved by the formation of an immune synapse. This is a highly organized subcellular interface between the NK and the target cell involving a number of communication processes leading to secretion of cytotoxic molecules and killing of the target cell. To address heterogeneity of NK cell populations we have developed two m i c ro ch i p - b a s e d approaches for high Schematic of a microchip made in silicon-glass resolution imaging mounted in a custom made holder. Black line of individual NK shows a trajectory of a NK cell (blue) interacting cells or NK cell-tarwith tumor target cells (yellow). get-cell conjugates over extended periods of time. The cells are restricted in space by miniature wells organized in an array format or an ultrasonic cage, which promotes and facilitates studies of cell-cell interactions. In order to characterize the intermittent movements of lymphocytes we implement a method that has previously been used in single molecule tracking studies. That makes it possible to automatically detect and quantify the periods of transient migration arrest that is typical for these cells. The method allows quick assessment and quantification of altered migration properties of cells during e.g. drug treatments and infections. Selected publications l Selected publications l l l Sandberg J K, Franksson L, Sundbäck J, Michaëlsson J, Petersson M, Achour A, Wallin R P, Sherman N E, Bergman T, Jörnvall H, Hunt D F, Kiessling R and Kärre K. T cell tolerance based on avidity thresholds rather than complete deletion allows maintenance of maximal repertoire diversity. J Immunol 165, 25, 2000. Sundbäck J, Achour A, Michaëlsson J, Lindström H and Kärre K. NK cell inhibitory receptor Ly-49C residues involved in MHC class I binding. J Immunol 168, 793, 2002. Chen, L, Sundbäck J, Olofsson S and Jondal M. Interference with O-glycosylation in RMA lymphoma cells leads to a reduced in vivo growth of the tumor. Int J Cancer 119, 1495, 2006. l l Sowinski S, Jolly C, Berninghausen O, Purbhoo M A, Chauveau A, Kohler K, Oddos S, Eissmann P, Brodsky F M, Hopkins C, Önfelt B, Sattentau Q and Davis DM. Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 10, 211, 2008. Benninger R K P, Vanherberghen B, Young S, Taner S B, Culley F J, Schnyder T, Neil M A, Wüstner D, French P M, Davis DM and Önfelt B. Live cell linear dichroism imaging reveals extensive membrane ruffling within the docking structure of natural killer cell immune synapses. Biophy J 96, L13, 2009. Manneberg O, Vanherberghen B, Önfelt B and Wiklund M. Flowfree transport of cells in microchannels by frequency-modulated ultrasound. Lab on a Chip 9, 833, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 37 Research Reports Infection Biology Infection Biology Research – broad, yet clearly focused to meet the emerging challenges ahead Infection biology includes all the disciplines of classical medical microbiology. Yet, medical microbiology of today tends to study microbes in their close relation to the host, host responses and in the perspective of the co-evolution of microbes with their hosts. Thus, apart from describing the pathogens themselves, infection biology also deals with events such as microbe-induced host cell apoptosis, signal transduction, actin polymerization and cell cycle control. As a result, infection biology has experienced a strong development into a multi- 38 and often a collaborative tradition at MTC. The fact that many research groups working within the subject area are involved in a number of national and international collaborations and the functional closeness to the Swedish Institute for Infectious Disease Control, all adds to the diversity and resources. disciplinary research field with obvious connections with immunology during the last decade, including innate immunity and inflammation, vaccinology and even carcinogenesis. Not surprisingly therefore, the research area of infection biology has found a rather fruitful platform for establishing itself within MTC. Topics covering bacterial, parasitic and viral pathogenesis, the activity of virulence factors, the formation of bacterial biofilms, molecular basis for resistance to antimicrobial agents, host responses and vaccine development have a long-standing Infection biology research at MTC Despite modern hygiene standards and treatment regimens, infectious diseases still pose a major threat to human health. Classical menaces such as malaria, pneumonia and Hannah Akuffo Jan Albert Tobias Allander Annelie Brauner Lars Engstrand Anneka Ehrnst Jan-Ingmar Flock Marta Granström Birgitta Henriques Normark Göran Kronvall Ewert Linder Åke Lundkvist Lars Magnius Tore Midvedt Roland Möllby Elisabeth Norin Staffan Normark Mikael Rhen Ute Römling Mats Wahlgren Britta Wahren Project leaders: Mats Andersson Andreas Bråve Fredric Carlsson Qijun Chen Christian Giske Maria Isaguliants Department of Microbiology, Tumor and Cell Biology – MTC Marianne Jansson Annika Karlsson Jonas Klingström Öjar Melefors Sarah Palmer Kristina Persson Laura Plant Katrin Pütsep Research Reports Infection Biology tuberculosis, as well as diarrhoeal diseases still prevail, while mankind is exposed to additional infectious entanglements such as HIV, emerging variants of influenza viruses and the rapid development of resistance to antimicrobials. As a whole, the coverage of the infection biology research at MTC is fairly broad, yet clearly focused to meet the emerging challenges ahead. A major focus of the bacteriologists at MTC is to understand how bacteria interact with the epithelial linings and professional phagocytic cells, and how these interactions affect the concomitant host response and the infection pathogenesis. Apart from defining the molecular basis of bacterial virulence, such studies have revealed microbial components suitable for vaccine development and pharmaceutical inhibitors that can block virulence factor activity. Another central line of research concerns the formation of bacterial biofilms, and role of novel second messengers in the regulation A particular interest is to characterize of biofilm formation. Furthermore, research many of the variable surface adhesins of the is conducted on molecular epidemiology parasite in terms of their antigenicity and and to understand the origin and spread of genetic regulation. antimicrobial resistance Studies on the human amongst bacteria. MTC immunodeficiency virus “Infection biology has also conducts research on (HIV) and the Epstein-Barr during the last decade a theme called biomedivirus (EBV) continue to be experienced a strong cal ecology that in part strong at MTC. Regarding development into a multiinvestigates the role of the HIV, the research covers disciplinary research field normal flora in human clinical epidemiology, with obvious c onnections health and disease. genome variability, viral with immunology, including evolution, molecular virolinnate immunity and inflamogy and vaccine developCreate vaccines mation, vaccinology and ment using a number of MTC houses a strong even carcinogenesis.” approaches. tradition in parasitology, Other lines of interest include studies and particularly in research devoted to Plason emerging viruses such as alphaviruses, modium falciparum. Here, again, the ambidengue virus and new influenza virus varition is to define the parasite components ants, as well as the use of Puumala virus as a that enable the completion and success of model to elucidate the infection pathogenesis the replication cycle in man in order to create of hemorrhagic fevers. vaccines or chemical compounds that could prevent or relieve states severe of malaria. Mikael Rhen Department of Microbiology, Tumor and Cell Biology – MTC 39 Research Reports Infection Biology Molecular analyses of host responses following Leishmania infection Leishmaniasis is a widely distributed disease affecting approximately 12 million people worldwide. The focus of our work is on understanding natural killer (NK) Hannah Akuffo cell interactions in the early phase of [email protected] Leishmania infection, mechanisms [email protected] of ulcer formation during Cutaneous leishmanisis (CL), and the effect of helminth infection on host response to vaccination against tuberculosis with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG). We confirmed that promastigotes, the developmental form of Leishmania parasites transmitted by the vector, induce cytokine secretion in NK cells (Lieke et al., 2008). Paradoxically, as a rapid consequence of this interaction, NK cell receptors show suppressive expression. We implicated the major surface molecules of promastigotes, glycoprotein (gp) 63, in these suppressive effects. Currently, we are following up the effects of Leishmania on NK cells by in vivo analysis by taking advantage of a clinical trial in Iran, using Leishmania major parasites for live vaccination. Mechanisms of ulcer formation remain unclear. We reported that Fas/FasL interactions are altered during CL and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration (Eidsmo et al., 2007). We proposed, using keratinocytes incubated with supernatants from L. major-infected cells that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL. We furthermore studied the expression of FasL and TRAIL in ulcerative and non-ulcerative CL and found higher FasL and TRAIL expression in ulcerative compared to non-ulcerative CL. These findings correlated with inhibition of ulcerative processes in a murine CL model following FasL and TRAIL neutralization. Currently, the only vaccine available against tuberculosis Leishmania promastigote (red arrow) is BCG which has variable meets natural killer cell (blue arrow). Picture by Thorsten Lieke. efficacy against pulmonary tuberculosis. Work in our group showed the chronic worm infection reduces the immunogenicity of BCG in humans which was associated with increased TGF-β production, but not with enhanced Th2 immune response. Selected publications l l l 40 Eidsmo L, Fluur C, Rethi B, Eriksson Ygberg S, Ruffin N, De Milito A, Akuffo H and Chiodi F. FasL and TRAIL induce epidermal apoptosis and skin ulceration upon exposure to Leishmania major. Am J Pathol 170, 227, 2007. Lieke T, Nylén S, Eidsmo L, McMaster WR, Mohammadi AM, Khamesipour A, Berg L and Akuffo H. Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation. Clin Exp Immunol 153, 221, 2008. Elias D, Britton S, Aseffa A, Engers H and Akuffo H. Poor immunogenicity of BCG in helminth infected population is associated with increased in vitro TGF-b production. Vaccine 26, 3897, 2008. Department of Microbiology, Tumor and Cell Biology – MTC HIV genetic variation and evolution The research in my group focuses on genetic and biological variability of HIV as well as the interactions between HIV and the immune system. We study how HIV evades humoral and cellular immunity. We also study the emergence Jan Albert and spread HIV variants that are resistant to [email protected] anti-retroviral drugs. We use molecular epidemiology to track the spread of HIV in Sweden and abroad. We take part in several EU projects such as EuropeHIVResistance and CHAIN and other international projects Our current research also focuses on the development and application of modern Bayesian methods for phylogenetic inference. HIV prevention still relies solely on information campaigns and other behavioral interventions. To guide prevention it is essential to have correct and detailed information about the local and global HIV epidemic. Phylodynamics is a rapidly developing interdisciplinary research field, which we use to document and time the HIV-1 outbreak among intravenous drug users in Stockholm, as well as the entire HIV epidemics in Latvia and Honduras. A second area of research is the application of massive parallel pyrosequencing (454-sequencing) to dissect HIV evolution with a previously unattainable resolution. 454-sequencing allows ultra-deep sequencing and statistically meaningful quantification of low-abundance genetic variants, such as rare sequence variants in an HIV quasispecies. Our preliminary data indicate that previously undetectable drug resistance can be detected down to 0.06% mutation abundance. We have generated more than one million HIV sequences within this project. Finally, we take part in an EU project called NGIN which focuses on neutralizing antibodies in HIV infection and aims to develop immunogens that Example of modeling of HIV spread by induce broad neutralization. sophisticated phylogenetic tree analyses. Selected publications l l l Skar H, Sylvan S, Hansson HB, Gustavsson O, Boman H, Albert J and Leitner T. Multiple HIV-1 introductions into the Swedish intravenous drug user population. Infect Genet Evol 8, 545, 2008. Vercauteren J, Wensing AM, van de Vijver DA, Albert J, Balotta C, Hamouda O, Kucherer C, Struck D, Schmit JC, Asjo B, Bruckova M, Camacho RJ, Clotet B, Coughlan S, Grossman Z, Horban A, Korn K, Kostrikis L, Nielsen C, Paraskevis D, Poljak M, Puchhammer-Stockl E, Riva C, Ruiz L, Salminen M, Schuurman R, Sonnerborg A, Stanekova D, Stanojevic M, Vandamme AM and Boucher CA. Transmission of drug-resistant HIV-1 is stabilizing in Europe. J Infect Dis 200, 1503, 2009. Murillo W, de Rivera IL, Parham L, Jovel E, Palou E, Karlsson AC and Albert J. Prevalence of drug resistance and importance of viral load measurements in Honduran HIV-infected patients failing antiretroviral treatment. HIV Med 11, 95, 2010. Research Reports Infection Biology Clinical virology and virus discovery The group is based at the Department of Clinical Microbiology at Karolinska University Hospital, and the research is focused on developing efficient diagnostic methods for virus infections. We have a special interest in Tobias Allander respiratory tract infections, but also study [email protected] gastroenteritis viruses and chronic virus infections. A main activity is identification of unknown viruses and clinical characterization of newly discovered viruses. The group has identified two novel human viruses, human bocavirus (HBoV) and KI polyomavirus (KIPyV). We have further established a relationship between human bocavirus and acute respiratory disease in infants and children. HBoV analysis is now routinely provided to health care. Virus discovery is done by combining a specially designed virus purification protocol with high-throughput sequencing and bioinformatics, in collaboration with the genome sequencing group at the Department of Cell- and Molecular Biology, Karolinska Institutet. We screen a wide variety of clinical samples for unknown viruses, from upper respiratory tract secretions to tumors. Virus infections impose a huge disease burden on humanity. Diagnosing infectious agents and searching for the cause for diseases with unknown aetiology is fundamental for many aspects of clinical medicine. A molecular diagnostic platform for daily rapid detection of 15 respiratory viruses Defending the uroepithelium – new strategies fighting urinary tract infections The urinary tract functions in close proximity to the outside environment yet must remain free of microbial colonization to avoid disease. Recently, we Annelie Brauner demonstrated that epithelial cells [email protected] of the urinary tract up-regulate the production of the human antimicrobial peptide cathelicidin upon infection with uropathogenic Escherichia coli. Cathelicidin aggressively fends off bacteria, thereby protecting the urinary tract from infection. However, if bacteria succeed to adhere to the urinary bladder, they form biofilms, which offer advantages to the bacteria, such as increased resistance to antimicrobial substances, mechanical forces and to nutrient depletion. Biofilms are mainly built up by different extracellular matrix components and substantially contribute to the complex interplay between the host and the invading microorganism. In spite of the effective defense system, urinary tract infection is currently one of the 10 most common reasons for outpatient visits. Up to 60 percent of all women develop the disease at least once in their life, and of these, 25% have recurrent infections. Children suffer less often from urinary tract infections but when the kidneys are involved up to 40% get renal scarring. Antibiotic-resistant bacteria are a growing and alarming problem and therefore call for immediate action to develop new treatment strategies. Our research interest focuses on patients with recurrent urinary tract infections, with special emphases on the mechanism of action between the microorganism, biofilm and innate immunity. Particularly, we concentrate on underlying mechanisms causing recurrent infections and possibilities to increase the endogenous production of cathelicidin and other novel antimicrobial peptides. We also focus on bacterial viruE. coli surrounded with curli fimbriae. The bacteria were isolated directly from lence factors enabling the fresh urine of a patient with urinary tract bacteria to withstand tradiinfection. tional antibiotic treatment. Selected publications l l l Allander T, Andreasson K, Gupta S, Bjerkner A, Bogdanovic G, Persson MA, Dalianis T, Ramqvist T and Andersson B. Identification of a third human polyomavirus. J Virol 81, 4130, 2007. Tiveljung-Lindell A, Rotzén-Östlund M, Gupta S, Ullstrand R, Grillner L, Zweygberg-Wirgart B and Allander T. Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses. J Med Virol 81, 167, 2009. Goh S, Lindau C, Tiveljung-Lindell A and Allander T. Merkel cell polyomavirus in respiratory tract secretions. Emerg Infect Dis 15, 489, 2009. Selected publications l l l Chromek M and Brauner A. Antimicrobial mechanisms of the urinary tract. J Mol Med 86, 37, 2008. Lüthje P and Brauner A. Putative link between the virulence-associated gene fluA and fluoroquinolone resistance in uropathogenic E. coli. J Clin Microbiol 48, 675, 2010. Kai-Larsen Y, Lüthje P, Chromek, M, Peters V, Wang X, Holm Å, Kádas L, Hedlund K-O, Johanson J, Chapman MR, Jacobson SH, Ute Römling U, Agerberth B, Brauner A . Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37. Accepted PLoS Pathogens 2010. Department of Microbiology, Tumor and Cell Biology – MTC 41 Research Reports Infection Biology Immunization strategies against HIV-1: immunogen design and optimization of vaccine delivery More than 25 years have passed since the identification and isolation of Human Immunodeficiency Virus type 1 (HIV-1). Although Andreas Bråve there is still no effective [email protected] vaccine available against the virus, recent results from a phase 3 trial in Thailand have indicated that protective immunity can be achieved by immunization. Due to the intrinsic nature of the virus, classical vaccine approaches, such as inactivated virus vaccines or protein-based vaccines, have failed and researchers are forced to turn to more novel vaccine-strategies, such as gene-based vaccination (e.g. DNA-vaccination). However, DNA vaccines have so far elicited weaker immune responses in humans than anticipated and, in most current clinical trials, plasmid vaccines are used primarily to “prime” the immune system for a subsequent boost with recombinant protein or a live recombinant attenuated viral vector, such as Modified Vaccinia virus Ankara (MVA) or Adenovirus. Our vaccine strategy against HIV-1 is to target multiple viral antigens deriving from various subtypes of the virus. The results from our clinical trials, in which HIV DNA plasmids are combined with a recombinant MVA, are encouraging with close to 100% of the volunteers Bifunctional vaccine-specific responding against HIV-1 antigens T cell responses as analyzed after immunization. We are now by FluoroSpot. T cells isolated developing the next generation of from DNA-immunized mice were stimulated with vaccine genetic HIV-1 immunogens and also antigen and respond by producfocusing on improving the delivery ing either IFN-g (green), IL-2 of the plasmid DNA vaccine by the (red) or both (yellow). use of electroporation. In vivo-electroporation is the application of short pulses of electric current immediately after, and at the site of, an injection with a genetic vaccine. We and others have shown that it is possible to significantly augment the subsequent vaccine-specific immune responses using this method. Now, the overall aim is to develop an immunization strategy that will allow plasmid DNA to be used as a stand-alone vaccine modality. Host-pathogen interactions in mycobacterial infection One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), which causes around 2 million deaths annually. To facilitate fundamental studies Fredric Carlsson of Mtb infection, safer and [email protected] mentally more amenable species are often used as models. Among these, the closely related Mycobacterium marinum (Mmar) is used increasingly to study mycobacterial pathogenesis. Mmar is a pathogen of fish and amphibians causing disease with many features of tuberculosis, and is also able to infect immunocompetent humans where it induces formation of dermal granulomas pathologically similar to those formed in tuberculosis. Thus, Mmar and Mtb likely share mechanisms of inducing and maintaining infection. The overall goal of our work is to significantly increase our understanding of how pathogenic mycobacteria interact with the host immune system to promote disease. One major interest in our lab is to understand how the mycobacterial Esx-1 (type VII) secretion system interacts with the host inflammasome, an important part of our innate immune response. Preliminary data indicate that Esx1-dependent activation of the inflammasome is host-detrimental during Mmar infection, suggesting that activation of this inflammatory pathway in mycobacterial infection is a manifestation of bacterial virulence rather than a manifestation of host response. Another focus in our lab is to elucidate the host-pathogen interactions whereby mycobacteria interfere with the onset and efficacy of the adaptive immune response in a novel mouse model of Mmar infection. It is well known that Mtb delays the onset of the adaptive immune response both in humans and in mice, but the underlying molecular events have remained unknown. This likely is central to the difficulties in generating an effective vaccine and in harnessing the human immune system for therapy. Our studies aim to provide fundamental knowledge to answer this key question. Bone marrow-derived macrophages from B6 mice infected with M. marinum. Blue: Macrophage nuclei stained by Hoechst stain. Green: Active caspase-1 stained by FAM-FLICA as a marker for inflammasome activation upon infection. Selected publications l l l 42 Bråve A, Boberg A, Gudmundsdotter L, Rollman E, Hallermalm K, Ljungberg K, Blomberg P, Stout R, Paulie S, Sandström E, Biberfeld G, Earl P, Moss B, Cox JH and Wahren B. A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8+ T cell and humoral responses in mice. Mol Ther 15, 1724, 2007. Sandström E, Nilsson C, Hejdeman B, Bråve A, Bratt G, Robb M, Cox, J, VanCott T, Marovich M, Stout R, Bakari M, Kisali Pallangyo, Ljungberg K, Moss B, Earl P, Michael N, Birx D, Mhalu F, Wahren B and Biberfeld G. Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara. J Infect Dis 198, 1482, 2008. Bråve A, Hallengärd D, Malm M, Blazevic V, Rollman E, Stanescu I and Krohn K. Combining DNA technologies and different modes of immunization for induction of humoral and cellular antiHIV-1 immune responses. Vaccine 27, 184, 2009. Department of Microbiology, Tumor and Cell Biology – MTC Selected publications l l l McLaughlin B, Chon JS, MacGurn JA, Carlsson F, Cheng TL, Cox JS and Brown EJ. A Mycobacterium ESX-1-secreted virulence factor with unique requirements for export. PLoS Pathog 3, e105, 2007. Carlsson F and Brown EJ. The art of making an exit. Science 323, 1678, 2009. Carlsson F, Joshi SA, Rangell L and Brown EJ. Polar localization of virulence-related Esx-1 secretion in Mycobacteria. PLoS Pathog 5, e1000285, 2009. Research Reports Infection Biology Interactions of malaria virulence factors and the human host The malaria parasite Plasmodium falciparum exposes various molecules at the surface of infected erythrocytes. Some of the surface-exposed molecules are parasite virulence factors associQijun Chen ated with severe malaria and immune [email protected] evasion. Our interest is to understand the genetic regulation and biological function of the virulence factors named P. falciparum erythrocyte membrane protein 1(PfEMP1) and Antigen 332 (also called Pf332). We have investigated the transcription and regulation of the var2csa gene that encodes a PfEMP1 associated with placental malaria. We established an approach combining real-time PCR allelic discrimination and discriminative RNA-FISH to distinguish between highly similar gene copies in P. falciparum parasites. We found that duplicated var2csa variants are simultaneously transcribed both on a population level, and, intriguingly, also in individual cells, with nuclear co-localization of the active genes and corresponding transcripts. This indicates transcriptional functionality of the duplicated genes, challenges the dogma of mutually exclusive var gene transcription and suggests mechanisms behind antigenic variation. Allelic discrimination, gene or transcript localization by FISH not only provide insights into transcriptional regulation of genes, but also suggest that this approach could be used for further investigation of genome dynamics and gene regulation. Another recent focus has been on the molecule that mediates erythrocyte invasion by the P. falciparum parasite. We found that the Pf332 molecule harbors a Duffy-binding like (DBL). The DBL domain of Pf332 is conserved among parasites isolated from different regions in the world. Importantly, affinity purified human antibodies as well as mAb to the DBL domain significantly inhibit invasion, indicating that the DBL domain plays a critical role for parasite invasion. The identification of the molecule paves a new way towards the development of a malaria vaccine. Characteristics of trans missible HIV-1 and co-receptor use Detection of var gene transcripts in single parasites by RNA-FISH. HIV mother-to-child transmission (MTCT) is preventable in industrialized countries, but remains an obstacle in most developing countries. Our Anneka Ehrnst research aims at improving the [email protected] prevention of mother-to-child transmission also in these countries. We study the role of HIV-1 subtypes and phenotypes for viral entry into cells and their role in transmission and disease progression. MTCT of HIV-1 could be decreased in Northern Vietnam, after introducing counseling, avoidance of breastfeeding, and anti-retroviral therapy (Ha et al., 2008), despite that women here usually escape from follow-up programs due to fear. We also determined the phylogenetic origin of their HIV virus. Combining the sequences of biological phenotypes of HIV isolates and those of molecular clones, we can pinpoint the exact transmitted virus. These are analyzed genetically and in neutralization to determine crucial properties in transmission. We also characterize co-receptor use of different subtypes HIV-1 binds to CD4 and to one of the and their corresponding co-receptors, CCR5 or CXCR4, when infecting a cell. The normal function of similar or different amino CD4 is to bind to MHC class II molecules acids in the critical V3 loop and the function of CCR5 and CXCR4 is of the env gp120 gene and to bind chemokines. change of co-receptor use in drug failure. An algorithm to determine co-receptor use is compared to other biometric methods and biological determination. A microchip analysis of HIV-1 peptides reacting with monomeric molecules of the major histocompatibility complex (HLA) class II (Gaseitsiwe et al., 2009) was developed, which confirmed several already known peptides and identified several new ones. Furthermore, we analyze the role of cytomegalovirus (CMV) in terminal illness of AIDS patients. When CMV was localized in the same area of the brain affected in Korsakoff ’s syndrome, which is ususlly caused by chronic alcoholism, Korsakoff ’s syndrome occurred in the absence of alcohol abuse (Matell-Pirskanen et al., 2009). The role of CMV in other central nervous system-related illnesses is under way, such as a CMV-related Addison’s disease, a rare endocrine disorder wherein the adrenal glands produce insufficient steroid hormones. Selected publications Selected publications l l l Chen Q. The naturally acquired immunity in severe malaria and its implication for a PfEMP1 based vaccine. Microb Infect 9, 777, 2007. Moll K, Chêne A, Ribacke U, Kaneko O, Nilsson S, Winter G, Haeggström M, Pan W, Berzins K, Wahlgren M and Chen Q. A novel DBL-domain of the P. falciparum 332 molecule possibly involved in erythrocyte adhesion. PLoS One 2, e477, 2007. Brolin KJ, Ribacke U, Nilsson S, Ankarklev J, Moll K, Wahlgren M and Chen Q. Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites. Genome Biol 10, R117, 2009. l l l Ha TTT, Tuan PL, Bao NH, Anh NM, Cam PD, Chiodi F and Ehrnst A. Successful reduction of mother-to-child transmission of HIV-1 by nevirapin and non-breastfeeding in Hanoi and Haiphon. Retrovirology 5, S1, P11, 2008. Gaseitsiwe S, Valentini D, Ahmed R, Mahdavifar S, Magalhaes I, Zerweck J, Schutkowski M, Gautherot E, Montero F, Ehrnst A, Reilly M and Maeurer M. Major histocompatibility complex class II molecule-human immunodeficiency virus peptide analysis using a microarray chip. Clin Vaccine Immunol 16, 567, 2009. Matell-Pirskanen R, Grützmeier S, Nennesmo I, Sandström E and Ehrnst A. Impairment of short-term memory and Korskakoff syndrome are common in AIDS patients with cytomegalovirus encephalitis. Eur J Neurol 18, 48, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 43 Research Reports Infection Biology The impact of the human gastrointestinal microbiota on health and disease The work of my group aims at creating a scientific environment with an interdisciplinary approach on the interface between epidemiology and the microbiological, Lars Engstrand immunological and genetic aspects [email protected] of chronic infectious diseases. One focus is Helicobacter pylori infection and to answer the question: why do some people who are infected become ill, while others do not? The long-term objective is to identify and characterize factors (both microbial and host related) that determine or modify the risk of disease outcome observed in people infected with this microorganism. Humans represent complex microbial ecosystems, which are postulated to contribute to both health maintenance and the development of chronic inflammatory diseases. Alterations in the human gut microbiota have recently been associated with diseases such as cancer, type II diabetes, inflammatory bowel disorder, allergy and obesity. Determining the microbial composition in patients and healthy controls may provide novel therapeutic targets. New highthroughput culture-independent molecular tools are now developed, allowing the scientific community to characterize and understand the microbial communities underpinning biological processes in unprecedented ways. Our ongoing projects have the potential to transform the situation of chronic inflammation in the gut from a purely descriptive to a predictive framework, in which ecological principles are integrated and exploited to biological systems and pathology. We interact with a number of clinical colleagues, including epidemiologists to initiate extended population-based studies. We are currently analyzing the human bacteriome in a number of cases and controls from such studies. The results will then be transferred to studies of host and microbial interactions, molecular epidemiology, biomarkers and prevention, clinical diagnostics and pathogenic mechanisms. Heatmap of genera present in human stomachs. Red: Individuals with high grade corpus atrophy, white: Controls, blue: Individuals treated with proton pump inhibitors and green: Individuals with dominance of Helicobacter sequences. Visualized using heatmap with MultiExperimental Viewer 4_5. Selected publications l l l 44 Andersson AF, Lindberg M, Jakobsson H, Bäckhed F, Nyrén P and Engstrand L. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS One 30, e2836, 2008. Giannakis M, Chen SL, Karam SM, Engstrand L and Gordon JI. Helicobacter pylori evolution during progression from chronic atrophic gastritis to gastric cancer and its impact on gastric stem cells. Proc Natl Acad Sci USA 105, 4358, 2008. Dicksved J, Lindberg M, Rosenquist M, Enroth H, Jansson JK and Engstrand L. Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls. J Med Microbiol 58, 509, 2009. Department of Microbiology, Tumor and Cell Biology – MTC Vaccination against streptococcal infections Recombinant surface proteins from streptococci and staphylococci can give rise to protective antibodies. We have now taken such studies beyond the stage of experimental mouse models and demonstrated Jan-Ingmar Flock vaccine efficacy on the target animal, the [email protected] horse. Streptococcus equi is a serious pathogen causing strangles in the horse, an upper respiratory tract infection. The bacteria adhere to the tonsils and spread via the lymphatic system. It is the most contagious and costly bacterial infection in the horse and causes severe problems for the horse industry. No safe and efficient vaccine is available. In collaboration with a team at Swedish University of Agricultural Sciences in Uppsala, we have vaccinated horses with various recombinant surface-located proteins from S. equi. Antigens to be tested in the horse are first tested for protective efficacy in a mouse model system. The vaccinated horses are subsequently experimentally infected with S. equi and protection is monitored by determination of body temperature, lymph node swelling, clinical symptoms, post mortem and histopathological analysis. A protection level at around 80% has been achieved. The present work is focused on finding additional antigen combinations, identification of protective epitopes, optimization of immunization route, dosage, and duration of protection. Currently, antibodies from sera and nasal washes from vaccinated horses are analyzed to further understand the mechanism of protection. Like Elevated body temperature is a typical sign many other bacteria, S. of strangles in the horse. The graph shows how experimentally infected horses, which equi has a very sophis- have been vaccinated with two different ticated system for which combinations (A and B) of recombinant S. immune evasion can equi proteins, are protected from infection. Mean values from six horses in each group here be studied in the are shown. The difference between vaccinatural host. nated and non-vaccinated horses is statistiS. equi and Strep- cally significant (p<0.0001). tococcus pyogenes, a human pathogen causing e.g. scarlet fever, are similar in genome sequence, mode of infection and clinical symptoms. Some antigens from S. equi used in the vaccine have orthologs in S. pyogenes, implying that the corresponding antigens from S. pyogenes could be used in a vaccine against this pathogen. Selected publications l l l Waller A, Flock M, Smith K, Robinson C, Mitchell Z, Karlström A, Lannergard J, Bergman R, Guss B and Flock J-I. Vaccination of horses against strangles using recombinant antigens from Streptococcus equi. Vaccine 25, 3629, 2007. Guss B, Flock M, Frykberg L, Waller A, Robinson C, Smith K and Flock J-I. Getting to grips with strangles: an effective multicomponent recombinant vaccine for the protection of horses from Streptococcus equi infection. PLoS Pathog 9, e1000584, 2009. Hulting G, Flock M, Frykberg L, Lannergård, Flock J-I and Guss B. Streptococcus equi encodes two specific IgG-degrading enzymes. FEMS Microbiol Lett 298, 44, 2009. Research Reports Infection Biology Multidrug-resistant Gram-negative bacilli During the last three years my research projects have concentrated on three major topics, all related to various aspects of multidrug-resistChristian Giske ant Gram-negative bacilli: [email protected] The epidemiology of extendedspectrum β-lactamase (ESBL) producing Enterobacteriaceae in Stockholm and factors influencing fecal carriage of ESBL-producing Enterobacteriaceae. The first epidemiological project has revealed a mainly polyclonal epidemiology with some minor strain clusters. A project where the dynamics of fecal carriage of ESBL-producing Enterobacteriaceae are being evaluated is ongoing, and currently around 80 patients have been included. A total of 100 patients will finally be included, and will be followed for at least one year. Strain and plasmid replicon typing will be conducted to understand more about the dynamics of carriage, and data regarding treatment and co-morbidities will be collected for all patients. Multilocus sequence typing as a tool for identifying clones of multidrug-resistant (MDR) Gram-negative bacilli with a high epidemic potential. The Klebsiella pneumoniae clone ST258, which is responsible for the majority of cases of carbapenem-resistant Enterobacteriaceae spreading in the US, and also in several European countries, was first described by our group. Also, we first described clones of Pseudomonas aeruginosa that have later been associated with epidemic spread of the metallo-β-lactamases CC111 and CC235. Further, we were the first to Multidrug-resistant Gram-negative bacilli – detect the new metalloa current public health threat. β-lactamase NDM-1, which is highly prevalent in India. Several projects related to laboratory methods of detection of new β-lactamases are currently being carried out. Partly, these projects explore phenotypic methods for confirming the presence of β-lactamases, and partly criteria for screening for such β-lactamases. Our results have attracted great interest both in Europe and in the US. Selected publications l l l Giske CG, Monnet DL, Cars O and Carmeli Y. Clinical and economic impact of common multidrug-resistant gram-negative bacilli. Antimicrob Agent Chemother 52, 813, 2008. Kitchel B, Rasheed JK, Patel JB, Srinivasan A, Navon-Venezia S, Carmeli Y, Brolund A and Giske CG. Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258. Antimicrob Agent Chemother 53, 3365, 2009. Samuelsen Ø, Naseer U, Tofteland S, Skutlaberg DH, Onken A, Hjetland R, Sundsfjord A and Giske CG. Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing plasmid-mediated KPC carbapenemase in Norway and Sweden. J Antimicrob Chemother 63, 654, 2009. Helicobacter pylori infection in Vietnam Our group has studied Helicobacter pylori infection in the context of a developing country, i.e. Vietnam, where peptic ulcer disease and gastric cancer Marta Granström are major public health issues. We could [email protected] show that for optimal performance of the serological assay local strains had to be used, a major problem for developing countries since the commercial kits available on the market have been made and evaluated for developed countries. We could also show that H. pylori infection was very common in Vietnam and significantly higher in Hanoi than in a rural area, probably due to the higher level of crowding in the city. The early infection in Vietnam also results on a high rate of peptic ulcer disease in children and the need to find the optimal treatment for them since there are no formal recommendations for eradication treatment even in developed countries. We conducted a large randomised, double-blind study to evaluate two main treatment strategies, one with and one without clarithromycin. The outcome was rather low eradication rates of around 60%, which is in contrast to the >80% obtained in developed countries. Antibiotic resistance testing revealed that the cause for the low eradication rate was an unexpectedly high rate of clarithromycin resistance of overall 50% in Vietnamese children, with the youngest children having even higher rates. An important finding was that twice-daily dosing of the proton-pump inhibitor significantly improved the eradication rate in the clarithromycin-resistant strains. The one year follow-up of children with previously eradicated H. pylori showed a high reinfection rate, with the highest rate found in the youngest age groups and then gradually decreasing until 9-11 years of age. The finding supports the notion of H. pylori infection being acquired at an early age due to unknown mechanism(s) of susceptibility. Pharmacy outside a pediatric hospital. Selected publications l l l Granström M, Lehours P, Bengtsson C and Megraud F. Diagnosis of Helicobacter pylori. Helicobacter 13 Suppl 1, 7, 2008. Nguyen TV, Bengtsson C, Nguyen GK and Granström M. Evaluation of a novel monoclonal-based antigen-in-stool enzyme immunoassay (Premier Platinum HpSA PLUS) for diagnosis of Helicobacter pylori infection in Vietnamese children. Helicobacter 13, 269, 2008. Nguyen TV, Bengtsson C, Nguyen GK, Hoang TT, Phung DC, Sörberg M and Granström M. Evaluation of two triple-therapy regimens with metronidazole or clarithromycin for the eradication of H. pylori infection in Vietnamese children: a randomized, double-blind clinical trial. Helicobacter 13, 550, 2008. Department of Microbiology, Tumor and Cell Biology – MTC 45 Research Reports Infection Biology Microbial vaccines as targeted cargo, genetic approach Strong immune response against primary resistance mutations can create a bottle-neck to HIV-1 forbidding the mutations to occur. Such response can be induced by genes encoding drugMaria Isaguliants resistant HIV enzymes. The idea is [email protected] unconventional and used only by our group. These DNA-encoded immunogens are not particularly strong, but immunogenicity can be enhanced if the enzymes are targeted to specific intracellular compartments. For this, their genes are fused with sequences encoding intracellular sorting signals. Targeting to proteasome/MHC class I pathway has been achieved by protein fusions to ornithine decarboxylase or its minimal proteasome targeting moiety (ODCsig). Mice DNA-immunized with chimeras of wild-type or drug-resistant reverse transcriptase (RT) with ODCsig mounted an enhanced immune response as compared to parental genes. Targeting to lysosome/MHC class II pathway was achieved by fusion to endosomal sorting signals. Potent cross-reactive immune response against both the wild-type and drug-resistant HIV RT was achieved after DNA immunization with RT chimeras fused to lysosome associated membrane protein I. Screening of immune response in animals is done by in vivo imaging (IVIS®) where the decrease of expression of a reporter gene co-injected with DNA-immunogen due to killing of expressing cells by the immune response is moniSubcellular localization of wild-type (A) and drug-resistant (B) HIV-1 tored. We have, thus, pioneered reverse transcriptase chimeras the use of drug-resistant HIV fused to lysosome associated memenzymes and their chimeras as brane protein I (LAMP-1). Staining of the cell nucleus in blue, LAMP-1 in DNA-immunogens and demgreen, reverse transcriptase in red, onstrated their potency. This in field overlays. motivates further progress towards human immunotherapy that would prevent the evolvement of drug resistant HIV. Another vaccine project, carried on the hepatitis C virus model, deals with increasing the efficacy of DNA immunization by applying different immunogen combinations, prime-boost regimens and novel adjuvants. Selected publications l l l 46 Petrakova N, Gudmundsdotter L, Yermalovich M, Belikov SV, Eriksson L, Pyakurel P, Johansson O, Biberfeld P, Andersson S and Isaguliants M. Autoimmunogenicity of a DNA-binding domain. Mol Immunol 46, 1467, 2009. Starodubova E, Boberg A, Ivanov A, Latyshev O, Petrakova N, Kuzmenko Y, Litvina M, Chernousov A, Kochetkov S, Karpov V, Wahren B and Isaguliants MG. Potent cross-reactive immune response against the wild-type and drug-resistant forms of HIV reverse transcriptase after the chimeric gene immunization. Vaccine 28, 1975, 2010. Masalova OV, Lesnova EI, Pichugin AV, Melnikova TM, Grabovetsky VV, Petrakova NV, Smirnova OA, Ivanov AV, Zaberezhny AD, Ataullakhanov RI, Isaguliants MG and Kushch AA. The successful immune response against hepatitis C nonstructural protein 5A (NS5A) requires heterologous DNA/protein immunization. Vaccine 28, 1987, 2010. Department of Microbiology, Tumor and Cell Biology – MTC HIV-1 and HIV-2 mediated patho genesis in relation to innate immunity and immune activation HIV-1 and HIV-2 are two related viruses with distinct clinical outcome. HIV-1 is more pathogenic and transmissible than HIV-2. HIV-1 Marianne Jansson is also spread worldwide [email protected] while HIV-2 largely remains endemic in West Africa. Disease progression of both infections is related to dysregulation of the immune system. However, the mechanism behind the divergent clinical outcomes is still to be revealed. In fact, the reason why HIV-2 in most cases is a controlled infection may give clues to how to modulate the pathogenesis of HIV-1. We have studied the impact of HIV-1 and HIV-2 on innate immunity function and chronic immune activation. Since microbial stimulation of Toll-like receptors (TLRs) triggers both anti-viral cytokines and initiate adaptive immunity we set out to analyze TLR responsiveness. We found that responsiveness to TLR7/8 stimuli is defective during HIV-1 infection while maintained during HIV-2 infection. Responsiveness to other types of TLR stimuli was shown to be impaired with loss of CD4+ T cells in both infections. Chronic immune activation is a marker of disease progression and it was recently suggested that immune activation in HIV-1-infected individuals results from microbial translocation into the blood after disruption of the gut mucosa. Our studies showed that microbial translocation, assessed as lipopolysaccharide in plasma, is independent of the infectious agent, HIV-1 or HIV-2, but correlates with severity of the disease. To better understand Microbial translocation correlates to severity of both HIV-1 and HIV-2 infections. how HIV interacts with innate immune cells for efficient spread, we also studied the ability of HIV to utilize dendritic cells (DCs), and the DC expressed molecule DC-SIGN, for transinfection of T cells. We found that HIV-1 variants emerging after AIDS onset display reduced ability to use DC-SIGN, while direct T cell infection efficacy is enhanced. This suggests that the ability of HIV-1 to use DC for efficient spread to T cells is more important during early and chronic phases of infection than at end-stage disease. Selected publications l l l Borggren M, Repits J, Kuylenstierna C, Sterjovski J, Churchill MJ, Purcell DF, Karlsson A, Albert J, Gorry PR and Jansson M. Evolution of DC-SIGN use revealed by fitness studies of R5 HIV-1 variants emerging during AIDS progression. Retrovirology 5, 28, 2008. Nowroozalizadeh S, Månsson F, da Silva Z, Repits J, Dabo B, Pereira C, Biague A, Albert J, Nielsen J, Aaby P, Fenyö EM, Norrgren H, Holmgren B and Jansson M. Studies on toll-like receptor stimuli responsiveness in HIV-1 and HIV-2 infections. Cytokine 46, 325, 2009. Nowroozalizadeh S, Månsson F, da Silva Z, Repits J, Dabo B, Pereira C, Biague A, Albert J, Nielsen J, Aaby P, Fenyö EM, Norrgren H, Holmgren B and Jansson M. Microbial translocation correlates to severity of both HIV-1 and HIV-2 infections. J Infect Dis 201,1150, 2010. Research Reports Infection Biology Immune escape and the potency of the cellular immune system against HIV-1 The cellular immune system includes T cells that can be divided into two arms: CD8+ and CD4+. They battle virusinfected cells by recognizAnnika Karlsson ing small peptides (epitopes) [email protected] of digested virus particles or proteins displayed on the human leucocyte antigen (HLA) alleles on an antigen presenting cell. The interactions between the T cell and the HLA-peptide complex are highly specific and thus extremely sensitive to variations. As the human immunodeficiency virus (HIV) is highly variable this poses one of the major obstacles in understanding the efficacy of a specific T cell response. Thus, vaccine and treatment failures have been attributed to the development of immune escape mutants and our inability to correlate viral control to pre-existing immune responses. A second obstacle is the variation of the human immune system; a multitude of HLA alleles and T cell receptors (TCR) recognizing the HLA-peptide complex. In my group, we study the interactions between the T cells and HIV using immunological, virological, and bioinformatic approaches. We hypothesize that a better understanding of the complex relationships between the virus, host genetic factors, and the immune response will provide valuable insights for the development of a vaccine. For an effective antiviral response, we believe that a multitude of viral epitopes would have to be matched by a multitude of human HLA alleles to be effective. The recent advances in bioinformatics have made it possible to evaluate some of the HLA-epitope-TCR interactions in greater details. Importantly, using this approach we have identified a limited set of HLA class I epitopes eliciting broad population coverage as preferred by a vaccine antigen. We believe that this knowledge can help us to identify regions to target in the next generation of HIV vaccines. Our goal is to identify epitopes within conserved regions that are recognized by as many patients as possible and from which the virus is unlikely to escape. CD8+ T cells cripple HIV replication by killing virus infected cells and production of antiviral cytokines (e.g. IFN-g, TNF-a). They recognize, via their T cell receptor (TCR), viral epitopes presented on the surface of HIV-infected cells by HLA class I molecules. Why do we get sick when voles don’t? Hantaviruses cause two severe zoonotic diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome Jonas Klingström (HCPS) in the Americas. There are no [email protected] treatments or vaccines available, and depending on the specific hantavirus up to 40% of patients die during infection. Puumala virus, carried by the bank vole, causes a relatively mild variant of HFRS, called nephropathia epidemica, in northern Europe including Sweden. There are several other hantaviruses that also cause disease in man, all of them have rodents as natural hosts. In rodents, infection causes a life-long, but asymptomatic infection, while humans who are infected will get the disease, but clear the infection. It is not known why humans but not rodents get HFRS/HCPS. HFRS/HCPS are believed to be caused by the immune response raised against the virus, rather than directly by the virus infection per se. By analyzing immune responses in patients and voles, we hope to identify possible differences that might explain why we get disease while rodents are protected. We are also trying Hantaan virus (green) infected cells show less to characterize how phosphorylated STAT1 (red) in nuclei (blue) than non-infected cells after IFN-g-stimulation. hantaviruses interfere with the normal activation and function, of innate and adaptive immune responses. We also want to investigate the differences in the interference with the immune system in humans and natural hosts. We have found that hantaviruses are rather insensitive to the antiviral action of interferons and human saliva. Hantaviruses also avoid to activate innate immune responses against the infected cells. A better understanding of the mechanisms behind the interference of hantavirus with the human immune system and its disease causing properties is of importance for the development of HFRS/HCPSspecific treatments and vaccines. Selected publications Selected publications l l l Karlsson AC, Chapman JM, Heiken BD, Hoh R, Kallas EG, Martin JN, Hecht FM, Deeks SG and Nixon DF. Antiretroviral drug therapy alters the profile of HIV-1-specific T cell responses and shifts the immunodominant CTL response from Gag to Pol. J Virol 81, 11543, 2007. Karlsson AC, Iversen AKN, Chapman JM, de Oliveira T, Spotts G, McMichael AJ, Davenport M, Hecht FM and Nixon DF. Broadening CTL responses in early HIV-1 infection are associated with viral escape. PLoS One 2, e225, 2007. Pérez C, Larsen MV, Gustafsson R, Norström M, Atlas A, Nixon D F, Nielsen M, Lund O and Karlsson AC. Broadly immunogenic HLA class I supertype restricted elite CTL epitopes recognized in a diverse population infected with different HIV-1 subtypes. J Immunol 180, 5092, 2008. l l l Stoltz M, Ahlm C, Lundkvist Å and Klingström J. Serum interferon (IFN)-l is decreased in Hantavirus-infected patients and in vitro established infection is insensitive to treatment with all IFNs and inhibits IFN-g-induced nitric oxide production. J Virol 81, 8685, 2007. Habjan M, Andersson I, Klingström J, Schümann M, Martin A, Zimmermann P, Wagner V, Pichlmair A, Schneider U, Mühlberger E, Mirazimi A and Weber F. Processing of genome 5’ termini as a strategy of negative-strand RNA viruses to avoid RIG-I-dependent interferon induction. PLoS ONE 3, e2032, 2008. Hardestam J, Lundkvist Å and Klingström J. Sensitivity of Andes hantavirus to antiviral effect of human saliva. Emerg Infect Dis 15, 1140, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 47 Research Reports Infection Biology Antimicrobial resistance – new threats and methodological progress Modern health care depends on the effective use of antimicrobials. Increasing rates of antimicrobial resistance noted all over the world poses a serious threat to health care Göran Kronvall systems. We have a longstanding [email protected] interest in these problems focusing on analyzing resistance and on methodological studies of susceptibility tests. In an investigation of antimicrobial resistance in relation to socioeconomic factors in an area in Peru we found higher resistance in high income groups compared to low income families. This was the most interesting finding, but other results also deserved attention. In these studies we used a modified rapid method to detect both resistance in dominant Enterobacteriaceae bacteria in the gut as well as total resistance including resistant bacteria in low numbers (Kristiansson et al., 2009). In molecular studies of integron-carried resistance in Escherichia coli we have discovered two new dfr-genes. Such genes give rise to trimethoprim resistance. The appearance of these resistance genes emphasizes the rapid development of new molecular types of resistance in a bacterial species, which is both a commensal in the gut and also sometimes acts as a pathogen (Grape et al., 2007). There has been a long series of studies in our laboratory of methods for antimicrobial susceptibility testing, focusing on the most common test, the disc diffusion method. Single strain regression analysis (SRA) and the M-test are two analytical methods designed from the results of these investigations. Normalized resistance interpretation (NRI) is the latest addition to this arsenal of analytical methods for studying antimicrobial resistance. The method is now patented in the USA (US Patent No. 7,465,559) and patents are pending in other countries. Its use has recently been expanded into the area of marine microbiology where lack of standards and scarcity of test results have made interpretation of susceptibility difficult. NRI was the solution to most of these problems (Smith et al., 2007). Parasitic infections are major obstacles to development especially in Africa. Large-scale initiatives to control major Ewert Linder parasitic infections have [email protected] shown that misdiagnosis – over-diagnosis in the case of malaria and under-diagnosis of schistosomiasis - is a severe but neglected problem. Basically this is due to poor resources: microscopy in endemic regions is not cared for and standardization and quality assurance of diagnostics are difficult to achieve. To overcome these difficulties, we are developing microscopybased diagnostics, independent of ordinary microscopy. The aim is to perform image capture of stool-, blood-, sputum- etc. specimens at the grass root level and to carry out diagnostics by bi-directional communication with a central server capable of computer vision, image analysis and instant feedback. As a first step towards these goals we have begun to explore the possibilities of virtual microscopy as an educational and quality assurance tool for parasitology (Lundin et al., 2009). We have established a website (http://www.webmicroscope.net/parasitology/), where parasite specimens for diagnostics can be viewed as in an ordinary microscope, and a network of European web servers has been developed to ensure stabile high speed data transfer (Linder et al., 2008). To develop virtual microscopy for diagnostics of parasitic infections, we aim at miniaturizing image capture making the ordinary microscope obsolete. We have managed to obtain images of helminth eggs with sufficient resolution for visual identification by placing specimens on ordinary microscope slides in contact with standard image sensors (see Figure). Our results suggest that helminth On-chip imaging using eggs such as those excreted in Schisstandard complementary metal oxide semiconductosoma mansoni infection can be tor (CMOS) image sensor detected by on-chip imaging. Further of digital camera (arrow), technical developments are needed in produces images of Schistosoma mansoni eggs in order to obtain a resolution sufficient stool specimen. Microscope for the detection of smaller pathogens slide with sample (a), such as malaria parasites and specific CMOS image sensor chip (b), resulting digital image of computer algorithms for image analythe sample (c and d). Schissis need to be worked out. Selected publications Selected publications Escherichia coli antimicrobial resistance against gentamicin, trimetoprim and trimeroprimsulfamethxazole between 2005 – 2008 at the Karolinska Hospital, Solna. l l l 48 Direct image capture on sensor chips and virtual microscopy for diagnostics of parasitic infections Smith P, Ruane NM, Douglas I, Carroll C, Kronvall G and Fleming GTA. Impact of inter-lab variation on the estimation of epidemiological cut-off values for disc diffusion susceptibility test data for Aeromonas salmonicida. Aquaculture 272, 168, 2007. Grape M, Sundström L and Kronvall G. Two new dfr-genes in trimethoprim-resistant integron-negative Escherichia coli. Antimicrob Agents Chemother 51, 1863, 2007. Kristiansson C, Grape M, Gotuzzo E, Samalvides F, Chauca J, Larsson M, Bartoloni A, Pallecchi L, Kronvall G and Petzold M. Socioeconomic factors and antibiotic use in relation to antimicrobial resistance in the Amazonian area of Peru. Scand J Infect Dis 41, 303, 2009. Department of Microbiology, Tumor and Cell Biology – MTC l l l tosome eggs sized about 50x100 micrometers (e). Linder E, Lundin M, Thors C, Lebbad M, Winiecka-Krusnell J, Helin H, Leiva B, Isola J and Lundin J. Web-based virtual microscopy for parasitology: a novel tool for education and quality assurance. PLoS Negl Trop Dis 2, e315, 2008. Lundin M, Szymas J, Linder E, Beck H, de Wilde P, van Krieken H, García Rojo M, Moreno I, Ariza A, Tuzlali S, Derviolu S, Helin H, Lehto VP and Lundin J. A European network for virtual microscopy-design, implementation and evaluation of performance. Virchows Arch 454, 421, 2009. Derda M, Winiecka-Krusnell J, Linder MB and Linder E. Labeled Trichoderma reesei cellulase as a marker for Acanthamoeba cyst wall cellulose in infected tissues. Appl Environ Microbiol 75, 6827, 2009. Research Reports Infection Biology Research on emerging viruses Our research is based on an interdisciplinary approach between molecular virology, immunology, genetics, epidemiology and diagnostic aspects of zoonoses, especially Åke Lundkvist highly human-pathogenic viruses. Our [email protected] major efforts are at present focusing on the following agents: Flaviviruses (tick-borne encephalitis virus, dengue virus), Bunyaviruses (hantavirus, Rift Valley fever virus) and avian influenza virus. Our hantavirus program has generated important results concerning novel animal models (monkey and rodents), virus-host interactions, pathogenesis, apoptosis and innate immunity. We have also found valuable, and sometimes surprising results on how, and under which circumstances, various Bunyaviruses can survive outside their vectors and hosts. The extreme increase of Epidemic Nephropathy caused by Puumala hantavirus infections in northern Sweden during recent years prompted us to investigate the presence and circulation of viral genetic lineages/sublineages in more detail. One of the major responsibilities of our group is microbiological preparedness. Microarray-based diagnostic techniques for detection of various emerging viruses have been developed. The awareness of highly pathogenic avian influenza virus repeatedly infecting man initiated also basic research aiming for a better understanding of the transmission and the dramatic changes in virulence. Based on a new animal model, we have studied how various avian influenza virus strains affect one of the major reservoirs, the mallard. Dengue fever pathogenesis and Rift Valley fever vaccine candidates are examples of new research areas. Based on a unique collection of virus isolates from dengue fever, dengue hemorrhagic fever, and dengue shock syndrome patients in Cambodia, we have recently initiated the search for markers hopefully explaining at least parts of the dramatic variation in dengue pathogenesis. New approaches, based on expression of viral structural proteins in plants, have been initiated for Rift Valley fever vaccine development. The Mallard (”gräsand” in Swedish) – an important natural reservoir of avian influenza virus. Genetic variability of important viral pathogens Viral hepatitis is a leading cause of liver cancer and a common reason for liver transplantation. We have previously described three new genotypes E, F and H of hepatitis B virus (HBV). MolecuLars Magnius lar epidemiology of HBV worldwide [email protected] detected virus isolates with identical S-genes common in Russia and Estonia. These isolates remained closely related, when complete HBV genomes were sequenced, but divided into five different clades three of which shared a monophyletic origin pointing towards possible iatrogenic transmission in the past (Tallo et al., 2008). Dendrogram obtained with UPGMA based on complete HBV genomes showing evolutionary relatedness and monophyly of three clades, I, II and IV, with identical S genes within each clade and the unrelated clade V also with identical S genes. Hepatitis E virus (HEV) was once considered a pathogen confined to tropical areas causing waterborn outbreaks often with thousands of cases. Collaborative research has revealed indigenous cases in Sweden and Denmark caused by HEV genotype 3. This genotype was prevalent not only in pigs, but also in wild boar which carried strains similar to the human cases (Norder et al., 2009). The route of transmission between pigs and man is under investigation. Enteroviruses infect millions of individuals with a wide range of clinical outcomes ranging from inapparent infections to acute flaccid paralysis. In parallel with the Center of Disease Control, USA, we established for the first time molecular typing for enteroviruses which yielded results consistent with serological typing enabling us to identify new enterovirus types. In line with these studies we have characterized enterovirus strains from cases of acute flaccid paralysis suspected to be caused by the polioviruses, other enteroviruses, in the Democratic Republic of Kongo. Thereby divergent isolates were found representing two new enterovirus types, EV-93 and EV-94 (Junttila et al., 2007). Selected publications l Selected publications l l l Hardestam J, Karlsson M, Falk KI, Olsson G, Klingström J and Lundkvist A. Puumala hantavirus excretion kinetics in bank voles (Myodes glareolus). Emerg Infect Dis 14, 1209, 2008. Klingström J, Stoltz M, Hardestam J, Ahlm C and Lundkvist Å. Passive immunization protects cynomolgus macaques against Puumala hantavirus challenge. Antivir Ther 13, 125, 2008. Jourdain E, Gunnarsson G, Wahlgren J, Latorre-Margalef N, Bröjer C, Sahlin S, Svensson L, Waldenström J, Lundkvist Å and Olsen B. Influenza virus in a natural host, the Mallard: Experimental infection data. PLoS ONE 5, e8935, 2010. l l Junttila N, Lévêque N, Kabue JP, Cartet G, Mushiya F, MuyembeTamfum JJ, Trompette A, Lina B, Magnius LO, Chomel JJ and Norder H. New enteroviruses, EV-93 and EV-94, associated with acute flaccid paralysis in the Democratic Republic of the Congo. J Med Virol 79, 393, 2007. Tallo T, Tefanova V, Priimägi L, Schmidt J, Katargina O, Michailov M, Mukomolov S, Magnius L and Norder H. D2: major subgenotype of hepatitis B virus in Russia and the Baltic region. J Gen Virol 89, 1829, 2008. Norder H, Sundqvist L, Magnusson L, Østergaard Breum S, Löfdahl M, Larsen LE, Hjulsager CK, Magnius L, Böttiger BE and Widén F. Endemic hepatitis E in two Nordic countries. Euro Surveill 14, 19211, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 49 Research Reports Infection Biology Molecular mechanisms controlling adaptation of bacteria Bacteria need to sense the environment in order to rapidly adapt to new and changing conditions, for example when they cause infections. We are especially interested in how the membraneÖjar Melefors bound two-component sensor BarA [email protected] can sense environmental factors and how this leads to phenotypic changes in the bacteria. It is not known exactly what these factors are, but we have recently demonstrated that growth rate plays a clear role in BarA activation in Escherichia coli. Activation of BarA leads to phosphorylation of the response regulator UvrY, which, in turn, controls transcription of the CsrB and CsrC sRNAs. These two sRNAs act by sequestering the CsrA regulatory protein. Free CsrA can bind to a number of target mRNAs, leading commonly to a block in translation and a decreased mRNA stability (or in some cases to the opposite effects). Earlier work has mainly identified target mRNAs involved in carbon metabolism. Using a microarray approach, we have recently shown that target mRNAs in E. coli often encode GGDEF/EAL domain containing proteins. We could, in a subsequent work, also show that CsrA targets a different set of GGDEF/EAL mRNAs in Salmonella typhimurium. Bacteria contain a large number of GGDEF and EAL domain proteins that act by controlling the levels of the intracellular second messenger cyclic di-GMP. This second messenger seems to be a key factor in the activation of virulence genes and in the decision of the bacteria to remain in a biofilm state or to become motile. We believe that a better understanding of these regulatory systems could reveal novel targets for antibiotic therapy in the future. Adaptation via the BarA system in Escherichia coli. Selected publications l l l 50 Jonas K, Edwards AN, Simm R, Romeo T, Römling U and Melefors, Ö. The RNA binding protein CsrA controls c-di-GMP metabolism by directly regulating the expression of GGDEF proteins. Mol Microbiol 70, 236, 2008. Jonas K and Melefors Ö. The Escherichia coli CsrB and CsrC small RNAs are strongly induced during growth in nutrient poor medium. FEMS Microbiol Lett 297, 80, 2009. Jonas K, Edwards AN, Ahmad I, Romeo T, Römling U and Melefors Ö. Complex regulatory network encompassing the Csr, c-diGMP, and motility systems of Salmonella typhimurium. Environ Microbiol 12, 524, 2010. Department of Microbiology, Tumor and Cell Biology – MTC Ecology of the intestinal Escherichia coli flora and diarrhoeal diseases The aim is to increase the understanding of the ecology of the intestinal flora and the role of diarrhoeagenic Escherichia coli (DEC). We have found that the majority of infant diarRoland Möllby rhoea in León appears to be caused by [email protected] E. coli that contains various virulence genes. There are different types of DEC, the best studied are the enterotoxigenic E. coli (ETEC) that may contain the gene encoding labile toxin (LT) and/or stable toxin (ST). During diagnostic, the virulence genes are detected by PCR-based methods using the collected sample, but not on individual isolates. Strangely enough, though, the various DEC types were almost as often detected in healthy infants as in infants with acute diarrhoea. Even more, when we investigated the individual bacterial isolates present in each individual by PhenePlate(R) typing, the strains diversity was similar in sick and healthy infants. Often only a small fraction of the E. coli bacteria investigated were found to contain the specific virulence genes. In contrast, E. coli strains containing the stable (entero) toxin gene estA, showed a low diversity, were only found Hypothesis of spread of virulence genes in true in a limited number of or false DEC. strains and dominated the E. coli flora in children with diarrhoea. These strains were sometimes very similar to old ETEC reference strains, isolated in other parts of the world more than twenty years ago. EstA carrying strains were able to transfer their genes through conjugation to non-pathogenic E. coli strains in the test tube. It is thus possible that there exist certain “Truly pathogenic E. coli (TPEC?)” that are very stable over time and that are able to dominate the intestinal flora of E. coli when they cause diarrhoea. Furthermore, these strains are “promiscuous” and give away their virulence genes in the intestine to various strains of the normal flora. These “Falsely pathogenic E. coli” are then detected in faeces and erroneously interpreted as the cause of diarrhoea. Therefore, new and more reliable methods for such investigations, from individual diagnostic to national surveillance projects, need to be developed urgently. Selected publications l l l Reyes D, Vilchez S, Paniagua M, Colque P, Weintraub A, Möllby R and Kühn I. Diversity of intestinal Escherichia coli populations in Nicaraguan children with/without diarrhoea. J Med Microbiol 58, 1593, 2009. Vilchez S, Reyes D, Paniagua M, Bucardo F, Möllby R and Weintraub A. Prevalence of diarrhoeagenic Escherichia coli in children from León, Nicaragua. J Med Microbiol 58, 630, 2009. Rahman M, Huys G, Kühn I, Rahman M and Möllby R. Prevalence and transmission of antimicrobial resistance among Aeromonas populations from a duckweed aquaculture based hospital sewage water recycling system in Bangladesh. Antonie Van Leeuwenhoek 96, 313, 2009. Research Reports Infection Biology The normal microflora All healthy mammals are born germfree, thus free of any microbes. Yet shortly after birth, microbes establish themselves with a growing complexity. Today we know that Elisabeth Norin the bacterial cells in the intestine outnumTore Midvedt ber the number of cells comprising the [email protected] whole human body. Thus, the microbes [email protected] live in a symbiosis with the host, mostly in a “balanced unbalance”, i.e. in a system based on continuous cross talk. There are three major actors, namely the host organism, the intestinal microflora and the environment. Any alteration of these actors, such as antimicrobial treatment, various diseases, surgery, physical-emotional stress, dietary alterations or probiotics, malnutrition or starvation may disturb the balance for a shorter or longer period. The intestinal microflora can be evaluated in several ways. We have chosen to study the flora under germfree and conventional conditions using the MAC-GAC concept. A MAC (Microflora Associated Characteristic) is defined as any anatomical structure, physiological, immunological or biochemical function in an organism that has been influenced by the microbes. When active microbes are absent, as in healthy newborn infants, sometimes after antimicrobial treatment and in germfree animals, these functions are defined as GACs (Germfree Animal Characteristics). Today we are involved in several projects. We follow the establishment of the intestinal flora in infants, healthy and diseased, aiming to investigate the role of exogenous factors of the development of the flora, health status and eventual allergy development. We study the intestinal flora in children at different ages from Italy, Germany, Spain, Scotland and Sweden. We are also involved in investigations regarding the role of the flora in celiac disease. Moreover, we investigate the role of dietary supplements (pre- and probiotics) given to both infants and elderly. Scheme of our gastrointestinal tract. Selected publications l l l Benno P, Ernberg I, Marcus C, Midtvedt T, Möllby R, Norin E and Svenberg T. MAGEN. Bakterier, buller och brak. Karolinska Institutet University Press 2008. Norin E, Jernberg C, Nilsson H-O and Engstrand L. Studies of the intstinal microflora by traditional, functional and molecular techniques. In; Lactobacillus, Molecular Biology – from genetics to probiotics. Eds: Ljung Å, Wadström T. Caister Academic press Norfolk, UK. p. 83, 2009. Sandin A, Bråbäck L, Norin E and Björkstén B. Faecal short chain fatty acid pattern and allergy in early childhood. Acta Paediatr 98, 823, 2009. Microbe-host interactions with a focus on Grampositive infections Our main research focuses on pneumococcal infections that are major causes of morbidity and mortality world-wide, annually killing 1–2 million individuals. PneumoBirgitta Henriques Normark cocci are also common colonizStaffan Normark ers of healthy children, a fact that [email protected] has prompted us to identify host staffan [email protected] and microbial factors deciding the outcome of disease. We found capsular serotypes with a high invasive disease potential behave as primary pathogens, whereas types with a lower relative risk of causing invasive disease affect primarily patients with underlying disease. Comparative genomic analyses identified a large number of accessory genetic regions present in subsets of clinical isolates. One such region codes for a pilus preferentially expressed by clones prone to colonize the nasopharynx. This pilus contributes to virulence and inflammatory host responses. On the host side, Toll like receptor 9 plays a central role in early pneumococcal defense promoting clearance by alveolar macrophages. Defensin-mediated neutrophil killing and bacterial capture in neutrophil extracellular traps (NETs) have been studied in detail. Pneumococci are not killed by NETs which are formed in pulmonary infections. NET trapping can be affected by the pneumococcal capsule and the surface charge of the bacteria. Furthermore, we have studied the role of antimicrobial effector proteins and peptides in host defence of the lungs and small intestine and described the first example of a complete developmental switch in innate immune effector expression and anatomical distribution. Moreover, we have characterized pili in enterococci, and studied Type III secretion system inhibitors in Chlamydia infections. Finally, our recent data suggest that cysteine Pneumococci (green) are trapped in NETs exclusion is an impor(elastase-red, DNA-blue). tant adaptive strategy among some Gram-positive bacteria against the challenges presented by oxidative environments. The practical implications of our data have facilitated antigen discovery for vaccines. Our studies on pilus epidemiology, composition and formation provide a basis for a pilus subunit vaccine and the structure/function of a pilus synthesizing enzyme opens novel approaches to prevent pilus formation. Selected publications l l l Sjöström K, Blomberg C, Fernebro J, Dagerhamn J, Morfeldt E, Barocchi M, Andersson M, Browall S, Henriques F, Rappuoli R, Normark S and Henriques-Normark B. Clonal success of piliated penicillin non-susceptible pneumococci. Proc. Natl Acad Sci USA 104, 12907, 2007. Henriques-Normark B, Blomberg C, Dagerhamn J, Bättig P and Normark S. The rise and fall of bacterial clones: with a focus on Streptococcus pneumoniae. Nat Microbiol Rev 6, 827, 2008. Daniels R, Mellroth P, Bernsel A, Neiers F, Normark S, von Heijne G and Henriques-Normark B. Disulfide bond formation and cysteine exclusion in Gram-positive bacteria. J Biol Chem 285, 3300, 2010. Department of Microbiology, Tumor and Cell Biology – MTC 51 Research Reports Infection Biology Characterization of HIV-1 populations in infected cells: role in HIV-1 evolution and persistent viremia Determining the genetic relatedness of HIV-1 populations in cells, plasma and tissue is important to understand the pathogenesis of HIV-1 and to target and eradicate persistent HIV viremia in patients Sarah Palmer on suppressive therapy. We [email protected] adapted unique techniques never used before in parallel, single-cell sequencing, single-genome sequencing, and single-copy assay, to characterize HIV-1 populations in single infected cells and plasma from HIV-infected patients. Analysis of cells and plasma from 9 HIV-infected patients revealed that a majority (80-90%) of infected CD4+ T cells contained a single viral DNA molecule indicating limited potential for recombination in virus produced by these cells. Sequencing revealed that the intracellular viral DNA from CD4+ T cells was phylogenetically related to contemporaneous plasma RNA implying ongoing exchange between these compartments throughout infection. Moreover, the genetic diversity and phylogenetic relatedness of intracellular and plasmaderived sequences from one patient remained unchanged after 6 months of suppressive therapy indicating a large pool of infected cells persists even after plasma viral RNA decrease. Analysis of 20002x105 CD14+/CD16+ monocytes from the same patients revealed no infected cells suggesting the frequency of infection is much greater in CD4+ T cells than monocytes. In collaboration with the US National Institutes of Health, we are phylogenetically comparing the HIV populations in plasma to those in infected cells extracted from peripheral blood mononuclear cells and cells located in the lymphoid tissue. These studies will further extend our understanding of HIV pathogenesis. With partners in Spain, Germany, Netherlands and USA, we characterize persistent HIV virus infection in patients on HIV treatment intensification to determine if additional therapy reduces and possibly eradicates residual virus in plasma or other compartments such as the central nervous system. Investigation of functionally important antibodies against malaria Malaria is a disease that kills one million people every year. Nearly all deaths are caused by the parasite Plasmodium falciparum. The merozoite form of P. falciparum Kristina E M Persson invades red blood cells and [email protected] plies within the cell, finally causing rupture of the red blood cell with release of daughter merozoites back into the circulation after 48 hours. Those who live in malaria-endemic areas and do not die from the disease at a young age, eventually develop immunity, but only slowly and after repeated exposure. Potential candidates for development of vaccines include antigens expressed on the surface of merozoites and/or proteins that are involved in erythrocyte invasion. Antibodies are an important component of acquired protective immunity. In our group, we are trying to understand which functions of antibodies are important for protection against malaria. Basic understanding of how these antibodies work is imperative for creating a functioning vaccine against malaria. We are studying the affinity of antibodies directed against different merozoite antigens, as well as the invasion inhibitory effect, and correlating this to protection. We are also investigating different invasion pathways in wild type isolates of parasites, and correlating this to symptoms and severity of disease. We have before developed a method for measuring invasion inhibitory antibodies, and we have also shown that variation in use of invasion pathways (involving the merozoite antigens EBA and Rh proteins) mediates evasion of inhibitory antibodies. The merozoite form of P. falciparum invades erythrocytes, but this process can be inhibited by antibodies directed against different merozoite antigens. Similar diversity in intra- and extracellular HIV compartments. Selected publications l l l 52 Kearney M, Maldarelli F, Shao W, Margolick JB, Daar ES, Mellors V, Rao V, Coffin JM and Palmer S. HIV-1 population genetics and adaptation in newly infected individuals. J Virol 83, 2715, 2009. Pereyra F, Palmer S, Miura T, Block BL, Wiegand A, Rothchild AC, Baker B, Rosenberg R, Cutrell E, Seaman MS, Coffin JM and Walker BD. Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters. J Infect Dis 200, 984, 2009. Dahl V, Josefsson L and Palmer S. HIV reservoirs, latency, and reactivation: prospects for eradication. Antivir Res 85, 286, 2010. Department of Microbiology, Tumor and Cell Biology – MTC Selected publications l l l Persson KE, McCallum FJ, Reiling L, Lister NA, Stubbs J, Cowman AF, Marsh K and Beeson JG. Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies. J Clin Invest 118, 342, 2008. Brolin KJ, Persson KE, Wahlgren M, Rogerson SJ and Chen Q. Differential recognition of P. falciparum var2csa domains by naturally acquired antibodies in pregnant women from a malaria endemic area. PLoS One 5, e9230, 2010. Persson KE. Erythrocyte invasion and functionally inhibitory antibodies in Plasmodium falciparum malaria. Acta Trop 114, 138, 2010. Research Reports Infection Biology Inflammatory responses to bacterial pathogens The ability of the host to defend itself against invasion by respiratory bacterial pathogens depends on the capacity to mount efficient immune responses. Our research focuses on their role in the Laura Plant induction of innate immunity and in [email protected] priming of adaptive immune responses to bacterial infection. We have recently published studies on the effect of the pneumococcal cytotoxin, pneumolysin, on human dendritic cell (DC) responses. Our studies revealed that the expression of haemolytic pneumolysin by pneumococci specifically inhibits the maturation of human DCs and the induction of proinflammatory cytokines and activation of the inflammasome. In addition, we have shown that clinical isolates with non-hemolytic pneumolysin are more proinflammatory and less apoptotic compared to clonally related strains with active pneumolysin. We have also been conducting studies onto the effect of the pneumolysin and pneumococcal produced hydrogen peroxide on T cell activation and survival. Despite accumulating evidence that chronic inflammation associates with prostate cancer the responsible causes of prostatic inflammation remain elusive. Epidemiologically, prostatitis and prostate cancer have been connected with infectious agents that evoke and maintain persistent inflammation. In a collaborative project we are evaluating the inflammatory potential of the prostate in relation to bacterial infections and bacterial components in order to determine whether bacterial infection can be implicated in events leading to prostate cancer. Intracellular localization of pneumococci in dendritic cells. Selected publications l l l Plant L, Wan H and Jonsson A-B. Non-lipooligosaccharide mediated signaling via toll-like receptor 4 causes fatal meningococcal sepsis in a mouse model. Cell Microbiol 9, 657, 2007. Lövkvist L, Sjölinder H, Wehelie R, Aro H, Norrby-Teglund A. Plant L and Jonsson AB. CD46 contributes to the severity of group A streptococcal infection. Infect Immun 76, 3951, 2008. Littmann M, Albiger B, Normark S, Henriques-Normark B and Plant L. Streptococcus pneumoniae evades human dendritic surveillance by pneumolysin expression. EMBO Mol Med 1, 211, 2009. Bacterial-host homeostasis in the gastrointestinal tract and oral cavity Mammals have evolved in the presence of bacteria. Hosts have developed symbiotic relations which, especially in the mouth and gastrointestinal tract, allow Mats Andersson beneficial bacteria to reside, while Katrin Pütsep preventing opportunistic [email protected] gens to gain foothold. The mechanism for this selection system is largely unknown. Our hypothesis is that antimicrobial peptides are a part of a system that controls this homeostasis. The intestinal mucosal epithelium consists of a single cell layer. This epithelium is rich in host derived antimicrobial peptides and proteins, which kill bacteria without involvement of strong inflammatory, tissue damaging reactions. We have shown that the repertoire and expression of antimicrobial peptides change following birth, increases in the mouse small intestine from gut to colon (Karlsson, Pütsep et al., 2008). The secreted intestinal antimicrobial components are retained by the mucus layer (Meyer-Hoffert et al., 2008), thus indicating a role for these peptides in shaping the microflora. The clinical relevance is stressed by the fact that many patients with inflammatory bowels diseases present with low levels of intestinal peptides and highly colonized mucus layers. To further elucidate the impact of innate defence on the natural microflora, we will analyze the intestinal bacterial metagenome in gene knockout models for innate defence molecules. The protection of the oral cavity is largely mediated by neutrophils and individuals with neutrophil dysfunction or severe neuImmunohistochemical localization of antibiotic peptides (CRS4C) in Paneth’s cells of tropenia are at high risk the mouse small intestine: Blue: cell nuclei. for periodontal disGreen: CRS4C. ease and tooth loss. We have earlier demonstrated that neutrophil granules of patients with chronic severe congenital neutropenia (SCN) are deficient in the antimicrobial peptide LL-37. This deficiency may serve as a specific biomarker for SCN. The lack of LL-37 was at a transcript level, although expression was readily activated by vitamin D in neutrophil precursors (Karlsson, Carlsson et al., 2008). Further studies aim to elucidate the mechanisms for this deficiency. Selected publications l l l Karlsson J, Pütsep K, Chu H, Kays RJ, Bevins CL and Andersson M. Secreted enteric antimicrobial activity localizes to the mucus surface layer. Gut 57, 764, 2008. Karlsson J, Carlsson G, Larne O, Andersson M and Pütsep K. Vitamin D3 induces pro-LL-37 expression in myeloid precursors from patients with severe congenital neutropenia. J Leukocyte Biol 84, 1279, 2008. Meyer-Hoffert U, Hornef MW, Henriques-Normark B, Axelsson L-G, Midtvedt T, Pütsep K and Andersson M. Regional variations in Paneth cell antimicrobial peptide expression along the mouse intestinal tract. BMC Immunol 17, 9, 2008. Department of Microbiology, Tumor and Cell Biology – MTC 53 Research Reports Infection Biology Microbial pathogenicity – a functional integration of virulence factor and bacterial house-keeping functions To enable colonization of hosts bacterial pathogens rely on selected genetic traits termed virulence factors. Amongst enteric bacteria, virulence factors are often coded for by horizonMikael Rhen tally acquired genetic elements in [email protected] the form of genetic continuums, termed pathogenicity island that became integrated into a conserved core genome during the evolution of the pathogen. Thus one may ask how such virulence factors became functionally integrated into functions coded for by the core genome of the bacterium. Others and we have shown that elemental virulence factors do not act as independent units but strongly depend on functions provided by the core genome. This integration occurs at two levels. First, the genetic regulation of virulence genes has to be integrated with the existing pattern of gene regulation. Second, the synthesis and assembly of virulence factors also rely on the biochemical constitution of the bacterial cell. We have used transcriptomic profiling of the classical bacterial model pathogen – Salmonella enterica – to define how the expression of individual genes on a whole genome scale becomes altered as the pathogen shifts between host-like environments. Such analyses defined significant alterations in virulence gene expression and revealed many core genome genes to be co-regulated with virulence genes. Inactivation of such core genes may not affect the in vitro well-being of the bacteria but may have a strong effect on the virulence in infection models. An example of this is thioredoxin 1, an evolutionary highly conserved oxidoreductase. Thioredoxin is needed for the activity of the Salmonella pathogenicity island 2, which in Salmonella in its phase as an intracellular bacterial pathogen. The image reveals S. turn steers bacterial typhimurium bacteria (green) replicating in an intracellular replica- eukaryotic cell with the aid of secreted virution and virulence. lence proteins (red). Host cell nuclei in blue. Our intention is to deepen such data mining and to probe for additional connections between virulence genes and ordinary house-keeping genes. The findings will illuminate how pathogens have evolved to cause disease and avoid host immune responses and possibly reveal new targets for pharmaceutical interference with virulence. Selected publications l l l 54 Hautefort I, Thompson A, Eriksson-Ygberg S, Parker ML, Lucchini S, Danino V, Bongaerts RJ, Ahmad N, Rhen M and Hinton JC. During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cell Microbiol 10, 958, 2008. Negrea A, Bjur E, Puiac S, Eriksson-Ygberg S, Åslund F and Rhen M. Thioredoxin 1 participates in the activity of the Salmonella enterica serovar Typhimurium pathogenicity island 2 type III secretion system. J Bacteriol 191, 6918, 2009. Puiac S, Negrea A, Richter-Dahlfors A, Plant, L and Rhen M. Omeprazole antagonizes virulence and inflammation in Salmonella enterica-infected RAW264.7 cells. Antimicrob Agents Chemother 53, 2402, 2009. Department of Microbiology, Tumor and Cell Biology – MTC Cyclic di-GMP signaling in biofilm formation and virulence of Salmonella typhimurium The small molecule cyclic di-GMP is a novel secondary messenger in many bacteria that regulates biofilm formation and virulence. Regulation Ute Römling of cyclic di-GMP metabolism [email protected] is complex, for example the genome of the enteric pathogen Salmonella typhimurium encodes eight putative di-guanylate cyclases and fifteen putative phosphodiesterases. Previously, we have shown that two di-guanylate cyclases contribute to the expression of biofilm formation in S. typhimurium. Recently, we demonstrated that four phosphodiesterases play a determinative role in the expression of biofilm formation. Thus, the effective cyclic di-GMP concentration is determined by the delicate balance between di-guanylate cyclase and phosphodiesterase activities. Virulence is another phenotype frequently affected by cyclic di-GMP signaling. We could recently show that cyclic di-GMP signaling affected invasion and induction of a pro-inflammatory cytokine in epithelial cells, basic phenotypes that are major determinants of S. typhimurium virulence. Invasion was mainly inhibited through the biofilm regulator CsgD and the biofilm matrix component cellulose. Inhibition of the pro-inflammatory response occurred through CsgD which inhibited the secretion of pro-inflammatory monomeric flagellin. The biofilm matrix component cellulose also affected microbehost interaction in the probiotic Escherichia coli strain Nissle 1917. Cellulose production in Nissle 1917 contributes to enhanced cytokine production and to adhesion of bacteria to a gastrointestinal epithelial cell line and to the mouse epithelium in vivo. Our fundamental research on bacterial signaling mechanisms demonstrated that cyclic di-GMP signaling is not only required for the regulation of biofilm formation, but also affected virulence properties in S. typhimurium. Future research will focus on the molecular mechanisms of cyclic di-GMP signaling in biofilm-formation and virulence. Salmonella typhimurium expressing cellulose. Selected publications l l l Simm R, Lusch A, Kader A, Andersson M and Römling U. The role of EAL containing proteins in multicellular behavior of Salmonella enterica serovar Typhimurium. J Bacteriol 189, 3613, 2007. Monteiro C, Saxena I, Wang X, Kader A, Bokranz W, Simm R, Nobles D, Chromek M, Brauner A, Brown Jr RM and Römling U. Characterisation of cellulose production in Escherichia coli Nissle 1917 and its biological consequences. Environ. Microbiol 11, 1105, 2009. Lamprokostopoulou A, Monteiro C, Rhen M and Römling U. C-di-GMP controls virulence properties of Salmonella enterica serovar Typhimurium at the mucosal lining. Environ Microbiol 12, 40, 2010. Research Reports Infection Biology Molecular pathogenesis of severe Plasmodium falciparum malaria We study the molecular pathogenesis of severe Plasmodium falciparum malaria with a focus on the molecules PfEMP1- RIFIN-SURFIN present at the infected red cell surface. RIFINs Mats Wahlgren are co-transported with PfEMP1 and [email protected] SURFINs to the parasitized erythrocytes surface but also accumulate in the parasitophorous vacuole and associate with the merozoite. By exposing shared polymorphic antigens on both parasitized erythrocytes and merozoites the parasite may coordinate the antigenic composition of these attachment-surfaces during growth in the bloodstream. Indeed, a recent analysis predicted that the RIFINs have undergone neo- or sub-functionalization and it was found possible to divide the RIFINS into sub-groups with differential developmental expression and localization patterns. The multiple genes encoding PfEMP1-RIFIN-SURFIN were identified to originate from duplications and transpositions of genomic segments onto multiple subtelomeres. The ubiquity of the segmental duplications, and the nature of genes within, suggests an important role in plasmodia speciation. Our team previously described P. falciparum rosetting, its association with severe malaria and the importance of the PfEMP1-family of proteins as adhesins. Rosetting depends on PfEMP1 and heparan sulfate expressed at the erythrocyte- and endothelial cell surfaces. Based on these findings we are presently developing a receptor-based drug together with Dilafor AB, a KIAB-company (www.dilafor.com). A significant milestone was recently passed with the completion of a Phase I study proving that the new compound is safe and well tolerated. Furthermore, an archetype anti-rosetting vaccine against severe disease based on the heparan sulfate-binding domain of PfEMP1 is presently being developed. Immunization of animals abolished PfEMP1-mediated sequestration. These results support the use of the PfEMP1 domain in the development of a vaccine targeting severe malaria. Staining of two distinct SURFIN in the parasitophorous vacuole of Plasmodium falciparum (red and green). Nuclei are stained blue. Towards protective and curative HIV immunity We are working on strategies to prevent or treat HIV infection by immunological means. For this purpose we have constructed and refined genetic vaccines representing several subtypes of HIV as Britta Wahren well as including most genes of the virus. [email protected] Compositions of genes are inserted in single plasmids. When a suitable composition, according to subtypes resident in the geographical region, is introduced into animal cells, they produce both free proteins and virus-like particles. Preclinical studies of the properties of these vaccine plasmids have demonstrated broad and strong cell-mediated and humoral reactivity systemically and at mucosal tissues. We have shown protection to HIV challenge in an experimental model, and even been able to eradicate infectious HIV by means of HIV-directed drug-labeled antibody infusion. Our clinical HIVIS program has set out to study the delivery of this genetic HIV vaccine boosted with a recombinant vaccinia vector in Sweden and Tanzania. Strong and broad cross reactivity against antigens from HIV subtypes HIV virus-like particle recovered A-E have been found as well from Vero cells transfected with DNA as good safety. Although the plasmids encoding HIV genes gp120, gp41, and gag. Anti V3 P4/D10+ staph HIV Vaccine ImmunogenicA labelled gold particle attaches to ity Study vaccine schedule surface of HIV-like particle (upper right was designed to elicit primaquadrant). Photomicrograph by B. Wahren, D. Hallengärd, A. Bråve, M. rily cell-mediated immunity, Liu, L. Xing and H. Cheng. broad antibody responses are observed in all volunteers after boosting. Therapeutic studies in Sweden and Italy have revealed induction of new cell-mediated immune responses as well as strengthening of previous reactions. Our next goal is to amplify primary responses by improved delivery systems, both systemically and locally at the mucosae of humans. Selected publications l Selected publications l l l Normark J, Nilsson D, Ribacke U, Winter G, Moll K, Wheelock CE, Bayarugaba J, Kironde F, Egwang TG, Chen Q, Andersson B and Wahlgren M. PfEMP1 motifs predict severity of Plasmodium falciparum malaria. Proc Natl Acad Sci USA 104, 15835, 2007. Chêne A, Donati D, Guerreiro-Cacais AO, Levitsky V, Chen Q, Falk KI, Orem J, Kironde F, Wahlgren M and Bejarano MT. A molecular link between malaria and Epstein-Barr virus reactivation. PLoS Pathog 3, e80, 2007. Mok BW, Ribacke U, Rasti N, Kironde F, Chen Q, Nilsson P and Wahlgren M. Default pathway of var2csa switching and translational repression in Plasmodium falciparum. PLoS ONE 3, e1982, 2008. l l Bråve A, Boberg A, Gudmundsdotter L, Rollman E, Hallermalm K, Ljungberg K, Blomberg P, Stout R, Paulie S, Sandström E, Biberfeld G, Earl P, Moss B, Cox JH and Wahren B. A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T cell and humoral responses in mice. Mol Ther 9, 1724, 2007. Sandström E, Nilsson C, Hejdeman B, Bråve A, Bratt G, Robb M, Cox J, Vancott T, Marovich M, Stout R, Aboud S, Bakari M, Pallangyo K, Ljungberg K, Moss B, Earl P, Michael N, Birx D, Mhalu F, Wahren B, Biberfeld G and the HIV Immunogenicity Study 01/02 Team. Broad immunogenicity of a multigene, multiclade HIV-1 DNA vaccine boosted with heterologous HIV-1 recombinant modified vaccinia virus Ankara. J Infect Dis 198, 1482, 2008. Roos AK, Eriksson F, Timmons JA, Gerhardt J, Nyman U, Gudmundsdotter L, Bråve A, Wahren B and Pisa P. Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment. PloS One 4, e7226, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 55 Research Reports Cell and Tumor Biology Cell and Tumor Biology Research Hanahan and Weinberg published the highly influential and visionary millennium article on the “Hallmarks of Cancer”, where 25 years of cancer research was summarized (Hanahan D and Weinberg RA, Cell 100, 57, 2000). Cancer was described as a multistep process involving activating/inactivating mutations in oncogenes and tumor suppressor genes (TSG), respectively, resulting in deregulation of fundamental cellular processes, thereby leading to cancer. Common hallmarks of cancer cells of different origins included Exactly ten years ago 56 self-sufficiency in growth signals, insensitivity to anti-proliferative signals, evading apoptosis, limitless replicative potential, sustained angiogenesis and tissue invasion and metastasis (see Figure). Despite tremendous progress to elucidate these processes, cancer metastasis is still poorly understood and will surely come more into focus. Ten years later, a number of new hallmarks can be added to the list as a result of the accelerating scientific and technological development (see Figure). One Marie Arsenian Henriksson Pontus Aspenström Yihai Cao Ingemar Ernberg Georg Klein Sonia Laín Lars-Gunnar Larsson Uno Lindberg Sven Pettersson Galina Selivanova Laszlo Szekely Department of Microbiology, Tumor and Cell Biology – MTC Eugene Zabarovsky such hallmark is block of differentiation, which characterizes most tumors. This may be related to the discovery of cancer stem cells or tumor-initiating cells with self-renewal capacity within the tumor mass. Another important hallmark of tumor cells which is under intense research is their potential of epigenetic reprogramming of the genome, which may explain some of their plasticity. A remarkable recent finding was that only Eva Klein four genes, including two oncogenes, could induce reprogramming of differentiated cells into pluripotent stem cells (iPS), an epigenetic phenomenon that may have relevance for cancer. Another hallmark is the ability of tumor cells to evade oncogene-induced cellular senescence, a state of irreversible growth arrest. This is probably related to another hallmark, the ability to cope with cellular stress causing DNA damage responses, one of the features of “oncogene addiction”. It has also become increasingly clear during recent years Rolf Ohlsson that communication between tumor cells and the environment, such as the tumor stroma Project leaders: is imperative to sustain growth and facilitate Gesan Arulampalam metastasis. Avoiding immune surveillance is LiFu Hu yet another hallmark. Stefan Imreh Much of the recent achievements Elena Kashuba mentioned above have been made possible through the very rapid technological development in genomics as well as in other “omics” enabling high-throughput measurements at a global level. This has paved the way for “systems biology”, where all these data are integrated, facilitating identification of key regula- Research Reports Cell and Tumor Biology tors that will be in focus as targets for cancer drug development. The development of RNAi technology and the expanding field of microRNA discovery greatly facilitate this work. From basic to translational research Laín, Marie Arsenian Henriksson, Ingemar Ernberg, Georg Klein, Elena Kashuba and Lars-Gunnar Larsson. Pontus Aspenström is studying the family of Rho GTPases and their role in cell signaling, as well as in cell and organelle movements. Uno Lindberg is performing basic research on the action of the microfilament system of the cell. Our tumor and cell biology research connects to several of the areas mentioned above and represents both research on fundamental biological questions to translational research. EBV, p53 and Myc in focus Rolf Ohlsson’s group performs world leading Since the days at the Department of Tumor epigenetic investigations on the organization Biology, EBV has been used as a tool to underof the nucleus and higher order chromatin stand oncogenic transformation of cells in structure. The groups of Eugene Zabarovsky the studies of Eva Klein, Georg Klein, Ingeand LiFu Hu are also carrying out epigemar Ernberg, Elena Kashuba, Laszlo Szekely netic characterizaand LiFu Hu. Significant tions of tumor cells. work in the area of angi“Enhancing the collaboraStefan Imreh, Eugene ogenesis in relation to tions between all the cell and Zabarovsky and Georg cancer and metastasis as tumor biology researchers Klein perform detailed well as to other diseases with all our diverse skills studies on identificais carried out by the and expertise will give MTC tion of new tumor group of Yihai Cao. Sven a very strong international suppressor genes on Petterson and Gesan platform for successful cancer chromosome 3. FuncArulampalam are studyresearch also in the future.” tional studies on tumor ing gut microbial signalsuppressor proteins and ing in the regulation of oncoproteins such as p53, Myc and oncogenes inflammation and metabolism and its influencoded by the Epstein-Barr virus (EBV) and ence on cancer, obesity and other diseases. their role in tumorigenesis are performed Strong efforts to identify and characterize by the groups of Galina Selivanova, Sonia small molecular compounds that reactivate or enhance the function of p53 or that inhibit the oncogenic and/or enhance the anti-tumorigenic functions of Myc is carried out by the groups of Galina Selivanova and Sonia Laín (p53) and Marie Arsenian Henriksson and Lars-Gunnar Larsson (Myc) through cellular and/or interaction screens. The pioneering work of Galina Selivanova in this field has brought the p53 reactivating molecule PRIMA to clinical trials. Laszlo Szekely is performing ground breaking combinatorial drug screens based on new advanced imaging technology. KICancer Network Last but not least, MTC scientists are interacting with other cancer researchers at KI through the KICancer Network that organizes several seminars and yearly meetings. Ingemar Ernberg is the enthusiastic driving force for these meetings and in the seminar serie on “What is life?” which has such an important influence on the intellectual environment and interactions at MTC and KI as a whole. Enhancing the collaborations between the cell and tumor biology researchers with all our diverse skills and expertise will give MTC a very strong international platform for successful cancer research also in the future. Lars-Gunnar Larsson Department of Microbiology, Tumor and Cell Biology – MTC 57 Research Reports Cell and Tumor Biology Gut microbial signaling in inflammation and metabolism The human gut harbors a large number of different commensal microbes, now recognized as determinants of growth, nutritional, immune and metabolic Velmurugesan Arulampalam thresholds of the host. Many of [email protected] these physiological responses are interdependent and are linked to intestinal health. The use of germfree animals, housed and maintained by our group, has facilitated the study of gut bacterial impact on host biology. The crosstalk between inflammation and metabolism has been apparent for nearly a century. The nuclear receptor Peroxisome Proliferator Activated Receptor g (PPARγ) is one of the most studied nuclear receptors in the context of inflammation and metabolism. This receptor plays a central role in mediating adipogenesis and regulating glucose homeostasis. More recently our laboratory observed that certain commensal bacterial strains can modulate the function of this receptor to promote a robust anti-inflammatory response. The multi-faceted properties of PPARg renders it an ideal candidate to study how gut microbial input can regulate metabolic thresholds. We are now studying the crosstalk between the gut epithelium and motile immune cells in the lamina propria, which provide one natural conduit for bacterial Can the balance between pro-inflammatory and anti-inflammatory signals emanating from the signals from the gut gut flora impact on cells of the lamina propria, to different organs. e.g. macrophages and dendritic cells, which We have postulated may determine the outcome of inflammationlinked metabolic lesions such as diabetes and that functions in other atherosclerosis? Is it possible to tilt this balorgans may be affected ance in favour of a healthier outcome, through by these microbial sig- manipulation of the gut microflora? nals and metabolites. The mechanisms behind these signals are enigmatic, and are thus relevant questions to be addressed, in relation to xenobiotic metabolism, inflammation, diabetes and even cognitive functions. Approaches to target the MYC oncoprotein to combat cancer Neuroblastoma (NB), a tumor that arises from neural crest cells in the developing sympathetic nervous system, is the most common and deadly solid childhood tumor outside Marie Arsenian Henriksson the CNS. NB cells show complex [email protected] patterns of genetic defects, including specific chromosomal aberrations and MYCN amplification. This occurs in 40-50% of high-risk cases and is associated with advanced-stage disease, rapid tumor progression and a survival rate of less than 15%. An alternative treatment option for these children is therefore urgently needed. Our research is focused on the MYCN oncogene in order to identify targets for development of novel anti-cancer therapies. To this end we will: 1) analyze the functional significance of MYCN-regulated microRNAs for NB pathogenesis; 2) characterize the role of MYCN in invasion and migration; 3) identify small compounds that selectively kill cells with high with high MYCN expression; 4) explore the normal function of MYCN during development of the nervous system. It was recently shown that MYCN regulates expression of microRNAs in NB cells. We have demonstrated that miR-18a and miR19a from the oncogenic miR-17~92 cluster target and subsequently repress the expression of estrogen receptor α (ESR1). Downregulation of mir-18a as well as ectopic ESR1 expression resulted in growth arrest and neuronal differentiation of NB cells. Importantly, we demonstrated expression of ESR1 in human fetal sympathetic ganglia suggesting a role during neuronal development. We propose that MYCN amplification may disrupt estrogen-signaling sensitivity in primitive sympathetic cells through downregulation of ESR1, thereby preventing normal neuroblast differentiation. Collectively, our studies will give miR-18a expression detected with Cy3 (red) at embryonic insights to the normal function of day E6 in a transverse secMYCN and its contribution to NB tion of the trunk region of the pathogenesis. This knowledge could developing chicken embryo. The image shows intense serve as basis for development of novel miR-18 staining in the nervcancer therapies for children with neu- ous system (neural tube and roblastoma as well as other diseases, dorsal root ganglia) and in the dermomyotome. Cell nuclei since MYC is activated in many different were counterstained with DAPI (blue). malignancies. Selected publications Selected publications l l l 58 Are A, Aronsson L, Wang S, Greicius G, Lee YK, Gustafsson JA, Pettersson S and Arulampalam V. Enterococcus faecalis from newborn babies regulate endogenous PPARγ activity and IL-10 levels in colonic epithelial cells. Proc Natl Acad Sci USA 105, 1943, 2008. Lundin A, Bok CM, Aronsson L, Björkholm B, Gustafsson JA, Pott S, Arulampalam V, Hibberd M, Rafter J and Pettersson S. Gut flora, Toll-like receptors and nuclear receptors: a tripartite communication that tunes innate immunity in large intestine. Cell Microbiol 10, 1093, 2008. Arulampalam V. Gastrointestinal inflammation: lessons from metabolic modulators. J Intern Med 263, 607, 2008. Department of Microbiology, Tumor and Cell Biology – MTC l l l Albihn A, Mo H, Yang Y and Henriksson M. Camptothecininduced apoptosis is enhanced by Myc and involves PKCδ signaling. Int J Cancer 121, 1821, 2007. Hydbring P, Bahram F, Su Y, Tronnersjö S, Högstrand K, von der Lehr N, Lilischkis R, Hein N, Wu S, Vervoorts J, Henriksson M, Grandien A, Lüscher B and Larsson LG. Myc/Ras cooperativity in transformation: Phosphorylation by cdk2 is required for Myc to repress Ras-induced senescence. Proc Natl Acad Sci USA 107, 58, 2010. Lovén J, Zinin N, Wahlström T, Müller I, Brodin P, Fredlund E, Ribacke U, Pivarcsi A, Påhlman S and Henriksson M. MYCNregulated microRNAs repress estrogen receptor α (ESR1) expression and neural differentiation in human neuroblastoma. Proc Natl Acad Sci USA 107, 1553, 2010. Research Reports Cell and Tumor Biology Signaling networks controlled by small GTPases The Rho GTPases are key regulators of cell morphogenesis and cell migration. Current cladistic analysis has shown that the family consists of 20 members, which can be further divided Pontus Aspenström into two classes, the classical and the [email protected] atypical Rho GTPases. The atypical Rho GTPases function in a distinct manner from the classical Rho GTPases since they reside constitutively in the active, GTP-bound, conformation, which has profound signaling consequences. We want to elucidate the signaling pathway downstream of Rho GTPases and Miro GTPases that control cell growth and cell migration during normal physiological conditions as well as during disease. We have employed the yeast two-hybrid system to identify novel binding partners for all members of the Rho GTPases. This way, we identified the non-receptor tyrosine kinase Pyk2 as a binding partner for the atypical Rho member Wrch-1. Our data show that Pyk2 collaborates with Src family kinases in order to impose the Wrch-1-dependent cellular effects. The Miro GTPases, which we identified a couple of years ago, have emerged as essential regulators of mitochondrial morphogenesis and motility along microtubules. The Miro GTPases function as calciumdependent sensors in the control of mitochondrial motility. We have identified novel binding partners to the Miro GTPases and we aim to study their roles in model organisms, such as zebrafish. Several lines of indications suggest that Miro GTPases are involved in neurodegenerative diseases. We intend to investigate if Rho GTPases and Miro GTPases can function as targets for medical treatments for diseases such as cancer and neurological pathologies. Miro-dependent aggregation of mitochondria. Transiently transfected Miro1 is localized to mitochondria in COS7 cells and does not influence the morphology of the mitochondrial network. In contrast, transient transfection of a constitutively active Miro1 mutant (MiroV13) result in mitochondrial aggregation caused by the deregulation of mitochondrial trafficking and fusion/fission. Angiogenesis in cancer and nonmalignant diseases Malignant and non-malignant human disorders share a common mechanism of switching on an angiogenic phenotype in pathological settings. Thus, antiangiogenic drugs can be used for the treatment Yihai Cao of cancer and non-malignant diseases. In [email protected] fact, antiangiogenic drugs have become an important component of the first-line therapy against age-related macular degeneration and a variety of human cancers. With this background, our research activity can be summarized as follows: 1. Cancer and metastasis. We have found that the growth factors FGF-2 and PDGF-B could synergistically induce angiogenesis, tumor growth and metastasis. We have demonstrated that growth factor VEGF could stimulate tumor cell dissemination, invasion, and hematologous and lymphatic metastasis. Impor- Blood vessel in brown adipose tantly, tumor-derived angiogenic tissue. factors could induce a systemic destructive effect on multiple tissues and organs, manifesting cancer cachexia, loss of body mass, and paraneoplastic, cancer-associated disease, syndromes, which significantly jeopardize the quality of life and shorten life time. We are one of the first to propose ”off-tumor” targets as one of the mechanisms of antiangiogenic drugs. 2. Angiogenesis and obesity. We are at the leading position to study the role of angiogenesis in adipogenesis and obesity. Using both genetic and epigenetic animal models, we have identified crucial angiogenic pathways leading to neovascularization in both white and brown adipose tissues (WAT and BAT). A recent example is the coldinduced angiogenesis during transition from WAT into BAT. 3. Cardiovascular diseases. We continue to work on the molecular mechanisms underlying arteriogenesis in ischemic muscles. We are the first to propose combinations of angiogenic and arteriogenic factors for the treatment of ischemic myocardium after heart infarction. 4. Retinopathy. We have developed an adult zebrafish retinopathy model, which allows us to study mechanisms of retinal angiogenesis under physiological and low oxygen concentrations. This model also allows us to identify new drugs for therapy. Selected publications l Selected publications l l l Ruusala A and Aspenström P. The atypical Rho GTPase Wrch1 collaborates with the non-receptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics. Mol Cell Biol 28, 1802, 2008. Ruusala A, Pawson T, Heldin C-H and Aspenström P. Nck is involved in formation of dorsal ruffles, cell migration and cell adhesion downstream of the PDGF b receptor. J Biol Chem 283, 30034, 2008. Reis K, Fransson Å and Aspenström P. The Miro GTPases: at the heart of the mitochondrial transport machinery. FEBS Letters 583, 1391, 2009. l l Nissen LJ, Cao R, Hedlund E-M, Wang Z, Zhao X, Wetterskog D, Funa K, Bråkenhielm E and Cao Y. FGF-2 triggers PDGF-Binduced angiogenesis and their reciprocal interplay synergistically promotes tumor neovascularization and metastasis. J Clin Invest 117, 2766, 2007. Xue Y, Religa P, Cao R, Hansen AJ, Lucchini, Jones B, Wu Y, Zhu Z, Pytowski B, Liang Y, Zhong W, Vezzoni P, Rozell B and Cao Y. Anti-VEGF agents significantly confer survival advantages of tumor-bearing mice by improving a cancer-associated systemic syndrome. Proc Natl Acad Sci USA 105, 18513, 2008. Xue Y. Petrovic N, Cao R, Larsson O, Chen S, Feldman HM, Liang Z, Zhu Z, Nedergaard J, Cannon B and Cao Y. Hypoxia-independent cold-induced angiogenesis in brown and white adipose tissues: mutually opposing roles of VEGF receptor-1 and -2 in modulating adipose metabolism. Cell Metabolism 9, 99, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 59 Research Reports Cell and Tumor Biology From viral molecular switches to human cancer The interest of my group is viral infections associated with cancer risk in man. Our prime system of study is the Epstein-Barr virus (EBV) infection. EBV infection is the most common Ingemar Ernberg virus infection in humans world-wide. [email protected] Under conditions of immune dysregulation, which can be due to infections with other pathogens, due to immunosuppression or genetic predisposition the EBV virus gets involved in carcinogenesis. About 200,000 new cases/year worldwide are estimated to primarily originate from an EBV virus infection. The EBV virus can cause different types of cancers with hematopeiotic or epithelial cell origin. We study primarily two molecular switches excerted by viral proteins that contribute to cancer risk. One is the switch between cell proliferation and rest in virus infected cells. The other switch is the switch between migration of epithelial cells induced by the viral protein LMP2a in interaction with the cellular tyrosine kinase Syk and their non-migration. We apply our studies at the molecular level on EBV-associated cancers: Hodgkins lymphoma, post-bone-marrow transplant patients and HIV-carriers with high lymphoma risk and nasopharyngeal carcinoma. We also use the EBV-infection of cells for a systems biology approach to cancer. The hypothesis is formulated as the “Cancer Cell Attractor” (se Huang et al., 2009). Functional studies on human chromosome 3 to understand tumor progression Virtually all tumors contain numerical and/or structural chromosome aberrations. During the microevolution of a malignant cell clone the selection for Stefan Imreh certain aberrant chromosome [email protected] constitution leads to the loss of certain chromosome regions that may contain tumor growth inhibitory genes or tumor suppressor genes or to the gain or amplification of other regions that may contain tumor growth promoting genes or oncogenes. We focus on the functional relevance of chromosome 3 aberrations in various malignancies. For this purpose we had previously developed a model system called “elimination test”, based on chromosome 3 transfer into tumor cells by microcell fusion. The microcell hybrids were tested for the elimination versus retention of specific chromosome regions after passages in severe combined immunedeficiency (SCID) mice. We identified a common eliminated region on chromosome 3 designated CER1 that was similar in both human chromosome 3/mouse fibrosarcoma and chromosome 3/human RCC microcell hybrids. It contains 33 genes whereby 6 of them were discovered and cloned by our group. Several of these genes may act as tumor suppressors. The group focuses on the functional analysis of 3 out of potentially 7 tumor suppressor genes in CER1: LF, LIMD1 and TMEM7. We are characterizing the CER1 and other chromosome 3 breakpoints. These breakpoint regions are “evolutionarily plastic” i.e. they are involved in the evolutionary rearrangements of the chromosomes, show intense transposon recruitment and contain segmental and/ or gene duplications. The puzzle of “evolutionary sorting” of tumor suppressor versus tumor breakpoint regions should be solved as a next step. Using EBV-infected cell lines to evaluate the Cancer Attractor Hypothesis. Selected publications l l l 60 Zhang X, Sanmun D, Hu L, Fadeel B and Ernberg I. Epstein-Barr virus-encoded LMP1 promotes cisplatin-induced caspase activation through JNK and NF-κB signaling pathways. Biochem Biophys Res Commun 360, 263, 2007. Birgersdotter A, Baumforth KR, Porwit A, Sjöberg J, Wei W, Björkholm M, Murray PG and Ernberg I. Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin’s lymphoma. Br J Cancer 101, 1393, 2009. Huang S, Ernberg I and Kauffman S. Cancer attractors: a systems view of tumors from a gene network dynamics and developmental perspective. Semin Cell Dev Biol 20, 869, 2009. Department of Microbiology, Tumor and Cell Biology – MTC Giemsa stained chromosomes. Selected publications l l l Kost-Alimova M and Imreh S. Modeling non-random deletions in cancer. Semin Cancer Biol 17, 19, 2007. Kost-Alimova M, Darai-Ramqvist E, Yau WL, Sandlund A, Fedorova L, Yang Y, Kholodnyuk I, Cheng Y, Li Lung M, Stanbridge E, Klein G and Imreh S. Mandatory chromosomal segment balance in aneuploid tumor cells. BMC Cancer 7, 21, 2007. Darai-Ramqvist E, Sandlund A, Müller S, Klein G, Imreh S and Kost-Alimova M. Segmental duplications and evolutionary plasticity at tumor chromosome break-prone regions. Genome Res 18, 370, 2008. Research Reports Cell and Tumor Biology Epigenetic changes in initiation and development of cancers Epigenetics refers to the study of heritable changes in gene expression without a change in DNA sequence. Epigenetic events are critical for normal development and growth of LiFu Hu cells. In cancer, silencing of tumor [email protected] suppressor genes (TSG) or activation of oncogene are main mechanisms for carcinogenesis. The change in gene expression is often caused by aberrant DNA methylation of CpG islands and histone hypoacetylation in early stages of cancer development, affecting various fundamental pathways, such as apoptosis, invasion and metastasis. We found that promoter hypermethylation of the cellular genes RASFF2 and CDH13 which work as TSGs is linked to clinical outcome of nasopharyngeal carcinoma (NPC) as a tumor model. On the other hand, hypomethylation activates the oncogene BAGE. Cancer related Epstein-Barr virus also uses epigenetic mechanisms to maintain virus latency and regulates the expression of both viral and cellular genes by the EBV encoded LMP1 oncogene. Aiming at an early diagnosis of cancer, we have identified novel TSGs by methylation and expression microarrays, and developed a sensitive “Multiplex Methylation Specific PCR (MMSP)” for NPC on mouth washing/swab samples. MMSP was patented in Australia 2008 and is in clinical trail. Based on the same strategy we designed tests to detect prostate/bladder cancer from urine and lung cancers from sputum samples. Micro RNAs (miRNAs) are short non-coding RNAs, which regulate gene expression in a sequence-specific manner via translation inhibition or messenger RNA degradation, which contributes cancer development and progression. Function of miRNAs in tumor progression, regulation by epigenetic alteration, and potential application for diagnosis of NPC are being explored. The EBV encoded, 170 bp long noncoding RNA EBERs, which are expressed abundantly and constitutively in the Epigenetic changes and disease. nucleus of all latently infected cells, are also studied. EBNA-binding cellular proteins and their role in cell transformation The project is aimed on elucidation of the role of cellular proteins that bind to Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) in cell transformation. Elena Kashuba We are using the EBV-induced B cell [email protected] immortalization as a model system to study the cell transformation. It is clear now that EBV exploits the normal signaling pathways of the B lymphocyte. The six growth transformation associated EBNAs promote cell proliferation and protect from apoptosis. Studying the influence of some EBNAs on the pRb – E2F1 pathway, we have shown that EBNA-6 interacts with a pRb-associated protein, MRPS18-2 (S18-2), leading to the liberation of E2F1. This stimulates the entry of the cell into the S-phase. Unexpectedly, overexpression of S18-2 protein in the primary rat embryonic fibroblasts (REFs) led to REF immortalization. The immortalized cells (18IM) lose contact inhibition, form foci, and are capable of anchorage-independent growth. Concurrently, mesodermal markers, such as vimentin, smooth muscle actin, and Fut4, disappear completely. 18IM cells express embryonic stem cell markers, such as SSEA-1, Sox2, and Oct3/4. In immunodeficient mice, 18IM cells formed small transiently growing tumors that have down-regulated SSEA-1 and showed pan-keratin staining. Over the last few years, we have discovered 15 previously unknown interactions between three transforming virus proteins, EBNA-3, EBNA-5 and EBNA-6 and cellular host proteins. We have used the yeast two-hybrid system, GST-pull down assay, immunoprecipitation with the following masspectrometry, and surface plasmon resonance (SPR) to find EBNA binding cellular partners and to monitor protein-protein binding. To clarify the mechanism of S18-2 induced immortalization we plan to use knock-out cells (p53, pRb etc) for transformation assay and the Danio rerio model for S18-2 silencing and overexpression. Also we are studying Immunostainings on primary rat embryonic the properties of other fibroblasts (REFs), c-Myc and Ras transproteins of S18 family, formed cells (MR) and S18-2 transformed cells (18IM) for mesenchymal (SMA, S18-1 and S18-3. vimentin) and stem (SSEA1) cell markers. Selected publications l Selected publications l l l Sun D, Zhang Z, Van Do N, Ernberg I, Huang G and Hu L. Methylation of CDH13 promoter in NPC could serve as a potential diagnositic biomarker. Oral Oncol 43, 82, 2007. Hu LF, Qiu QH, Fu SM, Magnusson K, Sun D, He B, Lindblom A and Ernberg I. A region on chromosome 5q carries a susceptibility locus predisposing for NPC. Eur J Hum Genet 16, 343, 2008. Nguyen-Van D, Ernberg I, Phan-Thi Phi P, Tran-Thi C and Hu L. Epstein-Barr virus genetic variation in Vietnamese patients with nasopharyngeal carcinoma: full-length analysis of LMP1. Virus Genes 37, 273, 2008. l l Kashuba E, Yurchenko M, Yenamandra SP, Snopok B, Isaguliants M, Szekely L and Klein G. EBV-encoded EBNA-6 binds and targets MRS18-2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes. Proc Natl Acad Sci USA 105, 5489, 2008. Savchenko A, Yurchenko M, Snopok B and Kashuba E. Study of the spatial architecture of p53, MDM2, and p14ARF-containing protein complexes. Mol Biotechnol 41, 270, 2009. Kashuba E, Yenamandra SP, Darekar SD, Yurchenko M, Kashuba V, Klein G and Szekely L. MRPS18-2 protein immortalizes primary rat embryonic fibroblasts and endows them with stem cell like properties. Proc Natl Acad Sci USA 106, 19866, 2009. Department of Microbiology, Tumor and Cell Biology – MTC 61 Research Reports Cell and Tumor Biology Role of EpsteinBarr virus in lymphomas Epstein-Barr virus (EBV) was discovered in Burkitt lymphoma (BL) 50 years ago. The virus carried by almost all humans is the causative agent of infectious mononucleosis, IM. Nine virally encoded proteins are expressed in B Eva Klein lymphocytes and transform, in concert with [email protected] cellular proteins, these cells into immortal cell lines in vitro. In vivo they are recognized and eliminated by the immune response. Under conditions of immunosuppression they may grow progressively. Several tumors that originate in other cell types were also found to carry the viral genome, but they do not express the full set of proliferation driving EBV proteins. The viral proteins that are expressed can induce phenotypic changes in the cell and influence its interactions with the microenvironment. We have discovered that soluble factors produced in the environment can promote the development of malignancies such as Hodgkins and NK lymphomas by modifying the expression of virally encoded proteins and/or by promoting cell growth. The response of humans to primary EBV infection is variable. It can pass without any disease or it can induce IM. Our in vitro experiments suggest that the symptomatology of IM reflects the activation of innate immunity. Leukotriene B4, known mainly for its role in allergy, contributes to the activation of innate immunity. We have also studied the hereditary EBV specific immunodeficiency X-linked lymphoproliferation (XLP). Boys affected with XLP carry a mutated SAP with the lack of the proapopototic function. The lack of SAP function contributes to the understanding of the clinical picture as activation of induced cell death is important for lymphocyte homeostasis. BL is driven by accidental chromosomal immunoglobulin/c-myc translocation and exists in both EBV carrying and EBV negative forms. Constitutive activation of myc that drives the cell to proliferate and induces apopotosis. As SAP is only expressed in EBV carrying but not in EBV negative BL SAP promoted apoptosis may be counteracted by the anti-apoptotic effect of EBNA-1 in the EBV genome carrying cells which would thus determine the fate of the Hypothetical scheme of the contribution of SAP Ig/myc transloca- and EBV to the fate of germinal center B cells with tion carrying B cells. Ig/c-myc translocation. Selected publications l l l 62 Liu A, Claesson H-E, Mahshid Y, Klein G and Klein E. Leukotriene B4 activates T cells which inhibit B cell proliferation in EBV infected cord blood derived mononuclear cell cultures. Blood 111, 2693, 2008. Nagy N, Matskova L, Kis LL, Hellman U, Klein G and Klein E. The proapoptotic function of SAP provides a clue to the clinical picture of X-linked lymphoproliferative disease. Proc Natl Acad Sci USA 106, 11966, 2009. Kis LL, Persson EK, Salamon D, Nagy N, Scheeren FA, Spits H, Klein G and Klein E. IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter. Proc Natl Acad Sci USA 107, 872, 2010. Department of Microbiology, Tumor and Cell Biology – MTC Epstein-Barr virus, oncogenes and tumor suppressor genes and immune and non-immune surveillance against cancer Georg Klein [email protected] The group works on the following questions: i) To analyze the transforming effect of EpsteinBarr virus (EBV) for human lymphocytes; ii) To study the tumor suppressor regions of human chromosome 3p; iii) To identify contact mediated microenvironmental control against tumor development. In the EBV field, we have identified, with Elena Kashuba, 10 new interactions between EBNA-3, EBNA-5, and EBNA-6 and cellular proteins. In addition, we have discovered that the cellular protein MRPS18-2 can transform normal embryonic fibroblasts into immortalized stem cell-like cells. We have shown, with Barbro EhlinHenriksson, that EBV influences chemokine receptors or chemokines and that these modulate the migration of lymphoblasts. In the search for tumor suppressor genes, we have identified, with Stefan Imreh and Eugene Zabarovsky, ”asymmetric” tumor suppressor genes (our term) that can inhibit tumor growth in vivo, without impairing the growth of the same cells in vitro. These genes are of great interest, since they may provide a lead for further investigations on the microenvironmental control of tumor growth. In a third area, I have defined four different forms of non-immune surveillance: genetic (DNA repair), epigenetic (stringency of chromatin structure), intracellular (apoptosis, elicited by illegitimately activated oncogenes) and intercellular (microenvironmental control). With Laszlo Szekely we have developed a high throughput in vitro system that permits the detection of normal fibroblast mediated inhibition of tumor cell growth. Plans: In the EBV project, we are looking for additional interactions of EBNAs with cellular proteins. In the tumor suppressor field, we wish to identify further ”asymmetric suppressors”. In the surveillance field, we wish to analyze the differences between the inhibitory action of normal cells taken from the different body sites, from people of different ages and different cancer risks, against prostatic carcinoma cell lines. GFP-S18-2 (green signal) shows predominantly cytoplasmic distribution. Upon EBNA-6 expression (blue signal, lower panel) GFP-S18-2 is translocated to the nucleus where it co-localizes with EBNA-6. Selected publications l l l Kashuba E, Yurchenko M, Yenamandra SP, Snopok B, Isaguliants M, Szekely L and Klein G. EBV-encoded EBNA-6 binds and targets MRS18-2 to the nucleus, resulting in the disruption of pRb-E2F1 complexes. Proc Natl Acad Sci USA 105, 5489, 2008. Klein G. Toward a genetics of cancer resistance. Proc Natl Acad Sci USA 106, 859, 2009. Ehlin-Henriksson B, Liang W, Cagigi A, Mowafi F, Klein G and Nilsson A. Changes in chemokines and chemokine receptor expression on tonsillar B cells upon Epstein-Barr virus infection. Immunology 127, 549, 2009. Research Reports Cell and Tumor Biology Discovery, characterization and optimization of bioactive small molecules through a p53 activation assay Our group is interested in finding new small molecule therapeutics as well as chemical tools for basic research using Forward Chemical Genetics, i.e. cell-based screens. So far Sonia Laín we have screened over 34,000 [email protected] compounds for their ability to increase the activity of the p53 tumor suppressor in cells using a robust and inexpensive primary assay followed by a carefully chosen series of secondary tests. p53 activation was chosen as a primary assay for two main reasons. First, p53 senses a wide variety of stress types and therefore, its activation can be used to identify compounds that affect a variety of cell signaling pathways involved in cancer as well as in other diseases. Second, there are numerous high quality reagents to study p53, which improves our chances of elucidating the mechanism(s) by which a compound works in cells. Several hits from our screening effort were selected for chemical optimization through or collaboration with Nick Westwood’s lab in Scotland. To date we have shown that two improved derivatives from our initial set of hits decrease tumor growth rate in vivo. One is a potent tubulin depolymerizing agent called JJ78:12. The other, named tenovin-6 (see figure), is the main focus of our current work. In order to elucidate the mechanism of action of tenovin-6 we performed a yeast genetic screen, biochemical assays and target validation studies in mammalian cells. In this way we showed that tenovin-6 inhibits the protein-deacetylase activities of SirT1 and SirT2, two members of the sirtuin family of NAD+ dependent deacetylases linking metabolism and gene expression. We are currently performing studies to further assess the selectivity of tenovin-6 and its derivatives for sirtuins in cells, to identify their binding site in the sirtuins, and to design derivatives that are more potent and/ Structure of tenovin-6. or specific for each of the sirtuin family members. In addition, we have recently started a new screen using an improved version of our p53 activation primary cell-based assay. Selected publications l l l Staples OD, Hollick JJ, Campbell J, Higgins M, McCarthy AR, Appleyard V, Murray KE, Baker L, Thompson A, Ronseaux S, Slawin AM, Lane DP, Westwood NJ and Laín S. Characterization, chemical optimization and anti-tumor activity of a tubulin poison identified by a p53-based phenotypic screen. Cell Cycle 7, 3417, 2008. Laín S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, McCarthy A, Appleyard V, Murray KE, Baker L, Thompson A, Mathers J, Holland SJ, Stark MJ, Pass G, Woods J, Lane DP and Westwood NJ. Discovery, in vivo activity, and mechanism of action of a small-molecule p53 activator. Cancer Cell 13, 454, 2008. Medda F, Russell RJ, Higgins M, McCarthy AR, Campbell J, Slawin AM, Lane DP, Laín S and Westwood NJ. Novel cambinol analogs as sirtuin inhibitors: synthesis, biological evaluation, and rationalization of activity. J Med Chem 52, 2673, 2009. The c-Myc oncoprotein: Function, regulation and targeting The myc oncogenes encode powerful transcription factors that control expression of a vast number of genes involved in, among other functions, cell growth, apoptosis, metabolism and Lars-Gunnar Larsson stem cell function. Deregulated MYC [email protected] contributes to the development of a high proportion of human cancers, in particular at the advanced stages of disease. We are studying the function and regulation of the Myc proteins, with the aim of identifying molecules targeting Myc protein activity. Recently, we uncovered regulation of cellular senescence, one of the main barriers of tumor development, as a new function of Myc. We found that induction of a Myc antagonist caused cellular senescence in Myc-transformed cells (Wu et al., 2009). Further, Myc represses senescence induced by other oncogenes such as Ras. Repression of senescence by Myc requires phosphorylation of Myc at Ser-62 by the cell cycle kinase Cdk2 (Hydbring et al., 2010). Cdk2 is also required to represses senescence induced by overexpressed Myc itself (Campaner et al., 2010). Importantly, inhibition of Cdk2 by small Bimolecular fluorescence complemenmolecule inhibitors induced tation (BiFC) assay showing interaction senescence in Myc-driven between c-Myc and the E3 ubiquitin ligase Skp2 in the nucleus of living cells tumor cells (Hydbring et al., 2010; Campaner et al., 2010). Our future research aims to elucidate exactly how Myc and Cdk2 regulates senescence and to evaluate the in vivo potential of Cdk2 inhibitors in Myc-driven mouse tumor models. Another potential way of combating Myc is to explore and utilize mechanisms of Myc protein destruction via the ubiquitin/ proteasome pathway, and we are continuing our work to identify new E3 ubiquitin ligases targeting Myc. Our recent research also involves identification of low molecular weight inhibitors of interactions between Myc and cofactors crucial for Myc function. Using novel methods for visualizing protein interactions in living or fixed cells, we have screened chemical libraries and identified several molecules selectively targeting Myc. The long-term aim of this work is the development of new drugs for cancer treatment. Selected publications l l l Wu S, Hultquist A, Hydbring P, Cetinkaya C, Öberg F and Larsson L-G. TGF-β enforces senescence in Myc-transformed hematopoietic tumor cells through induction of Mad1 and repression of Myc activity. Exp Cell Res 315, 3099, 2009. Campaner S, Doni M, Hydbring P, Verrecchia A, Bianchi L, Sardella D, Schleker T, Perna D, Tronnersjö S, Murga M, Fernandez-Capetillo O, Barbacid M, Larsson L-G and Amati B. Cdk2 suppresses cellular senescence induced by the myc oncogene. Nat Cell Biol 12, 54, 2010. Hydbring P, Bahram F, Su Y, Tronnersjö S, Högstrand K, von der Lehr N, Lilischkis R, Hein N, Wu S, Vervoorts J, Henriksson M, Grandien A, Lüscher B and Larsson L-G. Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation. Proc Natl Acad Sci USA107, 58, 2010. Department of Microbiology, Tumor and Cell Biology – MTC 63 Research Reports Cell and Tumor Biology Studies on the action of the microfilament system: Redox regulation of actin and the tropomyosins We study the structure and function of the actin microfilament (MF)-system. Lately, our focus has been on two aspects of its dynamic regulation in nonmuscle cells: namely processes Uno Lindberg related to changes in the redox [email protected] status of actin, and mechanisms coupled to the function of tropomyosin. We have found that redox control of actin by H2O2 can reversibly regulate polymerization and depolymerization of actin filaments. H2O2 in nanomolar concentrations is sufficient to activate cysteines of cytoplasmic actin and profilin for subsequent glutathionylation, which likely have drastic consequences for their cellular function. We have shown that a fraction of tropomyosin appears as soluble isoform-specific multimers seemingly involved in actin reorganization after cell stimulation. Tropomyosin can access and control the (+)-end of growing actin filaments where actin monomers bound to profilin are added to drive cell edge advancement. It appears that profilin and tropomyosin cooperate in fine-tuning this process which yet remains to be elucidated at the molecular level. Interesting in this context are the isoforms of tropomyosin where the variants of the protein that localize to advancing lamellipodia differ from those found along contractile actomyosin bundles further in from the edge. Of particular concern is the extensive alteration of expression of these isoforms in conjunction to malignancy. This supports the view of a close connection between the control of the MF-system and the altered behaviour of cancer cells, and suggests that alterations in the expression of the tropomyosin isoform pattern is part of this connection. Rat mammary adenocarcinoma cells (MTLn3) expressing variants of tropomyosin 5 (red and green), nuclei displayed in blue. Photo: Li-Sophie Rathje Selected publications l l l Lassing I, Schmitzberger F, Björnstedt M, Holmgren A, Nordlund P, Schutt CE and Lindberg U. Molecular and structural basis for redox regulation of β-actin. J Mol Biol 370, 331, 2007. Lindberg U, Karlsson R, Lassing I, Schutt CE and Höglund AS. The microfilament system and malignancy. Semin Cancer Biol 18, 2, 2008. Grenklo S, Hillberg L, Zhao Rathje LS, Pinaev G, Schutt CE and Lindberg U. Tropomyosin assembly intermediates in the control of microfilament system turnover. Eur J Cell Biol 87, 905, 2008. Three-dimensional epigenetics: The cancer connection The eukaryotic cell nucleus holds keys to many secrets of life. For example, the DNA blueprint is condensed more than 100,000fold from its linear shape to the very limited physical confines of the nuclear structure. Rolf Ohlsson Despite this tight packaging, the higher [email protected] order chromatin structure must allow regulated access to selected parts of the genome to enable specific transcription profiles during developmental windows. There must also be a substantial measure of precision in these processes, as chromatin features must be faithfully copied to re-enact conformations pivotal for the memory of a cell lineage, and to enable progressive restrictions in cell fates during development. The epigenetic reprogramming process governs the fate of the developing mammalian zygote by regulating the early allocation of the three germ layers and many other later pivotal processes during the lifetime of the individual. Upon abnormal environmental cues, such as viral infection, The image shows the close physical proximities between CTCF, a regulator of chromosomal netepigenetic instabil- works and replication timing patterns, with PARP1, ity may follow. This which is associated with DNA repair. We believe condition may both that the activity of PARP1, which is stimulated by CTCF, indirectly influences the ability of CTCF to initiate cancer and regulate replication-timing patterns (see also Farenable the selec- rar et al., Mol Cell Biol 30, 1199, 2010.) tion of meta-stable epigenetic marks sustaining abnormal proliferation at ectopic sites during the malignant process. It is becoming increasingly clear that the epigenome influences the expressivity of the genome in 3 dimensions within the nuclear architecture. Using novel techniques, the group is exploring reprogramming of chromosomal networks in human embryonic stem cells and in relationship with other epigenetic features, such as replication timing, replication origins and epigenetic marks of the primary chromatin fibres. We have thus been able to document that one replication origin, the H19 imprinting control region regulates not only genome wide patterns of interactions in a manner reflecting epigenetic reprogramming, but also the replication process of the sequences it interacts with. The overall aim of these projects is to understand the 3rd dimension of the epigenome and how it relates to both normal development and cancer. Selected publications l l l 64 Department of Microbiology, Tumor and Cell Biology – MTC Göndör A and Ohlsson R. Replication timing and epigenetic reprogramming of gene expression: a two-way relationship? Nat Rev Genet 10, 269, 2009. Göndör A and Ohlsson R. Chromosome crosstalk in three dimensions. Nature 461, 212, 2009. Sandhu KS, Shi C, Sjölinder M, Zhao Z, Göndör A, Liu L, Tiwari VK, Guibert S, Emilsson S, Imreh MP and Ohlsson R. Non-allelic transvection of multiple imprinted loci is organized by the H19 imprinting control region during germline development. Genes Dev 23, 2598, 2009. Research Reports Cell and Tumor Biology Gut microbiota: A key regulator of body function that sustains health Bacteria have a symbiotic relationship with their host and this interplay is involved in the setting of an organism’s homeostasis throughout life. Genetic variations and Sven Pettersson environmental cues that disrupt [email protected] this homeostasis are of paramount importance in influencing health. We have established an experimental platform including various animal models (also germ free mice) and access to clinical material to try to understand the imapct of gut micorbiota in health and disease. Simple questions like: How does bacteria connect and communicate with the host and which signaling pathways are used? Our hypothesis is that nuclear receptors (NR) act as second messengers in the communication between the host and its microbiota. NR, in addition, regulate and fine-tune most of the organs in the body. Identification of biomarkers connected to homeostasis. In collaboration with the Genome Institute of Singapore, we are exploring the impact of the incoming microbiota on host physiology in order to identify host biomarkers that are regulated by the resident flora in various organs in the body. Additional projects aim to identify biomarkers and mechanisms underlying colorectal cancer and inflammatory bowel disease (IBD). The Core Facility for Germ Free Research (CFGR). I am also the director of the KI sponsored core facility for germ-free research. Rodents are raised in an environment without gut microbiota and today we have developed state of the art technology to derive new lines on demand. The facility works in close collaboration with a number of domestic and international academic partners including the biotech industry. A working model of gut microbiota and effects on normal developmental programming. Environmental cues are known to impact the composition of the flora. The resultant altered flora may therefore be a risk factor in genetic predisposed inviduals. Pharmaceutical reactivation of p53 as a strategy to combat cancer p53 reinstatement leads to impressive regression of established tumors in mice, supporting the idea that restoring p53 is a good strategy in cancer treatment. My Galina Selivanova research focuses on the [email protected] ment of small molecules restoring the tumor suppression functions of p53, either by refolding mutant p53 to rescue its activity, or via preventing proteasomal degradation of p53 in tumors with non-mutated p53. One of our molecules, PRIMA-1MET, which can rescue the tumor suppressor function of mutant p53, is currently being tested in first-in-man Phase I clinical trial. We are addressing the fundamental question that need to be solved for the development of novel medicines, i.e. understanding of the mechanism of action of the candidate compounds, including target specificity in vitro and in vivo and possible off-target effects. Further, using small molecules as research tools, we address important questions of p53 biology. Applying systems biology we discovered important mechanisms which control the p53 choice between induction of apoptosis and growth arrest. We show that MDM2-dependant degradation of p53 cofactor hnRNP K and cdk inhibitor p21 switches Ablation of key oncogenic pathways by RITAthe p53 response reactivated p53. We found a potent inhibition of towards cell death crucial oncogenes by p53 upon reactivation by (Enge et al., 2009). small molecule RITA using microarray analysis (left). Inhibition of oncogenes by p53 reduces the Further, we found cell’s ability to buffer pro-apoptotic signals and that upon phar- elicits robust apoptosis (right). macological activation, p53 unleashes a transcriptional repression of anti-apoptotic proteins Mcl-1, Bcl-2, MAP4, and survivin, blocks the Akt pathway, which is central in the transmission of growth regulatory signals originating from cell surface receptors and c-myc oncogene on several levels and downregulates the oncogene product cyclin E and the transcription factor β-catenin (Grinkevich et al., 2009). Our study adds a new dimension to p53 regulation of physiological events, demonstrating that p53 reactivation triggers ablation of crucial oncogenes. The multitude of oncogenes inhibited by p53 and multiple levels on which they are targeted create external robustness of the p53 response (see Figure for more details). Selected publications Selected publications l l l Björkholm B, Bok CM, Lundin A, Rafter J, Hibberd ML and Pettersson S. Intestinal microbiota regulate xenobiotic metabolism in the liver. PloS One 4, e6958, 2009. Genander M, Halford MM, Xu NJ, Eriksson M, Yu Z, Qiu Z, Martling A, Greicius G, Thakar S, Catchpole T, Chumley MJ, Zdunek S, Wang C, Holm T, Goff SP, Pettersson S, Pestell RG, Henkemeyer M and Frisén J. Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression. Cell 139, 679, 2009. Dermot PB et al., 2009. Genome-wide association identifies 28 ulcerative colitis susceptibility loci. Nat Genet 42, 332, 2010. l l l Enge M, Bao W, Hedström E, Jackson SP, Moumen A and Selivanova G. MDM2-dependent downregulation of p21 and hnRNPK provides a switch between apoptosis and growth arrest induced by pharmacologically reactivated p53. Cancer Cell 15, 171, 2009. Grinkevich VV, Nikulenkov F, Shi Y, Enge M, Bao W, Maljukova A, Gluch A, Kel A, Sangfelt O and Selivanova G. Ablation of key oncogenic pathways by RITA-reactivated p53 is required for efficient apoptosis. Cancer Cell 15, 441, 2009. Zhao CY, Szekely L, Bao W and Selivanova G. Rescue of p53 Function by small-molecule RITA in cervical carcinoma by blocking E6-mediated degradation. Cancer Res 70, 3372, 2010. Department of Microbiology, Tumor and Cell Biology – MTC 65 Research Reports Cell and Tumor Biology High throughput live cell imaging in cancer research and therapy The greatest paradox of cancer therapy is that it has to be evidence based and individualized at the same time. In vitro chemosensitivity assays have been suggested to resolve this Laszlo Szekely paradox. However, early assays were [email protected] not able to keep the tumor cells alive for sufficiently long time. We have developed a new cell culture system (OmniSanguine) that is based on human whole blood extract that permits three days survival and a fluorescence multi-parameter functional analysis of primary tumor cells. We have built and programmed automated fluorescence microscopes (Hexascope) that can analyze cells on 384-well cell culture plates. Our assay can test the sensitivity for thirty different drugs on a robot printed drug plate on single cell level. We receive live tumor samples from several European oncological/hematological centers. We have initiated both retrospective and prospective studies to analyze different hematological malignancies (acute myeloid leukemias, acute and chronic lymphoid leukemias, post transplant lymphomas, multiple myelomas, late stage Hodgkin disease) as well as late stage carcinomas that grow in the form of ascites or pleural effusion. Our ambition is to provide drug sensitivity data for individualized assay guided therapies. We also use this technology to identify tumor types that could be targeted by newly developed drugs, as well as to screen chemical libraries on live tumor cells to identify novel anti-cancer agents. In addition we use color labeling of living cells – by chemical (fluorescent dyes) or genetic (transfection with fluorescent protein coding genes) means – to study the interaction of tumor cells with immune effector cells or normal stromal cells in mixed cell culture system in high throughput assays. Automated measurement of viability of primary human tumor cells using our newly developed imaging device – Hexascope. Novel methylation-dependant markers involved in the progression of cancers Recently we have developed and used NotI microarrays (NMA). NMA are the only existing microarrays giving the opportunity to detect simultaneously Eugene Zabarovsky and differentially copy number [email protected] and methylation changes. Thus they allow to check cancer cells for genetic and epigenetic abnormalities. At present, we analyzed over 400 samples representing various cancers: breast, kidney, cervical, colon, ovarian, lung, prostate, nasopharyngeal and esophageal carcinoma and leukemia. In the study 190 genes from human chromosome 3 were analyzed. For all studied cancers, we found genes specifically methylated in malignant cells. Deeper analysis of our data to find good markers for cancer and progression detection using cervical cancer samples revealed different methylation patterns at different tumor stages. Increased methylation during cancer progression was, for example, observed for the gene RBSP3 the expression of which was subsequently shown to be downregulated. With lung cancer we obtained similar data. Analysis of 20 prostate samples detected genes that were not only cancer specifically methylated such as LRRC3B, RBSP3 and GLCE, but the methylation pattern also allowed to discriminate aggressive from non-aggressive forms of prostate cancer. It is interesting to note that two such progression dependant genes, RASSF1A and RBSP3, had an exceptionally high incidence of single-base mutations. Altogether in 144 sequenced RASSF1A clones, 129 mutations Scheme of cancer development. were detected (mutation frequency (MF) = 0.23 per 100 bp). In 85 sequenced RBSP3 clones, 89 mutations were found (MF=0.10). The mutations diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. This was the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread. Selected publications l Selected publications l l l 66 Flaberg E, Sabelstrom P, Strandh C and Szekely L. Extended Field Laser Confocal Microscopy (EFLCM): combining automated Gigapixel image capture with in silico virtual microscopy. BMC Med Imaging 8, 13, 2008. Markasz L, Skribek H, Uhlin M, Otvos R, Flaberg E, Eksborg S, Olah E, Stuber G and Szekely L. Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes. J Immunother 31, 283, 2008. Stuber G, Flaberg E, Petranyi G, Ötvös R, Rokaeus N, Kashuba E, Wiman KG, Klein G and Szekely L. PRIMA-MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein. Mol Cancer 8, 23, 2009. Department of Microbiology, Tumor and Cell Biology – MTC l l Wang F, Grigorieva EV, Li J, Senchenko VN, Pavlova TV, Anedchenko EA, Kudryavtseva AV, Tsimanis A, Angeloni D, Lerman MI, Kashuba VI, Klein G and Zabarovsky ER. HYAL1 and HYAL2 inhibit tumor growth in vivo but not in vitro. PLoS One 3, e3031, 2008. Cheung AK, Lung HL, Ko JM, Cheng Y, Stanbridge EJ, Zabarovsky ER, Nicholls JM, Chua D, Tsao SW, Guan XY and Lung ML. Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Proc Natl Acad Sci USA 106, 14478, 2009. Kashuba VI, Pavlova TV, Grigorieva EV, Kutsenko A, Yenamandra SP, Li J, Wang F, Protopopov AI, Zabarovska VI, Senchenko V, Haraldson K, Eshchenko T, Kobliakova J, Vorontsova O, Kuzmin I, Braga E, Blinov VM, Kisselev LL, Zeng YX, Ernberg I, Lerman MI, Klein G and Zabarovsky ER. High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in cancer. PLoS One 4, e5231, 2009. Representatives of the technical and administrative staff at MTC. From left to right, back row: Sándor Feldötö, Greger Blomqvist, Thomas Böhlmark, John Sennett, Åsa Sjöman, Maret Johannesson, Agneta Sandlund. Front row: Hanna Hadzija, Lada Larsson, Margareta Hagelin, Barbro Ehlin-Henriksson, Anita Wallentin, Berit Ohlsson, Anna-Karin Persson, Milina Jovanovitch, Poonam Seth and Anna Lögdberg. Kai Eng, Gunilla Karlsson-Hedestam and Gerrry McInerney discuss their research. Photo: Lasse Skog PhD student Clemens Spinnler at his annual student seminar. Department of Microbiology, Tumor and Cell Biology – MTC 67 Avian influenza virus by Lennart Nilsson/Scanpix. What Happens Down in the Cellar? Lennart Nilsson at MTC Although the Karolinska Institutet (KI) is undermined by a network of supply corridors there is normally not much activity going on down there in the cellars. However, there are some exceptions. As this environment at MTC provides interference-free conditions it is the ideal working place for the worldfamous photographer Lennart Nilsson and his EOL high-resolution scanning electron microscope. Therefore, luckily for researchers at MTC, Lennart Nilsson has been working in the Department for more than 5 years. Almost naturally, this has lead to extended collaborations with some of the researchers “above”. However, spatial vicinity has never been a prerequisite for successful interactions. Lennart has had a long-standing tradition to collaborate with MTC researchers already before the Department was founded in 1993. He started his long-term collaboration with Hans Wigzell, at that time professor of immu- 68 Department of Microbiology, Tumor and Cell Biology – MTC nology, in the early ’70s. Natural killer (NK) cells of the immune system had just been discovered and characterized by researchers at the Department of Tumor Biology led by Georg and Eva Klein. Lennart was highly interested in the fight for life and death that is going on when killer cells attack and destroy tumor cells. They also looked at blood constituent parts, visualized viruses in the air and discovered fossilized bacteria. Lennart also captured infections of lymphocytes with tumor viruses together with Ingemar Ernberg, professor of tumor biology. Lennart has another long-standing interaction with Britta Wahren, professor in Clinical Virology. Lennart was fascinated by the beautiful vaccinia virus, especially how it was released from the host cells. Some results of the successful collaboration between Lennart and Britta can be viewed in an exhibition at the Swedish Institute for Infectious Disease Lennart Nilsson. Photo: Camilla Svensk Tumor cells attacked by spherical killer cells. By Lennart Nilsson/Scanpix. control (SMI), among them a high-resolution visualization of the bursting deadly virus produced by the dying host cell. Lennart Nilsson was among the first to visualize the three-dimensional structure of the highly pathogenic avian influenza virus H5N1 at high resolution with his electron microscope for the public. This achievement was possible through the close co-operation with Mia Brytting, chief microbiologist at SMI. Their collaboration started already in 2003 with a common interest in the Severe Acute Respiratory Syndrome (SARS) virus shortly after the first pandemic with this virus. To reach the general public How does a world-famous photographer, who already had made a world-wide career as a photojournalist and is a sought after celebrity and court photographer, get interested in the scientific world? Although Lennart Nilsson is grateful and very happy to work at KI, the collaboration with researchers is actually not his major driving force. To reach the general public, to make them see what has never been seen before, is actually what he is aiming for. This desire is perhaps most beautifully reflected in his famous book “A child is born” which was first published in 1965. To achieve his goals, Lennart has always used front-line visualization techniques. Starting with macro photography, he used the scanning electron microscope. Since the ’70s. Although his own microscope can reach a resolution of 700,000 x, Lennart is aiming for improvements. Hitachi has built a microscope with an even higher resolution. One is The genome seen inside the virus particle will give rise to millions of new particles, killing the infected cell. By Lennart Nilsson/Scanpix. located in Norway and Lennart hopes to be able to use this equipment in the future. Although “A picture says more than thousand words”, Lennart is also a highly appreciated lecturer who readily talks about his work and demonstrates the techniques he is using to anyone who is interested; may it be researchers or school classes. Perhaps Lennart reaches the widest audience with his films. His latest TV-production “A journey to the core of life”, which was produced by Mikael Agaton, was shown for the first time in Swedish television in February 2010. It has two parts “The Cell City” and “The Hunt for The Flu Virus”. Inspired by Ingemar Ernbergs book ‘The ruthless cell” (original title: Den hänsynslösa cellen), the film was mainly produced at MTC and SMI during 2008-2009 with the active participation of many scientists, among others Mia Brytting and Laszlo Szekely. Despite being 87 years old, Lennart Nilsson is involved in many new projects. Together with Britta Wahren and Anna-Lena Hammarin, he wants to look at the rabies virus, which has an interesting, unusual morphology. The dangers of smoking And most importantly, Lennart has ideas for another educational project. A new film is planned together with Georg Klein and his co-workers to describe the dangers of smoking leading to lung cancer, the most common cause of cancer-related death. Lennart wants to visualize the transformation of a normal lung cell into a cancer cell when exposed to tobacco smoke for many years. Lennart Nilsson’s fate at KI is directed by vibrations. He and his microscope have moved around the KI several times to find a vibration-free location. Wherever Lennart is, however, he continues his exciting collaboration with MTC researchers! Ute Römling Lennart Nilsson at KI n n n n n n n In the 1970-ties Lennart Nilsson starts to collaborate with researchers at KI. 1976 Honorary Doctorate at KI 1998 The Lennart Nilsson Award, sponsored by the Lennart Nilsson Foundation, honoring extraordinary photography of science was awarded the first time. 2003 The Lennart Nilsson lecture hall was inaugurated 2009 Personal Professorship by the Swedish Government 2010 “A journey to the core of life” is broadcasted on the Swedish Television 2010 The two stamps issued by the Swedish Post Office to celebrate the 200th anniversary of KI depict silicon and selenium, two of the elements discovered by Jöns Jakob Berzelius, one of the co-founders of KI. The material was taken from Berzelius own test tubes and photographed by Lennart Nilsson. Department of Microbiology, Tumor and Cell Biology – MTC 69 Foreign Adjunct Professors Sir David Lane p53 Forever – Focus on this Fascinating Protein I did my BSc in microbiology and my PhD in immunology both at University College London. My first post-doctoral positions were in virology and for most of my career I have worked on the biology of cancer. So MTC seems an ideal place for me to work and collaborate with as it combines these disciplines. Increasingly I feel that breakthroughs in research always come from crossing boundaries. It is amazing to me how easily we get trapped into particular systems or approaches and collaborating with others, who know different techniques and have hands-on experience is vitally important. Immunologists love flow cytometry and complex transplantation experiments. Molecular cell biologists transfect everything and never mix cells of different types together “Western blots and HeLa cells for us!” and microbiologists? Well, they are perfect of course and can do everything as long as it will grow in L Broth! So in MTC I see the great power of mixing disciplines. Growing human tumor cells in Zebra fish, examining the behavior of mice with altered gut flora and looking at the anti-parasite activity of new anti-cancer drugs are all being tried here. If the new system is two buildings away and established by someone you don’t know the casual but vital “let’s just try it” conversation over coffee with a colleague is less likely to happen. Of course, what everyone wants these days is to plan science and calculate outcomes in advance. How boring if we know what we are going to discover in advance! How will we really discover anything new? But of course once the initial observation is made then the work begins; the work to make the results certain, quantitative and reproducible by the community so that they have real value. Then we can write the paper. Its odd talking to some scientists now that the paper has become a painfully produced product. It should never be that way. When we have a great result the paper writes itself. My first contacts with MTC were, of course, through Georg and Eva Klein’s lab where Georg developed a great interest in the interactions of the EBV virus proteins with p53, the most frequently mutated gene in human cancer. At that time (the early 1980’s) it was becoming 70 Department of Microbiology, Tumor and Cell Biology – MTC clear that all of the DNA viruses might inactivate p53 function if successful in infection of transforming mammalian cells as the replication of viral DNA acts as a trigger to induce the p53 response and this potentially blocks infection and transformation. While great progress was made on this problem with discovery of p53 binding and inactivating proteins in the SV40 Adenovirus and Papilloma virus systems the results where much less clear in the case of other viral groups notably EBV, other Herpes viruses and Polyoma virus. Like so many others Georg “adopted” me and I was asked to act as opponent on the thesis exams of some now very notable MTCers. So many years later it is wonderful to be able to join the Department on a more formal basis. It is also very fitting that Georg and his team now seem to have solved the route by which EBV tackles p53. Since the role of p53 mutations in human cancer became apparent in the early ’90s work on this fascinating protein and the mechanisms by which it is controlled have been the main focus of my work though I love to play with other things too. We still have work going on RNA helicases, the proliferating cell nuclear antigen PCNA, eukaryotic translation initiation factor 4E and antibodies though not on SV40T antigen anymore. I love proteins and antibodies and trying to make things, so drug discovery attempts have been a big part of the last decade. I set up a biotech company (Cyclacel) that progressed three molecules to the clinic and I was heavily involved in science policy and development with A*Star, the agency for science, technology and research in Singapore and Cancer Research UK CRUK, a main funder of cancer research in Britain. But now I am back to looking at bands on gels, making and testing antibodies and screening libraries looking out for amazing surprises. So what do I hope for the interaction with MTC? Lots of great discussions over coffee, lots of new ideas and collaborations and lots of fun. I had great mentors when I was a student, especially my PhD supervisor Avrion Mitchison, who was very inspiring and extra ordinarily generous. So I would like to be like him and help all the students when I can. Current positions n n n n n Director p53 lab Singapore 2008– Chief Scientist A*Star Singapore 2008– Chief Scientist, Cancer Research UK 2008– Foreign adjunct professor at the Karolinska Institutet/MTC 2007– Honorary Professor, University of Dundee, 2009– Personal information n n Research interests: Original discovery of p53 and subsequent research on p53 all the way from a basic research to clinical application for three decades, over 340 publications among others in Nature, Cell and Lancet. Prizes (selected): Paul Ehrlich Prize, 1998; Knighthood, 2000; Sergio Lombroso Award in Cancer Research, Weizmann Institute of Science, 2005; Hon. Doctor of Science, University Paul Sabatier, Toulouse; University of Nottingham; University of Birmingham; University of Aberdeen and others. CV n n n n n n n n n n PhD in immunology, University College London, Great Britain 1977–1985 Lecturer, Imperial College of Science & Technology, London 1985–1990 Senior and Principal Staff Scientist, Imperial Cancer Research Fund, Clare Hall Laboratories 1990–1998 Department of Biochemistry, University of Dundee 1990–2009 Personal Chair in Molecular Oncology, University of Dundee. 1998–2009 Department of Surgery and Molecular Oncology, University of Dundee 1998–2009 Director of the Cancer Research UK Cell Transformation Research Group, The Cancer Research UK Laboratories at Dundee 2004–2007 Executive Director of the IMCB in Singapore on a three year leave 2006–2008 Founder of the Experimental Therapeutics Center (ETC) Singapore 2008–2009 Chair of Singapore’s Biomedical Research Council Foreign Adjunct Professors Margret Ann Liu MTC – Not Only a Scientific Experience! My work focuses on global health, particu- larly, on vaccines and immunotherapy for diseases such as AIDS and cancer. Being initially a Visiting Professor and now a Foreign Adjunct Professor at the Karolinska Institutet (KI) in MTC has provided a wonderful involvement in ongoing projects and engaging interactions with the faculty and students. My activities have ranged from teaching in courses, to co-supervising PhD students to being a thesis opponent, to participating in the various European and developing country networks to which MTC and the nextdoor Swedish Institute for Infectious Disease Control (SMI) belong. Applying for research grants is another activity which I had not done since my early days on the faculty at Harvard Medical School, and which has been quite a change from my days as the Senior Advisor in Vaccinology for the Bill & Melinda Gates Foundation overseeing many millions of dollars of funding for vaccine development! Vaccine development My primary affiliation is with the research group of Britta Wahren, currently Professor Emerita, and formerly Professor of Virology at MTC/SMI, developing a vaccine for HIV/ AIDS and immunotherapy for cancer, collaborating also with Prof. Håkan Mellstedt, Chief Physician, Departments of Oncology and Hematology, Karolinska University Hospital. These projects are particularly rewarding for me because through our efforts to improve DNA vaccines (an approach my former research group and I pioneered in the 1990s at Merck Research Labs), our KI group and KI clinicians are trying to benefit humankind by testing an HIV vaccine and a cancer immunotherapy in clinical trials. In fact, my appointment at the KI stemmed from this research as I was first introduced to Professor Wahren’s research and group at meetings, and then in 2001 gave an honorary lecture in the Karolinska Research Lecture series at the invitation of the Nobel Committee. The internationally collaborative research of the KI scientists has complemented and expanded the work that I do for a variety of international organizations focused on improving global health by developing vaccines needed by the world’s poorest people. For example, for the International Vaccine Institute (IVI), established by the UN Development Program, and located in South Korea, I was the long-time Chair of the Scientific Advisory Group, and now serve as Vice-Chairman of the Board of Trustees, coincidently under the Chairmanship of Prof. Ragnar Norrby, who recently retired from his position as Director General of SMI. The IVI recently enabled the production and licensure of a low-cost cholera vaccine to prevent untold suffering and mortality in developing countries. Likewise, when I have organized international meetings, whether in the US, Asia or Africa, KI faculty, students, and post-docs have been speakers and participants. One encounters Karolinska scientists throughout the world, so it is optimal to also work with them in Stockholm! Personal information n n n n Research interests: DNA based vaccine development, immunotherapy; inventor of six patents; organizer of a dozen of international meetings among them Cold Spring Harbor Laboratory meetings and Keystone Symposia. Co-Editor of the 3rd and 4th editions of “New Generation Vaccines”. Prizes: Recipient of an NIH Physician Scientist Award; Named 1 of “The 50 Most Important Women in Science” by Discover magazine, 2002. Memberships (selected): Vice-Chairman of the Board of Trustees, International Vaccine Institute; Director, Keystone Symposia Board; Trustee, San Francisco Conservatory of Music; Advisory Board, Elizabeth Glazer Pediatric AIDS Foundation. A new social life Being part of MTC and the KI has also resulted in friendships, cross-cultural sharing, and a huge improvement in my social life! I have been privileged to attend the incredible Nobel Prize ceremony and dinner as an affiliated faculty member of the KI. Also no parties elsewhere can compare with MTC parties. Who can forget Prof. Ingemar Ernberg playing the White Rabbit in an “Alice in Wonderland” satirical skit, complete with ears and hopping around the stage, demonstrating that a department chairman has many hidden talents. Another outcome of these parties and thesis celebrations is that my extremely limited Swedish vocabulary was expanded by singing drinking songs. Professor Wahren has welcomed even my family to stay in her home, providing us with an opportunity to discover the wonders of Sweden. What can I say, but “Tack så mycket!” for the incredible experience of being part of MTC and the KI. CV n n n n n n n n Current positions: n n ProTherImmune, Consultant in Vaccines and Immunotherapy for Biotech and Investment Companies, 2000– Foreign Adjunct Professor, KI, Stockholm, Sweden, 2007– n n 1977 B. A. in Chemistry, Colorado College; 1977 Teaching diploma for piano, Ecole Normale de Musique de Paris 1981 M.D., Harvard Medical School, Boston, MA 1981–1988 Internship, Residency in Internal Medicine and Clinical and Research fellow, Massachusetts General Hospital, Boston, MA 1984–1988 Visiting Scientist at the Massachusetts Institute of Technology, Boston, MA 1987–1989 Instructor in Medicine at Harvard Medical School, Boston, MA 1988–1997 Senior research fellow, later Director and Senior Director, Merck Research Laboratories, West Point, PA 1989–1995, Adjunct Assistant Professor of Medicine, University of Pennsylvania, Philadelphia, PA 1997–2000 Vice President, Vaccines Research, Chiron Corporation, Emeryville, CA 2000–2006 Vice Chairman, Transgène, Strasbourg, France 2000–2003 Senior Advisor in Vaccinology and Consultant, Bill & Melinda Gates Foundation Department of Microbiology, Tumor and Cell Biology – MTC 71 Foreign Adjunct Professors Alexander von Gabain Innovating Vaccines by Translational Entrepreneurship Vaccination is inarguably the most success- ful medical intervention which has become a mandatory part of many countries’ health care programs during the last century and has shown to be an effective instrument in the control of infectious diseases worldwide. Development and launch of novel vaccines has been triggered by the appearance of novel pathogens, by the apparent stagnation to control the rebound of global infectious diseases, by the pending threat of a pandemic flu and by the spread of multi-drug resistant pathogens in hospitals and the community. The encouraging scientific progress made in the areas of immunology, molecular biology, genomics, and host-parasite interaction, but also in the areas of novel manufacturing technologies, has facilitated the development of novel vaccines and enabled the field to expand the spectrum of the existing vaccine targets. Innovation in vaccine development is mostly driven by small and medium size biotech enterprises. The classical path giving rise to novel biotech firms has been the spinoff from an academic environment; e. g. university scientists stepping out of their institutes, converting into entrepreneurs and homing their scientific knowledge and research results into a business shell, aiming to translate the founding concept and vision into viable and badly needed products. This approach has led in single instances to the foundation of impressive companies, some of which have outgrown established pharmaceutical companies. The Company’s growth is nurtured by three pivotal platforms forming the fundament of Intercell: the Antigen Identification Program® enabling the discovery of the most protective microbial antigens, the vaccine adjuvant programs that increase the efficacy of existing and novel vaccines, and the patch delivery technology that pioneers a needlefree era in the vaccine field. Based on these platforms, the Company was able to build a coherent Research & Development pipeline that today comprises more than eight products in various stages, currently tested in Phase I to Phase III clinical trials. The Company’s most advanced product, a novel prophylactic vaccine against Japanese Encephalitis, was launched after its global registration in 2009. For more information please visit: www.intercell.com. Since my appointment as foreign adjunct professor, I have been engaged in numerous activities aiming to transmit and share my knowledge, networks and experience in microbiology, vaccinology, but also in biotech entrepreneurship and translational medicine with the MTC students, MTC colleagues and other Karolinska staff. Among other activities, I have supported the setup of collaborations between MTC and Intercell scientists on novel vaccines over the last decade, but also organized a teaching course on infectious disease biology including PhD students from Vienna and Stockholm. Furthermore, I am coaching students and faculty to spin off companies from the Karolinska Institutet (KI) with its impressive foundation of top class research. n von Gabain A. Innovating vaccines: from gene expression to translational entrepreneurship. Hum Vaccin 6, 1, 2010. Current positions n n n Chair of the Scientific Advisory Board and Strategic Advisor to the Board Intercell AG, 2009– Professor in Microbiology at the Max Perutz Laboratories, University of Vienna, Austria Foreign Adjunct Professor at the KI/MTC, 2007– Personal information n n n Research interests: Microbial gene expression, host-parasite interactions and immunology. Over 100 articles in peer-reviewed journals including Nature and Cell. Regularly speaks at and organizes conferences in the field of vaccinology. Prizes: Scrip Award with Intercell AB as Biotech Company of the Year 2008 Memberships (selected): Member of the Royal Swedish Academy of Engineering Sciences, the founding governing Board of the European Institute of Technology (EIT); board member of biotech enterprises including TVM Capital in Munich, Germany Foundation of Intercell Inspired by this paradigm and by the ambition to translate research into novel desperately needed vaccines, I stepped down from my academic Chair at the Campus Vienna Biocenter (VBC, for more information please visit: http://www.viennabiocenter.org) and, together with four colleagues, founded Intercell in 1998. As founder and in various board positions, I have been contributing to leading the Company into the top league of the 30 largest biotech firms worldwide, with today 450 employees in Austria, the UK and the USA, and technology provider to the five largest commercial vaccine players. 72 Department of Microbiology, Tumor and Cell Biology – MTC Selected publications n n n Giefing C et al., Discovery of a novel class of highly conserved scale antigenic fingerprinting of pneumococcus with human antibodies. J Exp Med 205, 117, 2008. Kamath AT et al., Protective anti-mycobacterial T cell responses through exquisite in vivo activation of vaccine-targeted dendritic cells. Eur J Immunol 38, 1247, 2008. Schlick P et al., Helices a2 and a3 of West Nile virus capsid protein are dispensable for assembly of infectious virions. J Virol 83, 5581, 2009. CV n n n n n n PhD in Genetics, University of Heidelberg 1979–1982 post-doc in the laboratory of Stanley N Cohen, University of Stanford 1983–1992 Associate Professor, University of Umeå and KI 1993–1998 Head of Department of the Microbiology and Genetics, University of Vienna, Campus Vienna Biocenter 1998–2005 Co-founder and CEO of the biotech company Intercell AG 2005–2009 CSO and chair of SAB, Intercell AG Foreign Adjunct Professors Yi-Xin Zeng Our valuable Collaboration with the Karolinska Institutet the immunotherapy of nasopharyngeal carcinoma, and together we have published several East Indian Company tradeship “Götheborg” important papers in the field. that had returned to China 265 years after its Another collaborative effort with great latest journey, I saluted Her Royal Highness impact is the China-Sweden Medical SympoQueen Silvia in Guangzhou. Meanwhile, the sium. In 2003, the 1st China-Sweden Medical 3rd China-Sweden Medical Symposium was Symposium was held in SYSU cancer center. held in the Sun Yat-Sen University Cancer In 2004, the 2nd China-Sweden Conference Center. At the opening ceremony, I had the was held in Stockholm. Accidentally, the honor to receive the official certificate for my Nobel Prizes were awarded during the time the appointment as a foreign adjunct professor at conference took place. As MTC, Karolinska Institutet a Chinese representative, (KI), from Professor Jan I was invited to particiCarlstedt-Duke, the Dean “I really cherish the great pate in the ceremony for of Research at that time. It honor as a foreign adjunct the Nobel Prizes, which was an exciting moment, professor of KI and will left a life-long memory and many distinguished work as hard as always to to me. In 2006, the 3rd guests witnessed and shared achieve more in scientific China-Sweden Medical my happiness, including research and medical Symposium was again Her Royal Highness Queen education and management held in the SYSU cancer Silvia, the Minister of as well.” center. During these Science and Technology of conferences, experts China, Professor Guanhua from both China and Sweden reported their Xu, and the Vice President of Chinese Acadnovel achievements and intriguing discusemy of Sciences, Professor Zhu Chen who is sions about different aspects of biomedicine now the Minister of Health in China. were initiated, which eventually promoted collaborations among scientists between the Many collaborative efforts two countries. Since 2003, when the 1st China-Sweden I really cherish the great honor as a foreign Medical Symposium was held in Guangzhou, adjunct professor of KI and will work as hard Sun Yat-Sen University (SYSU) has cooperas always to achieve more in scientific research ated with KI in a range of aspects in biomediand medical education and management as cal research. On March 27, 2004, the SYSU-KI well. This year will be the 200 anniversary of collaborative laboratory was established. The KI and I hope I can join the celebration and collaboration with KI has three goals: trainexpress my best wishes for KI. ing of PhD students or post-doctoral research who will most likely return to China; the application of joint grants and the formation Current position n Director of the State Key Laboratory of of joint projects of mutual interest with Swedish partners. As a foreign adjunct professor at Oncology in Southern China, 2004–. n Professor and President of the Sun YatKI, I have obtained a large number of benefits that arise from it, such as training PhD sen University Cancer Center, the largstudents. For example, Roujun Peng, one of est specialized Cancer center in Southern my PhD students, is doing her PhD research China, where cancer treatment, cancer on the subject of immunological genetics at prevention, research and training are inteKI right now. grated, 1997–. n Foreign Adjunct Professor, KI, Sweden, Another benefit is the cooperation in the area of cancer immunotherapy, which has 2007–. produced a great deal of achievements in the past three years. Jiang Li, a research scientist Personal information n Research interest: Cancer immunotherain my group, collaborated with Prof. Maria Masucci, professor in virology, focusing on py, Cancer genetics, nasopharyngeal carIn the summer of 2006, with the arrival of the n n cinoma and Epstein-Barr virus associated malignancies. Prizes: Award for Outstanding Young Teachers in Chinese Universities, Ministry of Education, 2002; First class award for Scientific Advancement, China Medical Association, 2003; Second class award for Natural Science Achievements, National Office for Science and Technology Awards, 2005; Award for National Young Scientists, 2006; He-Liang-He-Li Award for Science and Technology Advancement, 2007. Memberships (selected): Member of the Chinese Academy of Sciences; Member of the Academy of Sciences of Developing Countries, Member of the SFDA (Sino-Food & Drug Administration) Drug Evaluation Committee. Editor-inchief, Chinese Journal of Cancer; Editorial Board member of Cell Cycle (USA), Cancer Biology and Therapy (USA), Journal of Biological Chemistry (USA) and Journal of Translational Medicine (USA). CV n n n n n 1990 PhD at the Sun Yat-sen University of Medical Science, China 1990–1992 Research Assistant, GuangDong Provincial Peoples Hospital, China 1992–1994 Postdoctoral Fellow at the Institute of Medical Science, Tokyo University and Tokyo Metropolitan Institute of Gerontology, Japan 1994–1997 Research Associate, Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, USA 1997 Professor and Vice President, SunYatsen University Cancer Center, China Department of Microbiology, Tumor and Cell Biology – MTC 73 MTC International Evaluation of HIV Vaccines in Tanzania Development of promising vaccination strategies at MTC and SMI Karolinska Institutet (KI) and the Swedish Institute for Infectious Disease Control (SMI) have for many years had a Sida-supported research collaboration with the Muhimbili University of Health and Allied Sciences (MUHAS) in Dar es Salaam, Tanzania called the TANSWED program. This program comprises studies of various aspects of HIV infection in Tanzania. An important part of this program is the building of research capacity which includes PhD research training at KI of medical doctors from MUHAS. One of the main projects in the TANSWED program concerns “Evaluation of HIV vaccine candidates”. After more than 10 years of preparatory work a HIV vaccine trial (HIVIS03) supported by Sida and EU started in Dar es Salaam in 2007 using a combination of two HIV vaccines. The first vaccine is an HIV-1 subtypes A, B and C. In order to augment and broaden the vaccine-induced immune responses a prime-boost vaccine strategy has been implemented. This means that boosting immunizations were given with a modified vaccinia virus Ankara (MVA) vectorbased HIV vaccine representing subtypes A and E. The HIV MVA vaccine was obtained through collaboration with the National Institutes of Health HIV DNA vaccination of the first volunteer, a police officer, in the and Walter Reed Army InstiHIVIS03 vaccine trial in Dar es Salaam, using a needle-free injection system. tute of Research in the US. This HIV vaccine combination was first tested in a clinical trial of 37 DNA vaccine developed at KI/SMI which healthy non-infected vaccine recipients in consists of genes representing the major HIV Stockholm (HIVIS 01/02) coordinated by Eric structural proteins and three major HIV Participants in the annual HIVIS/TaMoVac HIV vaccine meeting in Bagamoyo in Tanzania including collaborators from Tanzania, Mozambique, Sweden, Germany, England and the US. 74 Department of Microbiology, Tumor and Cell Biology – MTC MTC International Sandström. The vaccine combination was shown to be safe and highly immunogenic. All vaccine recipients except one showed an HIVspecific cellular immune response. The positive HIV vaccine results in Stockholm have been confirmed in the HIVIS03 vaccine trial in Dar es Salaam which included 60 volunteers from a cohort of police officers. After 3 HIV DNA vaccinations followed by one HIV MVA vaccination all of the 35 vaccine recipients displayed HIV-specific cellular immune responses. It was found that a lower dose of HIV DNA vaccine given intradermally induced stronger immune responses after the HIV MVA boosting vaccination than did a higher HIV DNA dose injected intramuscularly. One reason for the excellent immunogenicity is that the delivery of the vaccine genes occurs via a jet stream using a needle-free injection system. The use of this technology involved collaboration with the Bioject company in the US. Placebo recipients showed no positive responses. Participants on the ”Genes and Genomes” in the Tropics course. Scientific Alliances in Africa are involved in numerous research and teaching efforts throughout Africa; courses are given and scientific alliances have been formed. We are fully connected and collaborate both for the sake of Swedish and African science. Makerere University in Uganda, (MUK) in particular, has an outstanding relationship with KI for many years. The signing of a contract of collaboration in 2002 by the heads of the two institutions formally established the importance of the inter-university links. We have as a consequence a joint degree for PhD students, unique in the world, with 43 graduates so far. Critical findings have been reported in outstanding scientific journals and our collaborative activities have changed both KI and MUK. At Makerere University a collaborative three-weeks PhD course named “Genes and Genomes in the Tropics” (www. ggtropics.com) is executed on an annual basis. It was created in the year 2000 in an attempt to increase the knowledge of front-line molecular- and cell biology of microbes but also to stimulate collaboration and networking between the two universities. 175 students of 15 nationalities, predominantly from KI and MUK have taken the course, but students and teachers have also come from Cameroon, Denmark, Ethiopia, France, Germany, India, Italy, Kenya, Mali, Nigeria, Norway, Portugal, Sudan, Tanzania and USA. Scientists at MTC Two new vaccine projects Funding has been obtained from the European and Developing Countries Clinical Trials Partnership (EDCTP) and Sida for two new big vaccine projects, TaMoVac I and TaMoVac II. These projects include building of HIV vaccine trial capacity and conduct of phase II HIV vaccine trials in Tanzania and Mozambique with the use of the HIVIS HIV DNA and MVA vaccines. These trials will evaluate ways of facilitating the injection of the HIV DNA vaccine and of enhancing the induction of cellular immune responses as well as antibody responses to HIV. A new concept will be introduced with the delivery of the HIV DNA genes by dermal electroporation. In this way we hope to further increase the vaccine gene influx into dermal cells and the expression of viral proteins. Preclinical studies have shown that this will considerably increase immunogenicity. The principal investigators of the TaMoVac I and II vaccine projects are two Tanzanian doctors who took their PhD degrees at KI, Muhammad Bakari, a clinician and Eligius Lyamuya, a clinical immunologist. Five Tanzanian and two Mozambican PhD students are presently engaged in these projects. Three of them are already registered at KI for PhD studies. The collaboration includes investigators from Tanzania, Mozambique, Sweden, Germany, England and the US. Gunnel Biberfeld Our research is focused on diseases such as malaria, Burkitt’s lymphoma, AIDS, tuberculosis and urinary tract infections. More than 20 separate studies have been carried out and published in collaboration between KI-MTC and MUK. For example, the ability of the malaria parasite Plasmodium falciparum to undergo antigenic variation has been investigated and a scheme was identified in the variable antigens pertinent to disease states and parasite phenotypes. In this and the other projects, PhD students and scientists from Stockholm and Kampala have worked at KI-MTC and MUK. Fellow students, “mirror students”, from Uganda or from Sweden devoted to the work with the same overall question and the same timeline have been engaged in the projects in order to install reciprocity in the support and in the work. This has allowed for parallel help and development from Uganda to Stockholm and vice versa. The close interaction seeks to avail opportunities for productive research and training of both young Ugandan and Swedish students. This has had both scientific and societal impact fulfilling social and economic needs by creating knowledge and empowerment. We live in a world developing at a rapid pace but research still needs to be a long-term activity. Mats Wahlgren Britta Wahren Department of Microbiology, Tumor and Cell Biology – MTC 75 MTC International Singapore skyline. Photo: bigfoto.com The Singapore Partnership for the Future The country of Singapore, located in the South East of Asia, with 4.5 million citizens is a vibrant modern cosmopolitan country with a strong economy and well developed infrastructure to support Research and Development (R&D). In addition, Singapore is a harmonic introduction and gateway to eastern culture and values. Since the end of 2002, I have been given the privilege to coordinate scientific research and education exchange activities between Karolinska Institutet (KI) and Singapore. In the early 2010, KI has well developed programs and tight bonds with the Universities of Singapore (NUS) and Nanyang Technology University (NTU) as well as the Minister of Trade funded Singapore Agency for Science, Technology and Research (A*STAR). Over the last five years Singapore has established itself as a major player in R&D both in South East Asia as well as internationally. Singapore can not compete in size and numbers with other major players such as India and China. Yet, its considerable investment in Life Science Research and its geographical location in the heart of South East Asia, make Singapore a very attractive partner for KI and Sweden. 76 Department of Microbiology, Tumor and Cell Biology – MTC Singapore and Sweden Singapore is an internationally competitive country eager to foster international collaborations with trustworthy and committed partners. Sweden is a solid partner for Singapore. Both countries are short of human capital – i.e. we are both in need of a constant influx of highly educated immigrants. Joint activities are therefore of mutual benefit. Partnership with Singapore is giving KI and Sweden access to intellectual capital, which already is becoming a strong competitive advantage at the global R&D arena. Furthermore, as English is the main spoken language, no communication barriers exist. Given the existence of good instruments to handle ethnic pluralism, Singapore has a lot to offer to KI and Sweden in terms of adaptation and learning about other ethnic values, information that is useful for our efforts to work and operate on the international arena, be it Europe, US or Asia. Singapore and KI Currently KI is engaged in PhD programs with the three major actors, A*STAR, NUS and NTU respectively. The most productive program so far is the one with A*STAR. For more information see: http://newshub.nus. edu.sg/ke and the KI homepage. Other examples of exchange programs are the possibility for PhD students from KI to visit A*STAR research institutes at Biopolis for up to one year. These programs are sponsored by the generous support from the universities and A*STAR. An overseas office for KI was opened in Singapore in 2004. This office has since then been used to establish a trust and respect for our mutual efforts to work and operate in Singapore. Large scale collaborative research activities are ongoing in the areas of breast cancer and Chronic Inflammatory Diseases (CID), health care problems of huge magnitude both in Sweden and in Singapore. Scanning the entire human genome for genes predisposing to CIDs is very costly, technologically demanding and requires a well defined clinical material. In recent years, the A*STAR funded Genome Institute of Singapore (GIS) and KI have successfully collaborated and identified biomarkers for multiple CIDs including rheumatoid arthritis, psoriasis and ulcerative colitis. It is expected that additional research exchange project in other MTC International areas will be launched during the next couple of years. Promoting Sweden - The Sweden Day To promote and foster interactions at the undergraduate level, we launched the “Sweden Day” in 2006. This event is held twice a year and is organized by undergraduate students from KI and other Swedish university students visiting Singapore. Their mission is to act like “mini diplomats” and promote higher education possibilities in Sweden in general and KI in particular. The students work closely with staff from the Swedish Embassy. For further information, see: http://swedenday.bloggplatsen.se/ where you can read more about our activities and the latest Sweden Day. Singapore 2010 and beyond Karolinska Institutet needs strong partners to be internationally competitive if we are to remain a major player in R&D in the field of Life Science. R&D today does not respect national borders, requires solid infrastructures and a long term commitment by the politicians to be competitive. Asia has huge unmet needs for better health care and the investment in medical research will therefore continue to increase in decades to come. This makes Asia very attractive to KI and Sweden. The build up of strong ties with Singapore is therefore essential as well as those currently under way with distinct regions in China and India. KI has a good reputation in Singapore and the KI brand is well respected for its R&D in translational research. International networks for high-quality graduate education MTC has strong international connections. The effective networking is also reflected in the graduate education profile of the Department. Within the field of Infection Biology, several research groups at MTC participate in two networks for graduate education within Europe: 1. EIMID – European Institute of Microbiology and Infectious Diseases – is a collaborative research initiative between five leading European institutions in the fields of Microbiology and Infectious Diseases; the Max-Planck Institute for Infectious Biology in Berlin, Germany; the Centre for Molecular Microbiology and Infection (CMMI) at the Imperial College in London, UK; the Institute Pasteur in Paris, France; MTC at the Karolinska Institutet (KI) and Novartis Vaccines and Diagnostics in Siena, Italy. EIMID conferences have been held at different geographic locations once a year since the start in 2004. 2. The European Research Training Group “IRTG 1273 Strategies of human pathogens to establish acute and chronic infections” is a collabora- tive network between Hannover Medical School (MHH), the TWINCORE Institute for experimental and clinical infection research in Hannover, the Helmholtz Centre for Infection Research in Braunschweig, and the Karolinska Institutet in Stockholm. The central aim of this program is to give young researchers from Germany and Sweden the opportunity to perform their PhD thesis in the field of infection biology and human microbial pathogens in a highly stimulating, competitive and international environment. The core element of IRTG 1273 is a joint research program centering on the question how human microbial pathogens achieve acute or persistent infection and how the host organism reacts to infection. Twenty-four research groups and twenty-seven graduate students from several institutions at KI have been involved in this program. Common meetings have been organized within the program at least once a year in Germany and Sweden and students have visited the other country to perform common projects in collaborative efforts. Birgitta Henriques Normark Mikael Rhen A long lasting friendship KI has made a firm commitment to its senior representatives in the Life Science program, to develop this partnership into a long lasting friendship. Long term objectives for both parties are to deliver first class research coupled with excellent education programs for students, scientists and for people working in the health care sector. The growing interest from many other Swedish universities to participate in our ongoing R&D activities in Singapore together with the well developed Swedish private industry operating in Singapore, open for more exciting investments in Singapore in the years to come. The upcoming bilateral national R&D exchange program between Sweden and Singapore, to be signed off in the near future, make this journey very exciting. Sven Pettersson Department of Microbiology, Tumor and Cell Biology – MTC 77 Scientific Networks Scientific Networks KICancer retreat September 2008 at Utsikten, Nynäshamn. KICancer KICancer is a global network for all research groups active totally or partly in cancer research at the Karolinska Institutet (KI), from clinical to preclinical science and from epidemiology to nursing care. Almost all groups working in the area have now joined the network, therefore KICancer gathers close to 130 groups. This accumulation of competence makes cancer research the largest research discipline of KI. KICancer was initiated in 2003 as the first translational network at KI; the need to closer connect clinical with preclinical competence and to establish common research projects was especially obvious in this research area. Now eleven networks covering the major research areas of KI are established. The idea of translational networks was already launched in 1996 by the dean of research and professor of virology, Erling Norrby, but it did not take off at that time. KICancer has adopted four major missions: 1. To offer “meeting places” in a broad sense, on the Web, http://researchnetworks.ki.se/ converis//area/182, in seminars, at conferences and the annual, much esteemed KICancer retreat, usually held at Djurönäset in the Stockholm archipelago. 2. To catalyze novel, translational research projects and initiatives. This was achieved by the award of grants such as the Strategic Grants; 8.5 million Swedish crowns were awarded to nine interdisciplinary, interdepartmental projects. In addition, eleven diagnose-based networks were initiated since 2008 to catalyze interdisciplinary collaboration around the major cancer forms. Douglas Hanahan, a member of the KICancer International Advisory Board, talks at the KICancer retreat. 78 Department of Microbiology, Tumor and Cell Biology – MTC 3. To organize the Research Training program in Oncology and Tumor Biology, offering around 15 courses/year for graduate students. 4. To assist in establishing networks and collaborations with major cancer centers in the world. Joint programs have been initiated with German Cancer Research Center (DKFZ) in Heidelberg, the Netherlands Cancer Institute (NKI) in Amsterdam, the Institute Gustave-Roussy (IGR) in Paris and the Harvard Cancer Center (including Danna-Farber) in Boston. Collaborations with Cancer Centers in Guangzhou and Tianjin, China have also been established. An international Advisory Board In 2009, the network was delighted to appoint a prominent International Advisory Board with David Livingston, Danna-Farber, Boston; Doug Hanahan, Lausanne; Mina Bissell, Lawrence-Berkeley and the Nobel laureate of 2008 Harald zur Hausen, Heidelberg, Germany. KICancer has catalyzed and inspired many new collaborations in the field. It has stimulated close interactions between cancer researchers at KI and provides a common voice to the KI leadership and the world outside of KI, such as the general public, companies and other universities. Ingemar Ernberg, Chairman Scientific Networks KI Inflammation and Immunology The KI Infection Board Members 2006 from left to right: Mikael Rhen, Sven Britton, Gunilla Karlsson Hedestam, Mats Kalin, Staffan Normark, Kristina Broliden, Markus Maeurer, Anna-Lena Spetz and Jan Albert. KI-Infection The KI-Infection Network has been up and running since 2005 and now has over 200 members including both established researchers and students at the Karolinska Institutet (KI). The Network aims to strengthen infectious disease research at the KI by promoting interactions between scientists working in all disciplines of infectious diseases. In addition the Network acts as a hub for infection biology-related information and highlights interesting news and events within the subject. A regular KI-Infection e-newsletter is sent to all members of the Network and the KI-Infection homepage (http://researchnetworks.ki.se/converis/area/187) is the central reference point for the Network’s activities. The KI-Infection Network has members from many departments, including MTC, the Center of Infectious Medicine (CIM), Department of Medicine and Clinical Microbiology at Karolinska Hospital, amongst others. In 2009 a new steering group was established consisting of clinicians and non-clinical researchers in multiple disciplines including bacteriology, virology, parasitology and immunology. The already existing and well functioning Postgraduate Program in Infection Biology, dedicated to the education of PhD students, has now become an integral part of KI Infection. The Study Counselor for the Postgraduate Program is Benedict Chambers (CIM) and the Program Director is Mikael Rhen (MTC). Each year the Postgraduate Program in Infection Biology and the KI Infection Network runs a retreat outside of Stockholm. In 2009, the retreat was held in Gothenburg, well attended by researchers within the Infection Biology Network at KI, as well as researchers within infection biology at Stockholm University, Uppsala University and the Hannover Medical School, Germany. This intensive collaboration between KI and Hannover exists as part of common international graduate program. In addition to the retreat, the Postgraduate Program in Infection Biology also coordinates an annual one-day retreat at Norra Latin in Stockholm where PhD students present their work. In 2010 the KI-Infection network will recruit new members with a particular focus on attracting clinical researchers and strengthen ties between the various subjects in infection research. Mikael Rhen, Chairman Laura Plant, Coordinator The Karolinska Institutet (KI) network in inflammation and immunology, KiiM, was formed in January 2005. The idea behind KiiM was to find a platform for interaction across department borders, to facilitate exchange of ideas and to inspire scientific discussions and collaborations, but also to serve a very practical site, namely to exchange methods and reagents. KiiM arranges seminars where internationally renowned scientists are invited, and where KiiM members meet and discuss science. KiiM also arranges a biannual highly appreciated retreat, which usually attracts 100 students, post docs and senior researchers. The latest one, held in October 2009, included, besides a few invited speakers, a very successful poster session and many impressive student presentations. In addition, a bench-to-bedside discussion on the anti-inflammatory effects of the lipid lowering statins was held. The retreat was a social and scientific success; many new collaborations were formed. KiiM is also building a project database and manages a very active email list which is used for search of reagents, collaborations and as a means of information about activities of interest for KiiMers. Many activities are arranged in collaboration with the KI Doctoral program Aii (Allergy Immunology and Inflammation) and with CfA (the Centre for Allergy Research). Together, these three organizations represent most KI researchers, from doctoral students to professors, within the field of immunology and inflammation. KiiM is headed by a steering committee of 15 people representing many different departments and areas of preclinical as well as clinical research. The steering group is chaired by Petter Höglund (MTC) and the network is coordinated by Louise Berg (MTC). Visit our website, which can be found at KI’s webpage, under research, research networks: http://researchnetworks.ki.se/converis/area/183. Petter Höglund, Chairman Louise Berg, Coordinator The KiiM annual retreat 2006 at Rosenön. Department of Microbiology, Tumor and Cell Biology – MTC 79 Emerging Collaborations The Royal Institute of Technology (KTH) The interactions between MTC and the Department of Applied Physics at The Royal Institute of Technology (KTH) continue to grow. At the moment, at least three major collaboration projects are up and running. KTH provide complementary tools such as microfabrication techniques, bioimaging, X-ray, laser technology and spectroscopy, to the solid biomedical competence at MTC. In these collaborations novel techniques that will be useful for future bio-applications are developed. Miniaturization of natural killer cell experiments A research consortium led by the immunologist Petter Höglund (MTC) and including Klas Kärre (MTC), Björn Önfelt (MTC/Applied Physics), Karl-Johan Malmberg (Center for Infectious Medicine, KI) and Ramit Mehr (Bar-Ilan University, Tel Aviv) study natural killer (NK) cell tolerance and education. The project concerns the molecular and cellular control of NK cell tolerance and education, the role of MHC class I molecules in this process and how NK cell education affects the antitumor effects of NK cells. The project includes several innovative aspects, such as the testing of a new model explaining NK cell education in quantitative terms with novel microchipbased imaging experiments developed in the Önfelt group at Applied Physics, KTH. Clinical and preclinical approaches will be fused in a translational effort. In addition, the team combines biology with biophysics and mathematics in an interdisciplinary setting. This research has a potential to lead to define the optimal conditions for NK cell-mediated elimination of leukemia and other tumors using adoptive cell therapy. Molecular interactions at the single cell level Petter Höglund and Jerker Widengren (Applied Physics) are using fluorescence correlation spectroscopy (FCS) to study protein-protein interactions on the surface of NK cells. FCS is an ultrasensitive spectroscopy technique for studying the mobility of single molecules. Sofia Johansson, a previous member of the Höglund laboratory at MTC, 80 Department of Microbiology, Tumor and Cell Biology – MTC The Royal Institute of Technology (KTH). currently holds a postdoctoral position in the Widengren laboratory, where she pursues this project and thus acts as a bridge between the laboratories. Novel imaging techniques applied to biological questions A research project led by Hans Hertz at Applied Physics is aimed at developing micro imaging in the x-ray regime. This technique enables imaging of thick samples in an aqueous environment with high resolution and contrast, paving the way for biological applications. In collaboration with Marie Arsenian Henriksson at MTC the technique is being investigated for its usefulness for small-animal imaging, especially for tumor detection. In initial studies, mice with defined human neuroblastoma tumors have been imaged with encouraging results. The project recently received a collaboration grant founded by the Swedish Research Council (Vetenskapsrådet). SciLifeLab Stockholm Science for Life Laboratory (SciLifeLab) Stockholm is a national resource center devoted to large scale bioscientific research. The center will focus on biomedicine includ- Photo: courtesy of KTH Photo Archive ing genomic and proteomic profiling, bioimaging and bioinformatics. SciLifeLab Stockholm was founded through a joined investment by the Karolinska Institutet (KI), KTH and Stockholm University and will be situated on new premises close to the Karolinska Campus in Solna. For more information visit the homepage http://www.scilifelab.se/. Hjalmar Brismar at the Department of Applied Physics at KTH was one of the main applicants and is currently involved in setting up the bioimaging part of the lab. Swedish network in bioimaging The newly formed Swedish BioImaging Network aims to facilitate collaboration between researchers in Sweden working in bioimaging and related fields. Björn Önfelt, Hans Hertz, Jerker Widengren and Hjalmar Brismar from the Department of Applied Physics are members of the network. If you work in the field of bioimaging and wish to become a member send an email to [email protected] with a short description of your bioimaging activity and interest. Read more on the homepage http://www.bioimaging.se/. Björn Önfelt Emerging Collaborations Center for Infectious Medicine (CIM) CIM was established in 2003 by former MTC scientist Hans-Gustaf Ljunggren within the Department of Medicine, Karolinska University Hospital in Huddinge. The center is actively engaged in immunology and infectious disease research. In 2007, the groups of Adnane Achour and Benedict Chambers, moved their laboratories to MTC. Adnane Achour arrival of my research group to MTC, has been extremely positive and resulted in excellent and exciting ongoing scientific collaborations with Birgitta Henriques Normark and Staffan Normark (on structural studies of components of the pilus of Streptoccus pneumoniae), Marie Arsenian Henriksson (structural studies of c-Myc), Martin Rottenberg, Markus Maeurer (development of a tuberculosis vaccine), Mats Wahlgren (studies of malaria-associated proteins) and Petter Höglund and Klas Kärre (natural killer cells) at MTC. The strategic localization of MTC within the north campus has also allowed us to deepen our previous collaborations with additional research groups at the Karolin- The ska University Hospital, Solna such as those belonging to Håkan Mellstedt and Jacob Odeberg (development of altered peptide ligand variants of tumor-associated proteins), Marie Wahren-Herlenius (modeling of MHC molecules), Robert A Harris (role of posttranslational modifications in autoimmunity) and Hans Grönlund (crystal structures of allergens). Presently, my group is composed of three PhD students, Adil Doganay Duru, Hannes Uchtenhagen (main supervisor being AnnaLena Spetz) and Eva Allerbring who will soon be joined Dr Tatyana Sandalova strengthening our skills and knowledge in structural biology, especially X-ray crystallography. Most of our research approach combines structural biology with biochemistry and immunology. Our focus has been on the molecular mechanisms underlying viral escape from immune recognition and/or the potential consequences of virus infections, Active site of the Streptococcus pneumoniaeassociated sortase C (Neiers et al., J Mol Biol 393, 704, 2009). such as the induction of auto-reactivity. We have recently initiated a series of cancerrelated studies in which we have developed/ adapted alternative ideas to design altered peptide ligands. The final aim is to increase the immunogenicity of wild-type peptides by introducing mutations that do not alter the conformation of the targeted peptides. Benedict Chambers The focus of my research is on both dendritic cell (DC) and natural killer cell (NK) biology and how these cells, together or by themselves, affect immune responses. DC release a variety of cytokines and chemokines upon infection that both attract NK cells to a site of infection and activate these to produce cytokines or to become cytotoxic. Part of my research is attempting to identify which DC subsets that are required for the activation of the NK cells. One way by which NK cells can control adaptive immune responses is to kill DC. This has to be avoided in a situation in which DC cells are used as part of a vaccine strategy. We have identified a variety of molecules on dendritic cells Live cell imaging (green) of NK-DC cell interactions. Many NK cells (blue) interact with DC (A). NK cells migrate towards DC (B), form a tight intercellular contact (immune synapse) with DC (C) and finally kill DC (D), seen as red staining of the cell nucleus. that are involved in NK cell mediated killing of dendritic cells. These findings are also being used in developing novel therapies to treat myeloid derived tumor cells which express many of the same ligands as dendritic cells. In viral infections we have found that NK cells appear to eliminate dendritic cells. The same was also observed with Toxoplasma gondii parasite infected DC. Interestingly in the latter case, the parasite could “jump” into the NK cells that had killed the dendritic cell. We are currently investigating how pathogens such as Toxoplasma, influenza and vaccina virus interact with NK cells. We have excellent collaborations with several research groups at MTC including those that belong to Klas Kärre, Martin Rottenberg, Gunilla Karlsson Hedestam and Mikael Rhen. Department of Microbiology, Tumor and Cell Biology – MTC 81 Translational Research Successful collaborations between preclinical and clinical scientists of new drugs and treatment principles to improve human health requires successful collaborations between preclinical and clinical scientists. The modern word for this process is “translational research”. MTC is primarily a preclinical department, and as such can provide novel ideas and biological insights useful for drug development. In order to stimulate this process, it is important to give good examples on how it can be successfully conducted. MTC has several research leaders engaged in successful translational research. Four such projects are highlighted here. We have asked the leaders to describe how the project was initiated, how they succeed in maintaining it and how they have overcome potential obstacles that lies in the challenges of communication between preclinical and clinical science. It is our hope that these short recollections will inspire the creation of more translational projects at MTC and other research departments, which will be beneficial for patients in the future. The development Petter Höglund Anna Nilsson Name: Yihai Cao Project title: Vascular endothelial growth factor (VEGF) in paraneoplastic syndrome. Collaborators: Dr. Weide Zhaong, Guangzhou, China. 82 Department of Microbiology, Tumor and Cell Biology – MTC project was initiated by us when the technological developments made it possible to immunophenotype T cells during the reconstitution phase after transplantation. We focus on the behaviour of EBVspecific T cells using tetramer technology and multicolor flow cytometry. How is the project maintained? Name: Ingemar Ernberg, MTC and Markus Maeurer, MTC and the Swedish Institute of Infectious Disease Control (SMI). Project title: Control of Epstein-Barr virus (EBV) after transplantation, with Special focus on the Pathogenesis and Control of EBV-positive lymphomas (SPACE). Collaborators: Olle Ringdén and Brigitta Omazic, (Transplantation Unit, Karolinska University Hospital Huddinge). Jacek Winiarski and Åsa Gustavsson-Jernberg (Pediatric clinic, Karolinska University Hospital Huddinge). How did you initiate the project and how is it maintained? We have had long-standing collaborations with the transplantation unit at Karolinska University Hospital Huddinge in projects relating to EBV infection and stem cell transplantation. The SPACE We have joint meetings every 5-8 weeks at the clinic when we go through recent data and perform a joint analysis. So far, close to 100 patients have been entered in the study. They are sampled at 1, 2, 3, 6 and 12 months after transplantation. Which obstacles have you encountered? Building a good, open database is difficult in projects containing clinical data and patient information. Which advice do you want to give regarding the translation of your project? Build relations. Be humble, show respect and understand that clinical work with patients is tedious and needs a good organization. The lab-work is very much routine, but still has to be performed at top level. How did you initiate the project? How is the project maintained? Our animal models had shown that high levels of VEGF in the circulation globally affects the vasculature in various tissues and organs, leading to severe anemia, hepatomegaly, splenomegaly, and endocrine disorders. Such paraneoplastic effects are called VEGF-induced cancerassociated systemic syndrome (CASS) and significantly shortens life time and lowers quality of life. We initiated this project to see if circulating levels of VEGF also in human cancer patients would be reversely correlated with hematocrit and other cancer-associated systemic disorders. I have continuous discussion with the clinicians involved, but we do not have regular meetings with them. Which obstacles have you encountered? Ethical approval of clinical studies is relatively complicated and time consuming. Which advice do you want to give regarding the translation of your project? Start to prepare ethical permission in good time. Otherwise, it can significantly delay your studies. Name: Galina Selivanova Project title: Rescue of mutant p53 function by the small molecule PRIMA-1. Collaborators: Klas Wiman (Cancer Center Karolinska, CCK), Sören Lehmann & Christer Paul (Department of Hematology, Karolinska Huddinge), Sten Nilsson (Department of Oncology-Pathology, Karolinska Solna). How did you initiate the project? Our preclinical discovery that the activity of mutant p53 could be restored by a synthetic peptide prompted us to perform a screen of the chemical library. This led to the identification of PRIMA-1 as an apoptosis-inducing drug and opened the door for clinical extensions. How is the project maintained? Together with colleagues Klas Wiman and Staffan Strömblad, and with the help of Karolinska Development AB, I started a small biotech company called Aprea AB. The first clinical trial with PRIMA-1 has recently started, led by Aprea AB. My group How did you initiate the project? Questions formulated in my scientific group at MTC are proposed to our clinical collaborators. Their experience, clinical and scientific, then helps to refine the questions and always propose relevant clinical angles and comments. After the project is outlined in detail we plan for relevant samples to be taken from patients provided that all ethical permissions are in place. How is the project maintained? Name: Francesca Chiodi Project title: B and T cell damage during HIV-1 infection. Collaborators: Bo Hejdeman, Sven Grützmeier (Stockholm South General Hospital, Södersjukhuset), Paolo Rossi (Hospital Bambin Gesu’, Rome Italy), Anna Nilsson (Department of Woman and Child Health, Karolinska Institutet). Important components to maintain the translational collaboration are: a) reciprocal respect of competence and culture in the pre-clinical and clinical environments; b) flow of information and contact during all stages of the project, including presentation of results to medical staff, both doctors and nurses; c) finalize papers with the active participation of clinical collaborators. It is also important to find common grant support to give legitimacy to the projects. Which obstacles have you encountered? is involved in discussions with Aprea, but mainly continue with basic research on the molecular mechanism of action of PRIMA-1. Which obstacles have you encountered? It has been a long road to where we are now. Along the way, I had to learn many new things, such as how to communicate with pharma people, clinicians and chemists. I also had to learn how to run this project and at the same time lead the basic research in my lab. Which advice do you want to give regarding the translation of your project? It is imperative to be dedicated to basic science and to be passionate about it. But if something that you have discovered can help people – put an effort into translating it into clinic! Nobody understands the implications of your discovery better than yourself! research are, in my experience, not really between the pre-clinical environment and the clinic. In my experience the major obstacle when working with translational projects in a pre-clinical environment is often to gain respect from colleagues only involved in animal experiments. Also publication in top ranked journals is some time difficult when working mostly with clinical specimens. But we are learning to bridge our questions both into the clinic and in parallel into the animal house. Which advice do you want to give regarding the translation of your project? If you get an idea for which you need clinical competence and samples, contact clinicians early to plan the project together. You may get relevant good suggestions which will increase the value and validity of your investigations. Involve clinical collaborators in all stages of planning and reporting. Never think about your clinical collaborators as a sample provider. Be patient if the flow of specimens to the lab is slow. The obstacles of conducting translational Department of Microbiology, Tumor and Cell Biology – MTC 83 Companies Founded by MTC Scientists Two examples of successful spin-offs Intervacc AB The major focus for Intervacc AB is on the development of vaccines against bacterial infections within the animal health sector. Top priority is presently given to a vaccine against strangles (kvarka in Swedish). This a very severe and contagious upper respiratory tract infection in the horse, caused by Streptococcus equi. It leads to severe suffering in the horse and economic losses for the business. The world market for a vaccine against strangles is estimated to around 200 million USD. We have recently proven in a number of studies on the horse, that a vaccine against S. equi does not have to be composed of attenuated or killed bacteria, such as those presently existing. Instead, the use of a well-composed set of recombinant proteins is both safe and efficient with no side effects and can meet the demands from the market for a modern vaccine. Intervacc AB is now in a stage where a proof of principle has been passed and the candidate vaccine is further optimized, tested for duration of protection, shelf life, and for Welsh Mountain Ponies were vaccinated and subsequently challenged with S. equi to assess a protective effect. Photo: Jan-Ingmar Flock the best route of immunisation. Such parameters can be tested directly on the target animal, the horse, which can be subjected to experimental infection. Experience gained from these vaccine trials is anticipated to facilitate in the development of a vaccine against the human pathogen Streptococcus pyogenes, having significant resemblance with S. equi. Several of the antigens used in the strangles vaccine are also found in Streptococcus zooepidemicus, another severe pathogen of the horse. Although there is no cross protection, a vaccine against S. zooepidemicus is also close DILAFOR AB Dilafor AB is a Swedish R&D company focused on developing pharmaceutical products from heparin derivatives with low anticoagulant activity (www.dilafor.com). 12 persons representing a broad scientific-, drug-development and business expertise founded the company in 2003 within Karolinska Development AB. Dilafor AB has a balanced product portfolio representing some highly promising ideas and innovations. Each of our projects addresses an important and unfulfilled medical need. All our drug development projects are based on the use of Low Anticoagulant Heparin (LAH). Although standard heparin has numerous biological functions, its potential development as a treatment for indications other than those currently approved is 84 Department of Microbiology, Tumor and Cell Biology – MTC The bite of an anopheles-mosquito can lead to malaria. limited by its anticoagulative properties. Dilafor has produced several forms of LAH. Two products are presently in development, tafoxiparin and DF02. Tafoxiparin, which is investigated for its effect on the prevention and normalization of protracted labor, has recently completed a “Proof of Concept” clinical trial in Phase II. Promising results were found in the trial, where 263 women were included, as protracted labor was prevented in the group given the drug tafoxiparin. Dilafor is also developing a new malaria at hand and steps have been taken towards such a vaccine. The vaccine development also involves a team at the Swedish University of Agricultural Sciences, Uppsala and Animal Health Trust in the UK. Intervacc AB is mainly funded by the Nordvacc Läkemedel AB and by private investments. A further spreading of shares is planned to secure funding for the remaining parts of the project and for additional developments. Information about Intervacc AB can be found at http://intervacc.se/ Jan-Ingmar Flock treatment – DF02 – that reduces the blocking of the capillaries in severe Plasmodium falciparum malaria. Today, there are no effective treatments against the most severe symptoms of malaria. A Phase I study was conducted with DF02 during 2009 on 33 healthy men. The study was randomized, double blind, and placebo. No severe adverse effects were reported. The results prove that DF02 is safe and well tolerated in all of the tested dose levels both at a single dose and when using multiple dosing (nine doses during three days). In addition, the results show that the drug is suitable for intravenous injection, which is the only way to treat patients with the most severe form of malaria. This will be followed by a proof of concept trial in Phase II. “New Chemical Entities” similar to tafoxiparin and DF02 are also being developed for yet other indications. Mats Wahlgren Future Faculty Career development – how to be successful in a large and complex organization a new seminar series for the future faculty (postgraduate level) at MTC was started. The main goals were to form an interactive group discussing questions such as career development, how to become a group leader, responsibilities and challenges, leadership and grant applications as well as to provide more insights into how to plan a future career in science. The first meeting was held in February 2009 in which professor Staffan Normark and Judit Wefer from the Grant Office at the Karolinska Institutet (KI) discussed important issues regarding grant applications, where and how to apply for resources and the evaluation of grant applications. In the next meeting the head of personnel at KI, Ylva Lindberg, and two human resource consultants, Emma Liffgarden och Niclas Asplund, talked about career development and work law. Three seminars were later held during fall 2009. The first one was held by the head During spring 2009 Francoise Barre Sinoussi, who discovered the HIV virus, has been one of the speakers at the Future Faculty seminars. Photo: Wikipedia of MTC, Marie Arsenian Henriksson, who informed about the organization of MTC and KI, and discussed the criteria to become a group leader. The second seminar was held by the Nobel Laureate in 2008, Francoise BarreSinoussi, who discussed premises on how to succeed in science with the title “Multidisciplinary science: a key issue in biomedical research”. To the last meeting during the fall a representative of both industry and academia was invited, adjunct Professor Alexander von Gabain. He was a scientist at KI more than 15 years ago and later co-founded a biotechnology company for development of vaccines, Intercell Biomedical Research & Development AG, in Austria. He described what it is like working in industry and how to develop a company when you come from an academic background. Plans for seminars during 2010 include economic responsibilities and obligations as a group leader, what is important in leadership and how to be successful in a large and complex organization like KI. Birgitta Henriques Normark Equality and Diversity Group of the ”Equality and Diversity group” represent scientists, PhD students, the technical and the administrative personnel within MTC. The main goal for this group is to promote equal and respectful treatment irrespective of gender, age, ethnicity, religion, disabilities or sexual orientation within all personnel categories. The group is also proactively involved in different aspects of diversity within the department. One important issue that has been discussed is the role of mentors for the PhD students. As a result of this discussion, the mentor function has been more clearly defined. We have also discussed guide lines for using English and/or Swedish within the department. During the last years, in order to focus on important issues that cover other The members subjects than science, the group has invited to a number of seminars, that have been well attended. In 2007, Stefan Einhorn, professor of molecular oncology at the Karolinska Institutet (KI), but also well known as an author of several popular science books, spoke about “The art of being kind”. His lecture was based on a book which has been a bestseller in Sweden and was translated into 12 languages. Angela Nilsson from Stockholm University presented her study on KI entitled “Innovative or conservative excellence”. In 2008, we invited Åsa Björndal and Laszlo Szekely working at MTC and the Swedish Institute for Infectious Disease Control, to talk about “Couples in Research. How to manage a family life in parallel with hard scientific work”. We have also invited Alexander Perski, who is leading the stress clinic at KI. He gave a seminar about “The Perfection Trap”. Hanna Akuffo preAlexander Perski had an engaged audience sented her Swedish in March 2009. experience “Just because you are paranoid does NOT mean they are not out to get you - on the other hand perhaps they are NOT out to get you”. In 2009, David Eberhard spoke about “Addicted by safety concerns” and Björn Klinge had a presentation entitled “Ethics”. All these lectures have been very appreciated and new events are in the pipe line. Elisabeth Norin Department of Microbiology, Tumor and Cell Biology – MTC 85 Undergraduate Education Teaching in medicine, biomedicine and optometry in undergraduate education within three study programs; medicine, biomedicine and optometry, in which we are responsible for the organization and implementation of courses and are, in addition, running parts of other courses at the Karolinska Institutet (KI). MTC also harbours, and is in charge of, one of the two teaching laboratories at Campus Solna, a class II laboratory equipped to have the capacity for laboratory work with both bacteria and sterile work with eukaryotic cells. Several undergraduate program have undergone/are undergoing extensive changes that have required revisions of course contents and structure. Teachers involved have spent a lot of time and devotion to form and implement these changes. At MTC there is a continuous effort to further improve the quality of undergraduate teaching to offer an education of highest quality. The medical studies are being reorganized aiming at a higher level of integration between disciplines and between preclinical and clinical subjects (LUT = läkarprogrammets nya utbildningsplan – the New Education Plan for Studies in Medicine). The former MMI course (Medical Microbiology and Immunology) at MTC has been replaced with courses within DFM1 (den friska människan – the healthy human being), DSM1 (den sjuka människan – the sick human being), and DSM2. These courses contain a mixture of lectures, and to a large part seminars in small groups, which have been much appreciated by the students. The courses consist of both basic immunology, microbiology and clinical microbiology. The content of the courses is discussed and evaluated on a regular basis by the course organisers and the teachers involved to further develop and maintain the high quality of the courses. MTC is involved The Bologna process With the introduction of the Bologna process for higher education in Sweden, the study program in Biomedicine has changed from being a 4-year program to a 3-year bachelor program and a 2-year Master program in Biomedicine, starting 2007. MTC is involved in several courses within the Bachelor program in Biomedicine. At semester 3, 86 Department of Microbiology, Tumor and Cell Biology – MTC MTC is responsible for giving the course in “Infection and Immunity” (15 credits). This course gives the students a basic knowledge in Immunology and Microbiology and in practical laboratory skills within these subjects. Teaching is based on lectures, seminars, labo- ratory work and literature project work. This course was given for the first time 2008, and it partly builds on the previous course within the old 4-year program. It is under constant evaluation and evolution to maintain high standards, and student input is an important part of improving the quality. At semester 5 a course in “Molecular Oncology and Biostatistics”, is given at the department of Oncology and Pathology. MTC is responsible for the advanced laboratory experiments, a major and central part of the course which is highly appreciated by the students. The Master program in Biomedicine is a 2-year international study program attracting students from all over the world. During the first semester, the course in “Frontiers in Translational Medicine” (22 credits) is intended to introduce the students to some major research areas at Karolinska Institutet. The course consists of four parts, MTC is in charge of two of them; Infection biology and Oncology. In collaboration with the Center for Infectious Medicine (CIM), Department of Medicine in Huddinge, MTC also takes part in teaching “Immunology and Infection biology” during the second year of the Master program in Biomedicine. Teaching is performed in close collaboration with the PhD courses given at MTC and CIM, and is an excellent opportunity to attract top students for future doctoral studies to MTC. Bachelor and Master programs MTC is also responsible for project courses and degree projects for students at the Bachelor and Master programs within Biomedicine and for incoming exchange students on these programs. The students do a research project with a research group at KI, any other university in the world, or at a company, and at the end of the course present this work orally and in a written report. The study programs in Biomedicine have introduced the 7 grade system A-F, according to the Bologna process. A lot of work is put into the development of how students are examined and graded in these programs and how to formulate course goals and grading criteria that can be assessed in 7 grades. In addition MTC is involved in the Study program in Optometry in which the students get tailor made lectures to cover important aspects of microbiology in this area for opticians. Jonas Sundbäck Jan-Ingmar Flock Thi Viet Ha Nguyen from Vietnam defends her thesis “Diagnosis and treatment of Helicobacter pylori infection in Vietnamese children” November 2009. Training for the Future – PhD Studies The third level of education, formerly designed as doctoral or postgraduate education, is very vivid at MTC, with about 150 students from 30 different countries guided by more than 65 supervisors. As usual at the Karolinska Institutet (KI), the main education takes place as scientific work in individual research groups under the guidance of at least two supervisors. There are several “sub-subjects” to register in, such as infection biology, immunobiology, cell biology and tumor biology, biomedical ecology and infectious disease control. The administration is handed by a full time employed administrator and a study counselor who, together with the Committee for Doctoral Education and the MTC leadership, assure that all regulations issued by KI are followed during registration for post-doctoral education, half time control and at the dissertation process. MTC has two special rules. All students give in connection with their yearly follow up, a 10–15 min seminar, and get questions from the audience and feedback from an independent senior scientist. An oral examination in the basic knowledge in microbiology and immunology or in cell and tumor biology is performed for each student by an independent senior scientist before the half time control. During the last years around 30 new students have registered per year and the same number have passed their dissertation. Two thirds are women, whereby among the Swedish students only one out of five is male (see Figure). This is an unusual gender imbalance compared to KI as a whole. Females foreign 25% Males foreign 27% Females Swedish 39% Males Swedish 9% The median age is 30 years, which is younger than for the average at KI and the median time from registration to dissertation is 4.6 years. The median age at dissertation is lower than that for KI total. 18 students (12 %) have a medical exam, half of which are from Sweden. In total only 18 students have had their basic education at KI, which shows the need for highly active external recruitment. A variety of scientific seminars, usually one per day, are offered at MTC. Furthermore, many research groups arrange their own seminar series. In addition, in 2009, MTC was involved in 15 postgraduate courses arranged by KI. Roland Möllby Department of Microbiology, Tumor and Cell Biology – MTC 87 Summer Research Schools – a fantastic opportunity for high school and undergraduate students to get a research experience at KI and MTC. Summer Research Schools An excellent opportunity for students to get a research experience three summer research schools at the Karolinska Institutet (KI): The Summer Research School in Biomedical Sciences for Swedish high school students, The Summer Course in Medical Research for KI undergraduate students and The Amgen Scholars Program for undergraduate students from all over Europe. The Summer Research School in Biomedical Sciences is open for all high school students that study either natural science or technical programs in Sweden. The interest is very high and 20 students are accepted each summer. Students from outside of Stockholm are offered a student room during their stay. The school is five weeks long and consists of lectures, study visits to a life science company and to the Nobel museum and a 4-week research project in a research group at KI. Many research groups at MTC hosted these students. There is also a social program for the students including a number of activities during the summer which is organized by undergraduate students from the KI. The school ends with student presenta- MTC administrates 88 Department of Microbiology, Tumor and Cell Biology – MTC tions and a farewell dinner. Many of these former students are now undergraduate students at the KI, with a strong interest in research! The Summer Course in Medical Research (10.5 credit points) is open only for undergraduate students at KI, to get research experience in a research group. 80-90 students from many of our study programs at the KI apply for the course which accepts 38 students. It starts in mid June with lectures in ethics, reading of research publications, learning to plan and create posters and powerpoint presentations and an inspirational lecture about creativity by Ingemar Ernberg, professor at MTC. The students take part in a six week project during the summer in a research group at KI, many at MTC, after which they present their results in a written report, a poster and an oral presentation during two days in August. The Amgen Scholars Program is new to Europe and KI since the summer of 2009. It is supported by the Amgen Foundation at Amgen, a major biotechnology company located in the US. Similar sister programs are located at the University of Cambridge, UK, and Ludwig-Maximilians University in Munich, Germany. It is an international summer research school for top undergraduate students from all over Europe, as defined by the Bologna process. In the first year 12 students where admitted. During the eight weeks of the program, the students got an introduction to research, how to present data, make posters and publish papers. The students also attended a few research presentations. Most of the time, however, was spent in research laboratories all over KI, including MTC. The program ended by student presentations and a final symposium at the Cambridge University which included all Amgen Scholars Programs in Europe in which the students presented their projects on posters. In summary, there is a fantastic opportunity for students to get a top-level research experience at the KI and MTC and vice versa an excellent opportunity for our research groups to get in contact with students who are highly interested in research and in future doctoral studies. Jonas Sundbäck Life as a PhD Student One of your greatest adventures! ing, supervision, language skills, teaching and presentation techniques. One of the main obstacles that I have found hardest to overcome is keeping the motivation and to continue working hard even though useful results can be absent for long periods of time. Since PhD students in a way are their own managers, their motivation is the only fuel that can run the ongoing projects. During those periods when the motivation has been failing me it has been very helpful to find it again elsewhere than in the lab, e.g. by seeking support from the supervisor, my mentor, colleagues, group members and from social activities. It is also To become a PhD student always good to once in a while remember the Supervision is crucial in the beginning since ultimate goal of the research project and the most PhD students are beginners within importance of it in a bigger picture, and not their fields! Finding a good supervisor that just as a cell culture in I got along with well your Petri dish. was one of my highWorking with est priorities when science in a research I started thinking group might involve about becoming a very intense periods PhD student. Eventuwith a lot of work, but ally, everything finally also freedom. A freefelt good at MTC in dom that gives you the every way! One could luxury to decide over say that the interestyour own work and ing research projects, your own time. The the highly compePhD student period tent supervision and has the capacity of people, the high qualbecoming one of the ity of the laboratories, Malin Stoltz performing an experiment. greatest adventures of the nice premises, your entire professional career. and the pleasant and stimulating research atmosphere all made me feel welcome and motivated to stay. Now when the dissertation The future is approaching rapidly I have not regretted The end of my PhD studies is approaching becoming a PhD student at MTC even once. and the question is: what will happen next? Knowing that the dissertation will come It feels like a PhD degree never can be wrong soon is of course a bit stressful, but a natural when applying for positions in the future. part of the process of being a PhD student. I have learned so much, and my whole way The path towards the dissertation is not of thinking has been totally changed during straight and is for sure full of hard work, hopethese years, even though I have not started fully giving the PhD student the opportunity writing the thesis yet! It seems to me that this to mature and gain more and more specific education has taken me to a point where I can knowledge and independence during the way. become whatever I want. In the end, except from the title, becoming a These years have given me a very solid PhD also involves personal development and ground to stand on during the rest of my achieving skills such as project leading, labocareer and I am grateful for that. ratory techniques, communication, networkMalin Stoltz Before writing this I counted backwards and realized that I have been working in the same group at MTC/the Swedish Institute of Infectious Disease Control (SMI) for almost five years and that I still like it! During 20042005, I attended Stockholm Research School in Molecular Life Sciences and did one of my three 10-week projects at MTC/SMI. Before this I was sceptical about conducting PhD studies and convinced that I would start in a pharmaceutical company. However, working in a highly stimulating research milieu at MTC/SMI made me change my mind! The MTC PUB – a lot of fun every month! One Friday every month there is a special night at MTC, a night when the party goes on until 2:00 or 3:00 in the morning – depending on how long the beers and cocktails last! Traditionally, MTC has one of the most appreciated pubs on the Solna campus and is actually considered one of the best party evenings you can find! The very experienced “Muppet Crew”, which ran more than 30 pubs, recently left the responsibility to organize this event in the hands of the grand “Viceversas Crew”. It was a challenge for the Viceversas to maintain the high level of quality and fun that the Muppet Crew had created. They were able to transform a normal pub into a real party, where they allowed everyone to travel, with their thematic pubs, all over the world, exploring the cuisine of every possible country... Bringing a new twist to the pub evenings, the Viceversas are doing a superb job reinterpreting movie themes, dressing in “particular”, eccentric ways and overall playing good music and preparing high quality sandwiches. It is clear that the trend remains; people prefer the MTC pubs to those that you find in the downtown area! Simone Pensioroso Department of Microbiology, Tumor and Cell Biology – MTC 89 Doctoral Theses 2007–2009 2007 297. Sandalova, Elena, Regulation of the proapoptotic protein Bim by T cell receptor triggering in human T cells. Opponent: Philippe Bouillet, Parkville, Australia. Supervisor: Victor Levitsky. The role of IL-7 in Lymphopenia and Bystander Apoptosis during HIV-1 infection. Opponent: MarieLise Gougeon, Paris, France. Supervisor: Francesca Chiodi. 298. Fluur, Caroline, Visualizing the dynamic interplay between the host and bacterial pathogens. A real-time study of renal infection. Opponent: Michael S Donnenberg, Baltimore, USA. Supervisor: Agneta RichterDahlfors. 305. Månsson, Lisa, 306. Hidmark, Åsa, Induction of type I interferons and viral immunity. Opponent: Bruce Beutler, La Jolla, USA. Supervisors: Gunilla Karlsson-Hedestam, Eva Nordström, Peter Liljeström. Carbapenem resistance in Pseudomonas aeruginosa. Opponent: Giuseppe Cornaglia, Verona, Italy. Supervisors: Bengt Wretlind, Göran Kronvall. 307. Corrigan, Gary, Evaluation of reverse transcriptase assay for viral load monitoring. Opponent: Elizabeth M Dax, Fitzroy, Victoria, Australia. Supervisors: Rigmor Thorstensson, Clas Källander. 300. Advani, Abdolreza, Epidemiological characterisation of Bordetella pertussis in Sweden, 1970–2004. Opponent: Per Olcén, Örebro, Sweden. Supervisors: Hans Hallander, Lars Engstrand, Patrick Olin. 308. Paulander, Wilhelm, Mechanisms of adaptation to the fitness cost of antibiotic resistance. Opponent: Ivan Matic, Paris, France. Supervisors: Dan Andersson, Sophie Maisnier-Patin. 299. Giske, Christian G, Genomic and transcriptomic variation in blood stage Plasmodium falciparum. Opponent: Hernando del Portillo, Barcelona, Spain. Supervisor: Mats Wahlgren. 309. Mok, Bobo, Regulation and characterization of antimicrobial peptides in man and mice. Opponent: Peter Hiemstra, Leiden, The Netherlands. Supervisors: Mats Andersson, Katrin Pütsep. 310. Karlsson, Jenny, 311. Bråve, Andreas, Prime-boost immunization strategies against HIV-1. Opponent: Tomás Hanke, Oxford, UK. Supervisors: Britta Wahren, Karl Ljungberg, Margaret Liu. Studies of cell signaling using bacterial toxins and organic electronic devices. Opponent: Thomas Borén, Umeå, Sweden. Supervisor: Agneta Richter-Dahlfors. 312. Kjäll, Peter, 301. Sjöström, Karin, Molecular epidemiology of pneumococcal carriage and invasive disease. Opponent: Karl Kristinsson, Reykjavik, Iceland. Supervisor: Birgitta Henriques Normark. Intercellular protein transfer and regulation of inhibitory NK receptor accessibility. Opponent: Francesco Colucci, Cambridge, UK. Supervisors: Petter Höglund, Klas Kärre, Maria Johansson. 302. Andersson, Katja, 303. Ries, Johannes, Pneumococcal pili and other cell surface properties affect the infection biology of Streptococcus pneumoniae. Opponent: Regine Hakenbeck, Kaiserslauten, Germany. Supervisors: Birgitta Henriques Normark, Staffan Normark, Eva Morfeldt. 304. Darai-Ramqvist, Eva, Involvement of evolutionarily plastic regions in cancer associated chromosome 3 aberrations. Opponent: David Gisselson, Lund, Sweden. Supervisors: Stefan Imreh, Maria Kost-Alimova. PhD student Xuan Xue follows the old tradition of nailing his thesis with a golden nail. 90 Department of Microbiology, Tumor and Cell Biology – MTC EBV gene variation and epigenetic alterations in Asian nasopharyngeal carcinoma and potential clinical applications. Opponent: Jaap M Middeldorp, Amsterdam, The Netherlands. Supervisors: LiFu Hu, Ingemar Ernberg, Phan Thi Phi Phi, Tran Thi Chinh. 313. Nguyen-Van, Do, 314. Liu, Anquan, Proinflammatory factor mediated lymphocyte activation – the pivotal role of leukotriene B4. Opponent: Dorothy H Crawford, Edinburgh, UK. Supervisors: Eva Klein, Laszlo Szekely. Chemokines and chemo kine receptors during viral infections in man. Opponent: John Gordon, Birmingham, UK. Supervisors: Anna Nilsson, Francesca Chiodi. 315. Mowafi, Frida, Characterization of c-diGMP signaling in Salmonella typhimurium. Opponent: Stephen Lory, Boston, USA. Supervisors: Ute Römling, Mats Andersson. 316. Simm, Roger, 317. Vahlne, Gustaf, Natural killer cell inhibitory and activating receptors – Regulatory role in effector functions against normal and tumor cells. Opponent: John Torgils Vaage, Copenhagen, Denmark. Supervisors: Maria Johansson, Klas Kärre, Petter Höglund. Modulation of the deubiquitinating system in viral infection, lymphoid cell activation and malignant transformation. Opponent: Ivan Dikic, Frankfurt, Germany. Supervisors: Maria G Masucci, Riccardo Gavioli. 318. Rolén, Ulrika, 319. van der Holst, Rutger, TRAPC a novel Triggering Receptor expressed on Antigen Presenting Cells. Opponent: Marco Colonna, St Louis, USA. Supervisors: Jonas Sundbäck, Klas Kärre. 320. Fernebro, Jenny, Genetic approaches towards understanding pneumococcal virulence and biology. Opponent: Tone Tonjum, Oslo, Norway. Supervisors: Birgitta Henriques Normark, Staffan Normark. 2008 Identification and characterization of novel virulence factors in Streptococcus pneumoniae. Opponent: Sven Hammerschmidt, Munich, Germany. Supervisors: Birgitta Henriques Normark, Staffan Normark. 321. Wartha, Florian, The Head Marshall Ying Zhao (foreground) from MTC leads the opening of the KI Conferment Ceremony May 2009. Photo:Ulf Sirborn 322. Beiter, Katharina, Recognition and clearance of Streptococcus pneumoniae by the innate immune system. Opponent: Malak Kolb, Nashville, USA. Supervisors: Birgitta Henriques Normark, Staffan Normark. 328. Tallo, Tatjana, Molecular epidemiology of 323. Svanh, Anita, Varicella immunity and vaccination. Opponent: Tomas Bergström, Gothenburg, Sweden. Supervisors: Annika Linde, Hans Gaines. 329. Reiner, David, 324. Meyer-Hoffert, Ulf, Antimicrobial peptides as defense molecules at the interface of the host and bacteria. Opponent: Artur Schmidtchen, Lund, Sweden. Supervisors: Birgitta Henriques Normark, Jens-Michael Schröder, Staffan Normark. 330. Andersson, Ida, Crimean-Congo hemorrhagic fever virus: Interferon-induced antiviral mechanisms and immune evasion strategies. Opponent: Richard Elliott, St. Andrews, UK. Supervisors: Ali Mirazami, Åke Lundkvist. 325. Juréen, Pontus, Molecular characterisation of antibiotic resistance in Mycobacterium tuberculosis. Opponent: Viesturs Baumanis, Riga, Latvia. Supervisors: Sven E Hoffner, Lars Engstrand. 331. Skoglund, Anna, 326. Kader, Mohammad Abdul, GGDEF-EAL domain proteins in bacterial physiology. Opponent: Iñigo Lasa Uzcudun, Navarra, Spain. Supervisors: Ute Römling, Mats Wahlgren. 332. Maljkovic Berry, Irina, hepatitis A, B and C in Estonia. Opponent: Magnus Lindh, Gothenburg, Sweden. Supervisors: Helene Norder, Lars Magnius, Ljudmilla Priimägi, Valentina Tefanova. Cell cycle and differentiation in Giardia lamblia. Opponent: Norbert Müller, Bern, Switzerland. Supervisors: Staffan Svärd, Frances Gillin. Mechanisms of genetic adaptation in Helicobacter pylori. Opponent: Arnoud van Vliet, Norwich, UK. Supervisors: Lars Engstrand, Margareta Krabbe och Britta Björkholm. Genetic aspects of HIV-1 evolution and transmission. Opponent; Andrew Leigh Brown, Edinburgh, UK. Supervisors:Thomas Leitner, Jan Albert. Pursuing the fever trail. Pathogenesis of blood-stage P. falciparum malaria & pregnancy. Opponent: Ross Coppel, Victoria, Australia. Supervisors: Mats Wahlgren, Qijun Chen, Craig Wheelock. 333. Rasti, Niloofar, Immunological consequences of Epstein-Barr virus replication. Opponent: Emmanuel Wiertz, Leiden, The Netherlands. Supervisors: Victor Levitsky, Maria Masucci. 327. Ortlieb Guerreiro Cacais, André, Department of Microbiology, Tumor and Cell Biology – MTC 91 334. Hardestam, Jonas, Hantavirus – shedding, stability and induction of apoptosis. Opponent: Göran Wadell, Umeå, Sweden. Supervisors: Åke Lundkvist, Jonas Klingström. 335. Aronsson, Linda, Microbial regulation of PPAR: Nuclear receptor networks important for colonic homeostasis. Opponent: Ian Sanderson, London, UK. Supervisors: Velmurugesan Arulampalam, Sven Pettersson, Jan-Åke Gustafsson. 336. Rölle, Alexander, Natural killer cell mediated recognition of herpesviruses – Mechanisms of viral immune escape. Opponent: Andrew McMichael, Oxford, UK. Supervisors: Klas Kärre, Cristina Cerboni, Adnane Achour. 337. Forsell, Mattias, Evaluation of new strategies to stimulate neutralizing antibodies against HIV-1. Opponent: Quentin Sattentau, Oxford, UK. Supervisors: Gunilla Karlsson Hedestam, Richard Wyatt. 338. Åkerström, Sara, SARS coronavirus – The role of occessory proteins and nitric oxide in the replication cycle. Opponent: Völker Thiel, St Gallen, Switzerland. Supervisors: Ali Mirazimi, Yee-Joo Tan, Åke Lundkvist. 339. Blomberg, Christel, Insight into the genetic characteristics of pneumococcal isolates. Opponent: Peter Hermans, Nijmegen, The Netherlands. Supervisors: Birgitta Henriques Normark, Eva Morfeldt, Staffan Normark. Innate immunity to intracellular bacterial infections. Opponent: Thomas Decker, Vienna, Austria. Supervisors: Martin Rottenberg, Hans Wigzell. Källman, Owe, Antibiotic resistance in Gram-negative bacilli – with focus on cephalosporin resistance mechanisms in Escherichia coli and Klebsiella pneumoniae. Opponent: Niels Frimodt-Möller, Copenhagen, Denmark. Supervisors: Barbro OlssonLiljequist, Christian G Giske, Mats Kalin. 341. 342. Simon, Melinda, Crimean-Congo hemorrhagic fever virus: Interactions with host cell structures in viral replications. Opponent: Einar Everitt, Lund, Sweden. Supervisors: Ali Mirazimi, Åke Lundkvist. Transcriptome based analysis of Hodgkin lymphoma: Insights into the microenvironment. Opponent: Volker Diehl, Cologne, Germany. Supervisor: Ingemar Ernberg. 343. Birgersdotter, Anna, 344. Tran, Thi Thanh Ha, HIV-1 genotype and vertical transmission in Northern Vietnam. Opponent: Birgitta Åsjö, Bergen, Norway. Supervisors: Francesca Chiodi, Phung Dac Cam, Anneka Ehrnst, Anna Nilsson. 345. Bucardo, Filemon, Pediatric rotavirus and norovirus diarrhea in Nicaragua. Opponent: Tomas Bergström, Gothenburg, Sweden. Supervisors: Lennart Svensson, Roland Möllby, Felix Espinoza. 346. Wang, Xiaoda, Characterization of Esche richia coli colonizing the gastrointestinal tract and urinary tract catheters. Opponent: Timo Korhonen, Helsinki, Finland. Supervisors: Ute Römling, Annelie Brauner. 340. Trumstedt, Christian, Surface antigens and virulence in Plasmodium falciparum malaria. Opponent: Alister Craig, Liverpool, UK. Supervisors: Mats Wahlgren, Gerhard Winter. 348. Rotzén Östlund, Maria, Viral respiratory infections: Diagnosis and Epidemiology. Opponent: Anders Widell, Lund, Sweden. Supervisors: Lena Grillner, Tobias Allander, Benita Zweygberg Wirgart. Molecular characterization of Mycobacterium tuberculosis complex from Kampala, Uganda. Opponent: Sebastien Gagneux, London, UK. Supervisors: Gunilla Källenius, Moses Jolaba, Tuija Koivula. 349. Asiimwe, Benon, 2009 350. Cagigi, Alberto, HIV-1 infection and loss of serological memory: the role of altered expression of B-cell chemokine receptors, timing of HAART and impaired antibody affinity maturation. Opponent: Rita Carsetti, Rome, Italy. Supervisors: Anna Nilsson, Francesca Chiodi. 351. Viklund, Ing-Marie, Identification and characterization of WASP and FKBP-like protein. Opponent: Maria Fällman, Umeå, Sweden. Supervisors: Sven Pettersson, Pontus Aspenström. Vaccination against drug-resistant HIV. Opponent: Fabrizio Manca, Genoa, Italy. Supervisors: Maria Isaguliants, Britta Wahren, Annika Karlsson. 352. Boberg, Andreas, 353. Ridderstråle, Karin, Myc oncoproteincofactor interactions; function, regulation and targeting. Opponent: Jonas Nilsson, Umeå, Sweden. Supervisors: Lars-Gunnar Larsson, Ola Söderberg, Natalie von der Lehr. 347. Normark, Johan, 354. Chêne, Arnaud, Impact of malaria on B-cell homeostasis and Epstein-Barr virus reactivation. Endemic Burkitt´s lymphoma pathogenesis. Opponent: Maria Manuel Mota, Lisbon, Portugal. Supervisors: Mats Wahlgren, Maria Teresa Bejarano, Adnane Achour. Signaling pathways controlling bacterial adaption. Opponent: Dieter Haas, Lausanne, Switzerland. Supervisors: Öjar Melefors, Ute Römling. 355. Jonas, Kristina, 356. Vudattu, Nalini Kumar, Alternative splicing of interleukin-7. Opponent: Rhodri Ceredig, Ireland. Supervisors: Markus Maeurer, Maria Masucci. Jenny Sjöberg from MTC receives her Doctoral hat at the Karolinska Institutet Conferment Ceremony Photo: Ulf Sirborn May 8th 2008. 92 Department of Microbiology, Tumor and Cell Biology – MTC Studies on regulatory networks governing virulence gene transcription in Staphylococcus aureus, Opponent: Paul Williams, Nottingham, UK. Supervisors: Patric Nilsson, Staffan Arvidson, Rune Andersson. 357. Gustafsson, Erik, Innate and adaptive immune responses in viral and chronic inflammatory diseases. Opponent: Vincenzo Banaba, Rome, Italy. Supervisors: Louise Berg, Klas Kärre. 358. Schepis, Danika, 359. Magalhaes, Isabelle, CD8α+ T cells and immune memory. Opponent: Pedro Romero, Lausanne, Switzerland. Supervisors: Markus Maeurer, Rigmor Thorstensson. Amoebiasis: Diagnosis and prevalence in León Nicaragua. Opponent: Agneta Aust-Kettis, Uppsala, Sweden. Supervisors: Ewert Linder, Jadwiga Winiecka-Krusnell, Roland Möllby. 360. Leiva, Byron, Dissecting the p53 pathway by means of small molecule-mediated reactivation and computational biology. Opponent: Michael Resnick, Research Triangle Park, USA. Supervisor: Galina Selivanova. 361. Enge, Martin, 362. Vivar, Nancy, Altered T cell homeostasis during HIV-1 infection: Consequences of lymphopenia and chronic T cell activation. Opponent: Franco Lori, Washington, USA. Supervisors: Bence Rethi, Francesca Chiodi. Gut microflora associated characteristics in children with celiac disease. Opponent: Knut Lundin, Oslo, Norway. Supervisors: Elisabeth Norin, Tore Midtvedt, Lars Stenhammar. 363. Tjellström, Bo, Molecular mechanisms of growth suppression by pharmacologically activated p53. Opponent: Jean-Christoph Marine, Gent, Belgium. Supervisors: Galina Selivanova, Joanna Zawacka-Pankau. 364. Hedström, Elisabeth, 365. Mphande, Fingani Annie, Towards understanding the SURFIN protein family and var genes in Plasmodium falciparum. Opponent: Jürgen Kun, Tubingen, Germany. Supervisors: Mats Wahlgren, Fred Kironde, Gerhard Winter. 366. Kis, Loránd Levente, The role of the micro- environment on the regulation of EpsteinBarr virus latent gene expression. Opponent: Lawrence S. Young, Birmingham, UK. Supervisors: Eva Klein, Laszlo Szekely. Display of recently finished doctoral thesis at the entrance of MTC. 367. Huitric, Emma, Drug resistance in M. tuberculosis and the characterization of a new anti-tuberculosis drug candidate. Opponent: Stewart Cole, Lausanne, Switzerland. Supervisors: Sven Hoffner, Dan Andersson, Koen Andries. 368. Gudmundsdotter, Lindvi, HIV-1 immune responses induced by natural infection or immunisation. Opponent: Harriet Robinson, Atlanta, USA. Supervisors: Britta Wahren, Frances Gotch, Nesrina Imami, Rigmor Thorstensson. 369. Benno, Peter, Biochemical parameters reflecting the intestinal ecology of healthy adults and alterations of these parameters in patients with ulcerative colitis or rheumatoid arthritis. Opponent: Per Falk, Princeton, USA. Supervisors: Tore Midtvedt, Elisabeth Norin. 370. Näslund Tanja, Viral vectors in innate and adaptive immunity. Opponent: Gerd Sutter, Munich, Germany. Supervisors: Peter Liljeström, Margaret Chen. 371. Negrea, Aurel, Pharmaceutical and mutational interference with virulence of Salmonella enterica serovar Typhimurium. Opponent: Josep Casadesus, Seville, Spain. Supervisor: Mikael Rhen. 372. Ribacke, Ulf, Parasite virulence and disease severity in Plasmodium falciparum malaria. Opponent: Manuel Llinás, Princeton, USA. Supervisors: Mats Wahlgren, Peter Nilsson. 373. Dellacasa Lindberg, Isabel, Dissemination of Toxoplasma gondii to the central nervous system – with special reference to in vivo bioluminescence imaging. Opponent: Geneviève Milon, Paris, France. Supervisors: Antonio Barragan, Mats Wahlgren. 374. Kilewo, Charles, Prevention of mother-to- child transmission of HIV-1 using antiretroviral drugs in Dar es Salaam, Tanzania. Oppo- nent: Thorkild Tylleskär, Bergen, Norway. Supervisors: Gunnel Biberfeld, Fred Mhalu, Andrew Swai. 375. Gaseitsiwe, Simani, Infections with HIV-1 and Mycobacterium tuberculosis; the Role of HLA Class II alleles and HIV-1 phenotypes. Opponent: Sven Britton, Solna, Sweden. Supervisors: Anneka Ehrnst, Markus Meurer, Andrzej Pawlowski, Max Essex. Genetic adaptions of Helicobacter pylori during gastric disease progression. Opponent: Stefan Bereswill, Berlin, Germany. Supervisors: Lars Engstrand, Britta Björkholm. 376. Kling Bäckhed, Helene, 377. Xue, Yuan, Mechanism of pathological angiogenesis in adipose tissue and tumor. Opponent: Paul Trayhurn, Liverpool, UK. Supervisor: Yihai Cao. Genetic content of clinical pneumococcal isolates and its relation to disease outcome. Opponent: Pentti Huovinen, Åbo, Finland. Supervisor: Birgitta Henriques Normark, Eva Morfeldt. 378. Dagerhamn, Jessica, Characterization of host and microbe interactions affect, adherence, clearance and systemic spread of S. pneumoniae. Opponent: Ingileif Jónsdóttir, Reykjavik, Iceland. Supervisors: Birgitta Henriques Normark, Staffan Normark, Barbara Albiger. 379. Dahlberg, Sofia, 380. Muschiol Sandra, Small molecule inhibitors of type III secretion and their effect on Chlamydia development. Opponent: Kenneth Fields, Miami, USA. Supervisors: Birgitta Henriques Normark, Staffan Normark, Agathe Subtil. 381. Nguyen, Thi Viet Ha, Diagnosis and treatment of Helicobacter pylori infection in Vietnamese children. Opponent: Peter Unge, Örebro, Sweden. Supervisors: Marta Granström, Gia Khanh Nguyen, Mikael Sörberg. Department of Microbiology, Tumor and Cell Biology – MTC 93 Core Facilities MTC FACS Facility To the forefront of flow cytometry technology core facility at MTC originates from the Fluorescence Activated Cell Sorting (FACS) unit at the former Tumor Biology Department established in 1983. It is thus the oldest core facility for flow cytometry in the Stockholm area. During recent years flow cytometry technology has developed rapidly. Instruments are now able to analyze large numbers of different fluorochromes simultaneously and in parallel there has been an expansion in the number of fluorescent dyes, directly conjugated antibodies and other reagents. This allows more advanced and accurate analysis of lymphocyte as well as other cell populations. MTC has during 2008–2009 moved to the forefront of flow cytometry technology through the support of the Knut and Alice Wallenberg foundation. We have obtained three new instruments. One advanced 11 color MoFlo XDP cell sorter which allows high-speed, four-way sorting, an LSR II cell analyzer with 18 color detection and a CyAn ADP instrument for 7 color analysis. The flow cytometry The FACS Facility at MTC has customers from more than 50 research groups at MTC and KI as well as external customers. Birgitta Wester operating the LSR-II flow cytometer, while a user is preparing samples. We have also obtained new computers and software for data analysis. To accommodate these new instruments we have expanded the facility. Altogether we now offer two instruments for cell sorting, five instruments for cell analysis and three computers for data analysis. We are now in the process of upgrading our CyAn ADP to 9 colors and equipping it with a plate-loader for automated analysis of samples in 96 well plates. Multicolor flow cytometry allows phenotypic and functional analysis of rare subpopulations of lymphocytes, it is thus possible to identify over 100 subpopulations in human blood. One may also study production of cytokines and degranulation via cell surface-mobilization of a marker for secretory lysosomes in small subpopulations of lymphocytes. The figure shows an example of functional analysis of subsets of mouse natural killer (NK) cells. Maria Johansson A 12-color flow cytometric analysis of cytokines (Mip1a and IFNg) and degranulation (CD107a) on subsets of mouse NK cells upon stimulation of the activating receptor NKp46. Courtesy Petter Brodin. 94 Department of Microbiology, Tumor and Cell Biology – MTC Birgitta Wester Mikael Jondal Core Facilities KI Visualization Facility (KIVIF) Providing researchers with frontline imaging technology Modern imaging technology finally made it possible to directly observe some of the most intricate details of the living material. Today we can visualize such delicate events as the movements of individual macromolecules or different cellular organelles in live cells or the subtle interplay between microbes and their host cells. With the right equipment and approach, biological imaging has become not only an illustrative tool but also a scientific method of its own. KIVIF at MTC was established in January 2004 and was officially inaugurated in November 2005 by KI President Harriet Wallberg Henriksson. The goal of KIVIF is to provide researchers at the Karolinska Institutet (KI) with frontline imaging technology in combination with technical support and imaging-related expert advice within the frame of their own scientific projects. The facility has around two hundred active users every year, mainly from within KI, but some researcher also come from other universities. KIVIF offers a broad range of imaging technologies; from basic fluorescence and phase contrast microscopy to advanced laser confocal live cell imaging. KIVIF also provides assistance with routine histology and sample preparation. The spinning disc confocal microscope in use at the KI Visualization Facility at MTC. Confocal microscopes At present the facility has the following instruments: n Three confocal microscopes: – a Perkin Elmer ERS system with microlens enhanced Nipkow spinning disc and 5 laser line illumination, equipped with a complementary laser engine for pixel precise burning capacity for FRAP, FLIP, acceptor bleaching FRET and photoconversion experiments. – a fully automated Perkin Elmer LCI spinning disc system with programmable XY stage and integrated image capture and on-the-fly image analysis capacity for extended field confocal microscopy, massively parallel filming and high throughput imaging. – a newly obtained Leica TCS SP5 X laser scanning confocal system with a continuous spectrum white laser for imaging of every possible fluorochromes and recombinant fluorescent proteins. n Three conventional fluoresConfocal live cell imaging of Human prostate tumor cells (PC3); cence microscope with cold CCD Z-stack projection at one timepoint. Green: b-actin (cytoskeleton), red: Histone 2A/m Nuclei. cameras for routine imaging. n An atomic force microscope (AFM) for studies of the surface morphology of cells, bacteria, viruses or even individual macromolecules e.g. DNA-protein complexes. n A laser micro-dissection microscope (Palm, Zeiss) for laser catapult capturing of small tissue pieces, individual cells, microbes, and chromosomes for subsequent PCR or proteomics analysis. n An IVIS biophotonic imaging instrument to trace luciferase labeled cells or microbes in live experimental animals. Laszlo Szekely and some of the members of his group have developed new instruments (programmable microscopes for automated screening of 384 well plates), new image analysis computer programs and a series of automated microscopy based assays for highthroughput live cell imaging. Using these assays it is possible to study the effect of different drugs, antibodies, extracellular matrix components on the viability, morphology and mobility of normal and tumor cells as well as interaction with different microbial pathogens, immune effector cells and/or stromal cells in multi-wavelength time lapse assays. Laszlo Szekely Emilie Flaberg Department of Microbiology, Tumor and Cell Biology – MTC 95 Core Facilities MTC Research Facility A cornerstone for research on infection and experimental cancer models As a central Karolinska Institutet (KI) core facility the MTC Research Facility has become a cornerstone for research on infections and experimental cancer models. In addition, experimental mouse models for angiogenesis, atherosclerosis, diabetes, rheumatism and other diseases are of increasing importance and are actively developed and explored. A large collection of immunodeficient, gene knockout, gene transgenic and other special mouse strains are also available for research purposes. The facility is widely used by researchers from MTC as well as researchers from other KI departments. Several external researchers, from other universities and from some private companies, most of them linked to KI, use the facility on a regular basis. In total, more than 70 research groups perform their animal experiments at the MTC Research Facility. Strict routines The facility has separate subunits for breeding, research, quarantine and biocontainment for experiments on infectious diseases. Researchers have full access to the research and biocontainment units after having passed an introduction program at the KI and locally. Breedings of special mouse strains is carried out in the breeding unit to which only a limited number of animal care takers have full access. Import and export of mice is handled via the quarantine/reception unit. Procedures for the daily handling of mice follow the “3 Rs” principle of animal research: “replacement, reduction and refinement” in order to improve research quality and increase animal welfare. Before any animal experimentation can start, all planned experiments have to pass ethical clearance. In an application to the local Ethical Committee, appointed by the government, a detailed scheme for the planned procedures is declared and explained. Researchers working at MTC have around 200 such individual ethical permits. For import and export of mice special routines are followed. This includes evaluation of health status reports and consulting KI’s veterinarians. 1–2 times every year the animal facility is inspected by representatives for the county’s animal welfare unit. Thanks to a strict entrance regulation policy, training sessions and a strict import policy for mice, the number of infections that risk disturbing research have been kept very low, especially in the breeding and research centers. Future development A detailed plan for a major reconstruction of the MTC Research Facility has been prepared to provide an even safer and more efficient handling of animals. This includes the construction of an air shower system for access to the breeding and research units. In addition, new washing machines for cages and flasks will be installed and individually ventilated cages (IVC) will be introduced in all animal rooms. New laboratories specially equipped for studies on infections and cancer development will be available. Hopefully the renovated animal facility will be in operation during 2011–2012. Anders Örn Petter Höglund The MTC Research Facility – some features n A “core facility” for researchers at MTC, KI and external users n High-quality laboratory technical assistance n A Cs-137 irradiation source for irradiation of cells and whole animals (transplantation studies) n Experimental infection models for virology, bacteriology and parasitology n Experimental cancer and angiogenesis models for studies of cancer pathogenesis and new therapies n One of Sweden’s largest collection of genetically modified animals (> 100 strains) n Humanized mice for studying human cells “in vivo” in a mouse model n Live-imaging to follow infections and/ or tumor growth in the living mouse (xenogenic-IVIS) n Visualization facility closely linked to the animal house (KIVIF) n A special unit for research on germfree animals Personnel The Research Facility’s IBL 637 irradiation unit. 96 Department of Microbiology, Tumor and Cell Biology – MTC 7Animal care takers 3Animal technicians 1Head of animal facility 1Compliance manager (Godkänd föreståndare) Core Facilities The BSL-3 laboratory is a KI-core facility and an important platform to develop HIV research at the Karolinska Institutet. HIV-laboratory An efficient platform to perform HIV research is a KI-core facility and an important platform to develop HIV research at the Karolinska Institutet (KI). There are several groups operating within the field of biology, virology and immunology of HIV infection and these groups are located in pre-clinical and clinical environments. More than 150 PhD-theses have been presented on the subject of HIV/AIDS from KI during the last 20 years. Considering the fact that there are so many established research groups at KI and the Karolinska University Hospital devoted to HIV work this core facility will most likely be needed for many years! Due to safety aspects HIV work is conducted at the BSL-3 laboratory level; it is in fact desirable to have a laboratory devoted exclusively to work with known HIV-contaminated material. The major hazard to laboratory personnel working with HIV infected blood and body fluid is contamination of hands and mucous membranes of the eyes, nose and mouth. The MTC-BSL-3 facility is a safe and efficient technical platform for HIV research, for training and capacity building within the frame of international collabo- The BSL-3 laboratory rations existing at the KI and Karolinska University Hospital. The users of this facility are already involved in several international collaborations with countries heavily affected by HIV infection and AIDS. These collaborations are supported by the Swedish International Development Cooperation Agency (Sida), the World Health Organization, the European Commission and the Karolinska International Research and Training Committee (KIRT). In addition to the academic partners, also a few companies use the BSL-3 facility. The users of the BSL-3 facility pay user fees per hour. The fee is meant to cover material, equipment service and the salary of a half time technician. The security rules established to work within the BSL-3 laboratory ensures that the HIV work is conducted safely. The approval to work in the BSL-3 laboratory is periodically reviewed and authorized by the Swedish Work Environment Authority. Admittance to this facility is allowed only following contact with responsible personnel, for teaching of related safety issues and discussion and for practical introduction to the facility. Before admission to the BSL-3 laboratory, all users need to attend a course of three hours. To fresh up the competence, each user attends a two hours course on the simulation of accidents during work with HIV each year. For convenient access, the regulations and rules are summarized in the booklet “Safety regulations at the BSL-3 safety lab at MTC” and are the basis for the work in the HIV laboratory to be followed by all users. In general, the methods used in the BSL-3 facility aim at isolating and characterizing HIV and studying immune responses to this virus. Many of these analyses have relevance to follow the course of HIV pathogenesis, for the development of an HIV vaccine and improved antiviral therapy. The most common methods are: Isolation of HIV from biological specimens; isolation and culture of different cell subpopulations from blood and biological specimens from HIV infected patient; HIV Neutralization assay to evaluate the titers of neutralizing antibodies present in biological fluids of HIV infected patients; HIV susceptibility and resistance to different drugs. Francesca Chiodi Department of Microbiology, Tumor and Cell Biology – MTC 97 Interactions with the Outside World During 2007–2009, MTC had a very active and broad “out-reach” activity including appearance in TV and radio, in the general press, by giving public lectures, by taking part in public theatre and TV-productions and by the writing of popular science books. ”Research in Focus” (Forskning i Fokus) is an evening activity for the general public interested in our research areas and their implications for society and its citizens. It is announced in daily newspapers and on websites. Usually the audience fills a large lecture hall. In the autumn 2007 we had an unusual experience. One of our topics on “Gut feeling – the gut and its microbes” raised a crowd. As a result some 100 persons could not be accommodated and we repeated the event one month later – with the same results! So we had a third evening with the same theme in early 2008 in the largest lecture hall of Karolinska Institutet (KI). These three occasions attracted a crowd of close to 800 persons – a new record for this activity. Another type of activity is the “What is life”-lectures organized together with the Books written by MTCers Magen Peter Benno, Ingemar Ernberg, Claude Marcus, Tore Midtvedt, Roland Möllby, Elisabeth Norin, Torgny Svenberg Meteorer Georg Klein Framtidens farliga smitta Britta Wahren, Patrik Wahren 98 Department of Microbiology, Tumor and Cell Biology – MTC KI Cultural Board. This series is open to the general public and has become an important arena for going beyond our everyday scope in science. During 2007–2009 we had 27 invited speakers including Craig Venter, among the first to sequence the human genome; Peter Gärdenfors, professor of cognitive science and one of Scandinavia’s most notable living philosophers; Svante Pääbo, director at the Max Planck Institute for Evolutionary Anthropology; James Watson, codiscoverer of the DNA structure; Matthias Uhlén, leader of the Human Protein Resource Project; Göran Burenhult, a Swedish archeologist and Edward O Wilson, a socioecologist. From Cirkus Cirkör’s “Inside out”. Photo Mattias Edwall. MTC faculty members also regularly teach natural science classes at the high school level in two schools normal flora of the gut (the “2 kg-gruppen”). outside Stockholm. For some students this In addition, Georg Klein has written a book includes project work with MTC scientists. In about brilliant, eccentric scientists. addition, during every year the students have A number of public appearances have a thematic week together with the KI faculty. been organized at the public libraries of First year was “Medicine through history”, Stockholm. Most noticeable were the three second year “World health” and third year discussion evenings at the Stockholm City “Cancer”. library during spring of 2009 organized together with the Karolinska University Press AB under the thematic name “Research Outstanding cancer scientist important for life”. In connection with the George and Eva Klein Award 2008 a public lecture was given by the outstanding cancer scientist Robert A. WeinCirkus Cirkör berg, Whitehead Institute, Cambridge (US) MTC has been involved in the Cirque Noveau on “How do we deal with the cancer epidemgroup “Cirkus Cirkör” – to provide ideas and ics”. Another public lecture was given by the knowledge in modern biology, which contribP.R. China Minister of Health, former cancer uted to their two shows “99% unknown” 2007 scientist Professor Chen Zhu on the adoption and “Inside-Out” 2009, performed at the of traditional Chinese medicine to modern “House of dance” in Stockholm but also tourmedicine and the coming Chinese health care ing world-wide. reforms which he is launching. During 2008 and 2009 several of our MTC faculty participates actively in the faculty members and students were involved Karolinska University Press AB popular book in the Lennart Nilsson-Mikke Agaton producproduction, including co-authorships in the tion “A journey to the core of life”, a 2 hour TV following books (titles translated): Prostate production which was shown in 2010. IngeCancer, Breast Cancer, Dangerous infecmar Ernberg was the scientific advisor of this tions of the future, The gut and its bacteria project. (”Magen- bakterier, buller och brak”). The Ingemar Ernberg, chairman latter was initiated from MTC projects on the of the KI Cultural Board Professor Robert A Weinberg and HRH Crown Princess Victoria together with Eva and Georg Klein. Photo: Ulf Sirbjorn Professor Robert A Weinberg received the Visiting Professor Award from the hands of HRH Crown Princess Victoria. Photo: Ulf Sirbjorn Georg and Eva Klein Foundation In support of innovative research The “Georg and Eva Klein Foundation” The board of the foundation has eight members representing both science and society. The board convenes 4–5 times per year and had the following composition during 2009: (GEKS) was established in 2005 initiated by researchers from MTC in order to celebrate the 80th birthdays of Georg and Eva Klein, the founders of the Department of Tumor Biology at the Karolinska Institutet in 1960. Ebba Lindsö, Entrepreneur Georg and Eva’s ground-breaking reCarl Fredrik Sammeli – Founder, Prime search, from the ’60s until today, contributed Group a number of very important scientific findAlf Svensson – Member of the EU parliament ings in the areas of tumor immunology and Magnus Uggla – Executive Vice President, tumor biology. To mention a few, the capacHandelsbanken ity of the immune system to recognize and Marie Arsenian Henriksson – Head of MTC react against certain tumors, the role of EBV Ingemar Ernberg – Professor in lymphoid malignancies, the importance Klas Kärre – Professor of chromosomal translocations for tumor Anders Örn – Professor (Chairman) development, identification of tumor suppressor genes and important aspects of EBVhost interactions for viral latency and tumor Those who have and have had the privilege to development. be trained and to mature scientifically under The main objective of the foundation the auspices of Georg and Eva Klein often talk is “to support biomediabout the unique culture created cal research in the spirit in their laboratories. To capture of Georg and Eva Klein.” the “Klein spirit” in words is not Funds are collected in oreasy. One important factor is the der to support research actual mentoring process that on cancer and cell biology, is performed in a very personal immunology, genetics and manner, where curiosity is an microbiology. The focus is important driving force. www.gekleinfoundation.org on: Another success factor is to let the most junior students explain n Basic research with the potential to deand discuss their research projects directly with very experienced visiting scientists. This velop knowledge that can lead to medical exposure fosters confidence and triggers a applications n Knowledge sharing through international curiosity-driven attitude to science. The spirit of learning by doing is also scientific exchange and networking n Science with a humanistic perspective important where trial and error(s) are neces- sary components. The “Klein spirit” also tries to support researchers whose projects contain more than data collection and potentially can lead to synergy between disparate fields of science. World famous research In 2008 two scientific prizes were awarded by the foundation. The “Visiting Professor Award” was given to Robert A Weinberg working at MIT, Cambridge, USA. His world famous research is focused on the genetic basis of cancer development and metastasis. In addition to his research achievements Robert Weinberg has written a popular textbook with the title “The Biology of Cancer”. During a hectic week in Sweden he gave a most appreciated series of lectures for researchers and students. In addition, he gave an outstanding presentation on “The future of cancer – research and treatment” for a layman audience. Dr Weinberg also held several highly interactive seminars with junior scientists on various themes in cancer biology. Dr Theresa Vincent received the “Junior Scientist Award” at the same occasion for her excellent research on the effect of cell signaling on tumor cell behavior. This stipend partly supported her research at the Cornell University in New York. The actual prize ceremony was held at the Nobel Forum, KI in the presence of HRH Crown Princess Victoria as well as many researchers and some of the generous donors to the foundation. Anders Örn Department of Microbiology, Tumor and Cell Biology – MTC 99 MTC Organization MTC has a flat organization with around 35 research groups, each led by a group leader, a professor or a senior researcher. The group leader is responsible not only for the scientific work but also for the economy and the staff in the group. Marie Arsenian Henriksson has been the Head of the Department since October 2006. She has since then been appointed by the KI President for a second three-year period. The Head of Department has assigned a management group consisting of the two vice chairmen, the head of administration (AC) and four group leaders. Professor Staffan Normark and Professor Klas Kärre are vice chairmen and Åsa Edström has been the head of Administration since August 2008. Joint Influence Group Equality and Diversity Group Department Council Future Faculty Management Group during 2009 Work Environment Group Marie Arsenian Henriksson (Chair) Åsa Edström Annelie Brauner Klas Kärre (Vice Chair) Staffan Normark (Vice Chair) Francesca Chiodi Gunilla Karlsson-Hedestam Pontus Aspenström Faculty Advisory bodies Importantly, there are a number of advisory bodies, including the Department Council, the Faculty (the group leaders), the Post Graduate Research Committee, the Work Environment Group, the Equality and Diversity Group and the Undergraduate Council. The Department also has a Joint Influence Group, a Future Faculty, and a Student Council. In addition, there is an external Scientific Advisory Board (see box), which interacts with the management of MTC to discuss issues of importance for the long-term development of the department. Support for the research groups The main focus of the Administration and The Service Unit is to support the research groups when it comes to economy, staff administration and different service functions. In order to streamline the service and infrastructure at MTC previous units for service, IT and shops for laboratory material were merged into one, the Service Unit, during 2009 . A head for this new unit was subsequently recruited, Petra Hartley. The department also has several core facilities Including KIVIF, the FACS facility, the Research Facility and the HIV laboratory supporting the ongoing science. Åsa Edström 100 Department of Microbiology, Tumor and Cell Biology – MTC Third Level (Postgraduate) Research Committee Student Council Second Level (Undergraduate) Research Committee Scientific Advisory Board Xin Lu, professor and director, Ludwig Institute for Cancer Research, Oxford, Great Britain. Peter H. Krammer, professor and Head of the Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany. Olle Stendahl, professor, Linköping University, Linköping, Sweden. MTC Staff Technical staff Professor Xin Lu. Female Male Total 39 11 50 Administrative staff 18 1 19 Postgraduate students 90 55 145 Postdocs 20 13 33 4 6 10 11 9 20 Research assistants Researchers Senior lecturers 1 1 2 Professors 4 16 20 187 112 299 TOTAL Economy Result (in kSEK) Income of governmental grants Income of fees Income of external grants Internal incomes 2009 2008 2007 2006 63 735 56 735 55 329 58 613 4 786 4 989 5 399 6 737 110 714 102 258 101 330 103 804 12 986 8 481 21 113 18 900 SUM OF INCOMES 192 221 172 463 183 171 188 054 Personnel costs -111 246 -101 151 -99 537 -102 802 Premises costs -32 434 -31 178 -30 964 -31 509 Operational costs -47 600 -38 786 -38 200 -34 237 -3 714 -3 215 -3 639 -3 867 -194 994 -174 330 -172 340 -172 415 Financial incomes 216 1 895 1 179 396 Financial costs -79 -59 -49 -37 FINANCIAL NET 137 1 836 1 130 359 Change of capital -2 636 -31 11 961 15 998 Depreciation SUM OF COSTS Development of MTC incomes Income according to contributor in 2009 kSEK 200 Foundations and organizations 150 Internal income Foreign research grants Other govermental contributions 100 KI govermental grants 50 National research councils 0 94 95 96 97 98 99 00 01 02 03 04 05 06 07 08 09 Sum of incomes Internal incomes Income of contributions Income of fees Income of governmental grants Department of Microbiology, Tumor and Cell Biology – MTC 101 Contributors and List of Financiers We would like to thank our financiers for their valuable support to our research! The eight major external contributors 2007 The eight major external contributors 2009 ksek 21 649 Swedish Research Council (VR) 40 390 European Union (EU) 15 214 Swedish Cancer Society (CF) 19 613 Swedish Cancer Society (CF) 15 144 European Union (EU) 16 872 10 531 Swedish Foundation for Strategic Research (SSF) 11 500 Swedish Institute for Infectious Disease Control (SMI) Swedish Institute for Infectious Disease Control (SMI) 10 580 The Swedish Agency for Innovation Systems (VINNOVA) 4 207 3 500 The Swedish International Development Cooperation Agency (Sida) 3 981 The Swedish Childhood Cancer Foundation (BCF) 3 397 The Swedish Childhood Cancer Foundation (BCF) 3 226 Torsten and Ragnar Söderberg Foundation (TRSS) 2 432 Torsten and Ragnar Söderberg Foundation (TRSS) 3 000 The Swedish International Development Cooperation Agency (Sida) – – – – – – – – – – – – – Accuro AB Actar AB Active Biotech AB AFA Insurance Affibody Biotechnology AB Åke Wiberg Foundation Alex and Eva Wallström Foundation Animal Protection Agency Aprea AB Arla AB AstraZeneca AB Athera Biotechnologies AB Aventis Pasteur SA – – – – – Bill & Melinda Gates Foundation Binex Company BioInvent International AB Biostapro AB Biovitrum AB – Cancer and Allergy Fund – Cancer Association in Stockholm – Cancer Research Funds of Radiumhemmet – Cancer Research Institute – Capio Research Foundation – Cardoz AB – Carl Trygger Foundation – Cederberg Foundation – Clas Groschinsky Memory Foundation – Clinical Gene Networks AB – DAKO AB – Diamyd Therapeutics AB – Dilafor AB – Ekhaga Foundation – Euro-Dignostica AB – European Union (EU) – – – – – 102 ksek Swedish Research Council (VR) German Cystic Fibrosis Foundation Glaxo AB Golje Memorial Fund Göran Gustavsson Foundation Harvard University – Henning and Ida Persson Research Foundation – High Q Foundation – Human Frontier Science Program – – – – – – IMED AB Immune Tolerance Network Index Pharmaceutical AB Independent Pharmaceutica AB Ingabritt och Arne Lundberg Research Foundation Innogene-Kalbiotech Company INTAS International Association International AIDS Vaccine Initiative IPULS AB – – – – Jeansson Foundation Jerring Foundation Jochnick Foundation Julin Foundation – – – – Karo Bio AB – Karolinska Institutet Foundations and Funds – Knut and Alice Wallenberg Foundation – King Gustaf V Jubilee Foundation – Knowledge Foundation (KK) – Knut and Alice Wallenberg Foundation – Konsul TH C Berghs Foundation – – – – – Länsförsäkringar Alliance Lars Hierta Memory Foundation LipoPeptide AB LTB4 Sweden AB Ludwig Institute for Cancer Research – Lundberg Research Foundation – – – – – – Mabtech AB Magn. Bergwalls Foundation Mälardalen University Mary Beves Foundation Maths O Sundqvist Foundation Max-Planck Institute Department of Microbiology, Tumor and Cell Biology – MTC – Nancy Lurie Marks Family Foundation – Nanna Svartz Fund – National Association for Kidney Diseases – National Board of Health and Welfare Foundation – Norwegian Institute of Public Health – National Institutes of Health (NIH) – Nobel Foundation – NOLabs AB – NordVacc AB – Novo Nordisk AB – O.E. & Edla Johansson Veterinary Foundation – Olle Engkvist Foundation – Oncoreg AB – Petrus and Augusta Hedlund Foundation – PhPlate Microplate Techniques AB – Physicians against AIDS Research Fund – Q-Med AB – Robert Lundberg Memorial Fund – Royal Institute of Technology (KTH) – Royal Swedish Academy of Sciences (KVA) – – – – – – – – – – S*Bio Ltd Scandinavian Clinical Nutrition AB Scripp Research Institute Sigurd and Elsa Goljes Foundation Swedish Agency for Innovation Systems (VINNOVA) Swedish Board of Agriculture Swedish Cancer Society (CF) Swedish Childhood Cancer Foundation (BCF) Swedish Diabetes Association Swedish Foundation for International Cooperation in Research and Higher Education (STINT) – Swedish Foundation for Strategic Research (SSF) – Swedish Heart-Lung Foundation – Swedish Institute (SI) – Swedish Institute for Infectious Disease Control (SMI) – Swedish International Development Agency (Sida) – Swedish Medical Association (SLS) – Swedish National Board of Forensic Medicin – Swedish Nutritional Research Foundation – Swedish Orphan AB – Swedish Research Council (VR) – Swedish Research Council FORMAS – Swedish Rheumatism Association – Swedish Society for Medical Research (SSMF) – Swedish Society of Medicine – Swedish University of Agricultural Sciences – Thymed Company – Tobias Foundation – Torsten och Ragnar Söderberg Foundation (TRSS) – – – – – Unimed Innovation Company United Way International University of Bergen University of California University of Rome, Tor Vergata – Vaccine Research International Plc – Virtual Genetics AB – Viscogel AB – WennerGren Foundation – World Health Organization (WHO) Location of MTC Picture: John Sennett Mailing Address Visiting Address Phone Website MTC, Karolinska Institutet FE 280 SE-171 77 Stockholm Sweden Nobels väg 16 or Theorells väg 1 KI Campus, Solna +46 (8) 524 8 66 78 http://ki.se/mtc Clinical Microbiology Laboratory building L2:02 Karolinska University Hospital, Solna SE-171 76 Stockholm Sweden Karolinska University Hospital, Solna Sjukhusringen 13 Laboratory building L2:02 +46 (8) 517 735 88 Visitors to MTC From Arlanda Airport take the airport coach to Sankt Eriksplan and bus 3, 77 or 507 to bus stop “Karolinska Institutet” (4th stop). Walk across the campus to Nobels väg. From the Central Train Station take bus 47 to Tomteboda (end stop). Walk across the campus via Berzelius väg and Nanna Svartz väg to Nobels väg. By car from E4 North: follow the signs to Karolinska University Hospital (Karolinska sjukhuset). Take exit 165 and turn immediately left. Follow Karolinska vägen until it becomes Tomtebodavägen. Take then the first street to the left and find Nobels väg 16 on the left after 50 meters. From the city: follow Torsgatan and Solnavägen and turn left to Tomtebodavägen. By car from E4 South: Take the right lane towards S:t Eriksplan. Continue onto Norrtull. Take the right lane at the Norrtull exit and turn right onto the bridge to Solna. Continue for about 600 metres and turn left into Tomtebodavägen and then left into Nobels väg. Department of Microbiology, Tumor and Cell Biology – MTC 103 Fungal cellulase binds to Acanthamoeba cell wall. Artistic interpretation by Ewert Linder adapted from: Appl Environ Microbiol. 75, 6827-30, 2009.
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