symposium of molecular medicine - programme

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29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine Introduction

During the last decade, we have witnessed a true success story regarding the degree programmes of Molecular Medicine, Biomedicine, and Human Biology in Germany. Not only have these degree programmes accomplished to break through many of the boundaries which traditionally separate medical and science studies, they have also succeeded in attracting many highly gifted, young people to this exciting and fast growing field of science. Year by year, the number of gymnasium graduates applying dramatically exceeds the number of university admissions to the studies of Molecular Medicine, e.g. in Erlangen by a factor of >20. As evident from the excessive number of applications turned down, more needs to be done: In particular, the restrictions imposed by the administrative courts on Molecular Medicine programmes should urgently be reconsidered.

The Annual Meetings of the Study Group of Molecular Medicine, initiated in 2006 by student members of the German Society for Biochemistry and Molecular Biology (GBM, Gesellschaft für Biochemie und Molekularbiologie), are something special: Now in their fourth year, these meetings bring together students from degree programmes of Molecular Medicine, Biomedicine, and Human Biology, to attend scientific presentations from internationally leading scientists. Just as these programs have succeeded in positively interfacing the medical and science studies, the annual meetings on Molecular Medicine also provide a platform to break any barriers between established scientific meetings and educational presentations. Organized by student hosts of the Erlangen programme in Molecular Medicine, the 2009 meeting again offers an exciting scientific programme, this time focussing on ‘Translational Approaches to Molecular Medicine‘. This theme closely reflects the scientific characteristics of Molecular Medicine as a strong link between molecular research and clinical practice. I am confident that this year‘s programme will again attract the interest of participants from all of the German programmes in Molecular Medicine and Biomedical Science. As in the years before, I am also confident that during this meeting many new contacts will be made that will prove fruitful for your further studies.

Welcome to Erlangen!

Cord-Michael BeckerCoordinator, Programme of Molecular Medicine at University of Erlangen-Nürnberg;Speaker, Study Group of Molecular Medicine of the German Society for Biochemistry and Molecular Biology

Molecular Medicine: Breaking barriers!

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29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine Table of contents

1 Introduction

2 Table of contents

3-4 Programme overview

5 Keynote Lecture

6 Commemorating Lecture

7-9 Neurosciences session

10-12 Regulation of Transcription session

13-15 Pain session

16-18 Innovative Therapies session

19-21 Virology session

22-24 Immunology session

25-27 Imaging session

28 Map of Erlangen

29 Organization Team

30 Notices

Page Content

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Programme

29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine

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Programme

29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine

Notes...

“Nature is always the same, and yet its appearance is always changing. It is our business as artists to convey the thrill of nature's permanence a l o n g w i t h t h e elements and the appearances of all its changes

Paul Cezanne(ca. 1900)

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Keynote Lecture

18:45 - 19:30

Thursday29 Oct 2009

Mesangioblasts for the cell therapy of muscular dystrophy

Giulio Cossu – Milan

Mesoangioblasts are recently characterized progenitor cells, associated with the vasculature and able to differentiate in different types of solid mesoderm, including skeletal muscle. When mesoangioblasts were delivered intra-arterially to dystrophic muscle of α-sarcoglycan KO mice or Golden Retriever dogs they resulted in a significant functional amelioration of the dystrophic phenotype. Human adult mesoangioblasts were isolated and expanded in vitro from muscle biopsies and shown to correspond to a subset of pericytes.

Based on these results, a monocentre, prospective, non-randomised, open-label phase I study of cell therapy with HLA-matched donor human mesoangioblasts in DMD patients will start in June 2009, with a one year preliminary study (involving 30 DMD patients, aged 5-10), required to validate outcome measures. Three out of these patients will undergo successive transplantations at escalating doses of cells under a continuous regime of immune suppression. Safety will be the primary objective of the study. However it is expected that transplantation of mesoangioblasts will result in a detectable increase in muscle strength and consequent clinical amelioration or stabilization.

Problems still facing this approach and possible strategies to overcome them will be discussed.

This work was supported by grants from the European Community, Duchenne Parent Project, MDA, AFM and the Italian Ministry of Research.

Chair: Stephan Ellmann

Notes...

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Notes...

Commemorating Lecture

Human T-cell leukemia virus type 1 (HTLV-1) was first discovered in 1980. This retrovirus is the causative agent of an aggressive T-cell leukemia named Adult T-cell leukemia (ATL). To date the mechanism of how HTLV-1 transforms T-cells is not fully understood; however, it is accepted that the virus encodes an oncogenic protein, Tax. In this presentation, I will review some of Ralph Graßmann’s contributions to HTLV-1 research and update our current insights into the transforming routes employed by Tax to cause check point abrogation, DNA damage, and the creation of genomic instability.

19:30 - 20:00

Thursday29 Oct 2009

Evening

HTLV-1 research after three decades: Insights into cellular transformation mechanisms for ATL (adult T-cell leukemia)

Kuan-Teh Jeang – NIH, Bethesda in memoriam Prof. R. Graßmann (1957 - 2008)

Chair: Stephan Ellmann

Discover Erlangen's Clubs, Bars and Pubs and join our Pub Crawl!

“Do not quench your inspi rat ion and your imag ina t i on ; do no t become the slave of your model.

Vincent Van Gogh

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Neurosciences

The spontaneous mouse mutant oscillator (spdot) with a deletion in the a1 subunit of the glycine receptor is lethal around postnatal day 21. Lethality is due to a 7 bp frameshift deletion resulting in two variants of the a1 protein in oscillator mice, one truncated a1 polypeptide and a long a1 variant with a missense C-terminal part. Both proteins lack the glycine receptor amino acid sequence information between transmembrane domains 3 and 4, transmembrane domain 4, and the extracellular C-terminus. We could show that coexpression of the lacking part, called the “tail” domain, together with truncated a1 protein resulted in a rescue of ion channel function. We identified residues and domains within the loop between transmembrane domains 3 and 4, which are important for rescue efficiency to reach almost wildtype levels. Using spinal cord neuronal cultures of homozygous oscillator mice as an in vivo model, we could demonstrate the rescue of the glycine receptor a1 expression by a significant increase at the transcript as well as the protein level of spdot-trc in vivo. Here, the oscillator rescue of GlyRa1 depends only on the missing C-terminal tail domain that is lost due to the oscillator deletion. Our data strikingly pinpoint to the importance of the TM3-4 loop for biosynthesis, clustering, and pharmacology of the GlyR and give implications for novel directions concerning gene-therapeutical signs.

8:30 - 10:45

Friday30 Oct 2009

Glycine receptor domains and their importance for protein functionality en route to molecular therapy

Carmen Villmann – Erlangen

Chair: Martin Eberhardt

Notes...

“Biology points out the individuality of every being, and at t h e s a m e t i m e reminds us of the brotherhood of all.

Jean Rostand (1962)

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Neurosciences

Neuromodulators are a highly divers group of signaling molecules that regulate properties of neuronal synapses. Neuropeptides such as endogenous opioid or tachykinin peptides are stored in large dense core vesicles and are released from synaptic terminals together with the primary neurotransmitter. Endocannabinoids represent retrograde signaling molecules that are synthesized at postsynaptic sites in an activity-dependent manner and that act on presynaptic receptors. Some neuromodulators, such as D-serine are produced by astrocytes. Our laboratory is using genetic mouse models to study the role of these neuromodulators in animal models of psychiatric disorders, with a special emphasis on drug addiction, stress-related disorders, nociception and schizophrenia. These mouse models enable us to investigate, step-by-step the molecular, cellular and neuronal mechanisms that connect a gene to a specific behavioral phenotype. I will present examples of mice that cannot produce certain endogenous opioid peptides, or are deficient in cannabinoid receptors. These animals have striking behavioral and physiological deficits in their responses to drugs of abuse and in their response to stressful or painful stimuli. Elucidation of the pathways leading to these deficits will enhance our understanding of the etiology and pathomechanisms of psychiatric disorders.

8:30 - 10:45

Friday30 Oct 2009

Neuromodulators as regulators of mouse behavior

Andreas Zimmer - Bonn


Notes...

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Neurosciences

Increased activity of acid sphingomyelinase (ASM) is present in a variety of neuronal diseases, like Alzheimer`s disease, seizure disorder, Parkinson`s disease, chronic alcohol dependence and major depression. For endotoxic shock syndrome and cystic fibrosis, pathologic participation of ASM was already confirmed and could be reduced by ASM inhibition. The pathologic consequences of an increased ASM activity are described within the “rheostat concept”, which characterizes the bioactive properties of ceramide and sphingosine-1-phosphate as proapoptotic and proproliferative, respectively.

Since increased ASM activity results in an elevation of ceramide concentrations, ASM activation induces apoptosis upon cellular stress. To further validate the importance of this lysosomal glycoprotein within the etiopathology of neuronal alterations, we established a cell culture based assay to identify ASM regulation by antidepressants and other commonly used drugs.

This study revealed, that potent ASM inhibitors are significantly enriched in three therapeutic groups, namely: The group of psycholeptics, psychoanaleptics and the group of antihistamines for systemic use. Furthermore it is obvious, that substances capable of ASM inhibition are blood-brain barrier permeable.

8:30 - 10:45

Friday30 Oct 2009

Antidepressant drugs adjust dysregulated phospholipid metabolism

Philipp Tripal - Erlangen


Notes...

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Regulation of transcription

Connective tissue growth factor (CTGF, CCN2) is a secreted multifunctional protein. The biological activity of CTGF is regulated both at the level of intracellular signaling leading to gene expression and by its extracellular interaction partners, which determine the functional outcome of CTGF action. Multiple signaling pathways have been related to CTGF gene expression, some of which, such as transforming growth factor (TGF)beta – Smad signaling, seem to work in different cell types, or are shared by multiple stimuli, such as activation of RhoA - Rho-kinase signaling. Other pathways leading to CTGF induction seem to be rather specifically detected in a restricted number of cell types. This presentation will concentrate on the impact of the activation of one of the major signaling pathways, which is operative in all cells, namely phosphatidylinositol-3 kinase (PI3K) – AKT signaling on CTGF gene expression.

In smooth muscle cells, fibroblasts, and epithelial cells, inhibition of this pathway either reduced CTGF expression or was not involved in CTGF gene expression depending on the stimulus used. In endothelial cells by contrast, activation of PI3K – AKT signaling was inversely related to CTGF expression: Inhibition of PI3K – AKT signaling was sufficient to increase CTGF expression in resting cells and further enhanced CTGF synthesis in cells treated by a variety of stimuli including TGFbeta, hypoxia or shear stress. In different types of endothelial cells, FoxO transcription factors, which are negatively regulated by AKT, were identified as potent activators of CTGF gene expression.

Our data indicate that FoxO proteins are key regulators of CTGF gene expression which determine the effect of PI3K – AKT activation in terms of CTGF regulation in different cell types.

10:45 - 12:45

Friday30 Oct 2009

Cell type-dependent regulation of Connective Tissue Growth Factor gene expression

Margarethe Goppelt-Strübe – Erlangen

Chair: Manuela Hugle

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Notes...


Nuclear domains 10 (ND10), alternatively termed PML nuclear bodies (PML-NBs) or PML oncogenic domains (PODs), have been discovered approximately 15 years ago as a nuclear substructure that is targeted by a variety of viruses belonging to different viral families. We have shown that replication of human cytomegalovirus is considerably enhanced in cells depleted of individual ND10 components suggesting that the respective proteins are able to silence viral gene expression. However, this silencing is counteracted by specific viral effector proteins: while the structural protein pp71 is able to degrade hDaxx in a proteasome-dependent manner, ND10 accumulations are disrupted by the action of the regulatory protein IE1.

Thus, accumulating evidence argues for an involvement of ND10 in host antiviral defenses either via mediating an intrinsic immune response against specific viruses or via acting as a component of the cellular interferon pathway.

10:45 - 12:45

Friday30 Oct 2009

Interplay between human cytomegalovirus infection and host defense by PML nuclear bodies

Thomas Stamminger – Erlangen

Chair: Manuela Hugle“And the end of all our exploring will be to arrive where we started and know the place for the first time.

T.S. Eliot (1942)

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Proper myelination of nerves is essential for rapid saltatory conductance throughout the vertebrate nervous system and relies on the presence of myelinating glia cells. In peripheral nerves, myelination is carried out by neural crest derived Schwann cells. During development, neural crest cells become specified to Schwann cell precursors that progress through several consecutive stages and as myelinating Schwann cells finally undergo terminal differentiation by establishing a one-to-one relationship with axons and myelinating the axonal fibres.

The High-Mobility-Group-domain-containing transcription factor Sox10 is expressed in all migrating neural crest cells. In mice that carry a spontaneous or a targeted mutation of Sox10, Schwann cell precursors are never formed in development. This confirms a Sox10 requirement at early stages, but also makes it impossible to study additional functions of Sox10 at stages of Schwann cell development during which Sox10 is still expressed.

In order to investigate Sox10 function at these later stages we generated mice that selectively lost Sox10 in the Schwann cell lineage during two distinct time points of development. The use of a Cre-recombinase under control of Desert Hedgehog regulatory sequences allows Sox10 deletion which starts in Schwann cell precursors. To investigate Sox10 function during Schwann cell terminal differentiation we employed a Krox20::Cre-recombinase mediated approach.

Mice carrying the Desert Hedgehog::Cre-mediated Sox10-deletion show a severe neuropathologic phenotype of the PNS limiting life span to a maximum of 6 weeks. Schwann cell development in these mice is arrested at the immature Schwann cell stage with major differences in the expression pattern of characteristic Schwann cell markers and a total lack of myelination.

Sox10-deletion during Schwann cell terminal differentiation using a Krox20::Cre-mediated approach results in a milder phenotype with remaining expression of most Schwann cell markers. Strikingly, ultrastructural analysis revealed a significant reduction of the numbers of myelinated axons and the extent of myelination although the animals are viable beyond 4 months of age.

The mouse models thus prove for the first time the continuous significant impact of Sox10 function throughout Schwann cell development and are excellent models for the investigation of Schwann cell-based peripheral neuropathies in human patients.

10:45 - 12:45

Friday30 Oct 2009

The transcription factor Sox10 is required for multiple steps in Schwann cell development

Markus Finzsch – Erlangen

Chair: Manuela Hugle / Cord-Michael Becker

12:45 – 14:00: Lunch Break13:15 – 14:00: GBM Meeting of the study group „Molecular Medicine“

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Pain

Persistent postoperative pain has an incidence of up to 50%, depending on the type and extent of surgery, and is linked to an increased risk for the development of chronic pain. Continuous inflammatory processes or accidental or hazarded intraoperative nerve injuries contribute to the pathobiology of persistent pain. Risk factors for persistent pain are pre-existing pain, repeated surgery and severe postoperative pain. Largely unknown is the influence of intra- and postoperative applied anesthetics and analgesic drugs, genetic factors, and psychological susceptibility.

This talk will focus on the interaction of anesthetic agents with the nociceptive system, in particular with the TRP channel family. In addition to their clinically known action, local anesthetics (LA) and other anesthetic agents can exhibit pro-nociceptive effects by interaction with members of the TRP channel family. LA activate TRPV1 and, to a lesser extent, TRPA1. LA also induce a TRPV1-dependent release of CGRP. LA sensitivity of TRPV1 requires segments of the putative vanilloid-binding domain and involves [PI(4,5)P2] signalling. TRPV1 and TRPA1 may serve as putative key elements of anesthetic-induced nociceptor excitation and may play a role in anesthetic-induced neurotoxicity. The modulation of nociception by anesthetic agents is subject of current studies.

If these studies are able to substantiate long-lasting side effects of anesthetics on the nociceptive system that might contribute to persistent pain, then TRPV1 and/or TRPA1 antagonists might prove to be powerful analgesic adjuncts for the prevention and treatment of post-operative pain.

14:00 - 16:15

Friday30 Oct 2009

Determinants and modulators of postoperative pain

Carla Nau – Erlangen

Chair: Carl-Philipp Hackstein

Notes...

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Pain

Maladaptive plastic changes in the spinal dorsal horn contribute critically to the development and maintenance of chronic pain syndromes. Besides long-term potentiation-like increases in synaptic transmission between primary afferent nociceptors and spinal projection neurons, diminished synaptic inhibition has been recognized as a major contributing factor in pathological pain. Peripheral inflammation, nerve injury and intense input from primary nociceptive C fibers lead to diminished synaptic inhibition in the spinal dorsal horn and, as a consequence, to increased pain sensitivity. Conversely, compounds facilitating synaptic inhibition in the spinal dorsal horn can normalize pathological pain states (Knabl et al., 2008).

Recent research has shed light on several distinct molecular pathways which are triggered by inflammation, nerve damage or intense nociceptor activation and which lead to diminished GABAergic or glycinergic synaptic inhibition (Harvey et al., 2004; Coull et al., 2005; Pernia-Andrade et al., 2009). This presentation will review these recent findings.

References:●Pernía-Andrade AJ et al. (2009): Spinal endocannabinoids and CB1 receptors mediate C

fiber-induced heterosynaptic pain plasticity. Science 325, 760-764.●Knabl J et al. (2008): Reversal of pathological pain through specific spinal GABAA

receptor subtypes. Nature 451, 330-334.●Coull JA et al. (2003): Trans-synaptic shift in anion gradient in spinal lamina I neurons as a

mechanism of neuropathic pain. Nature 424:938-942.●Harvey RJ et al. (2004): GlyRα3: An essential target for spinal PGE2 mediated

inflammatory pain sensitization. Science 304, 884-887.

14:00 - 16:15

Friday30 Oct 2009

Mechanisms of spinal pain sensitization

Hanns Ulrich Zeilhofer – Zürich


Notes...

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Pain

Chronic pain represents a severe, debilitating condition. Although up to 10% of the world-wide population is affected, current treatment strategies are often inadequate. Voltage-gated sodium channels (VGSC) are essential for the fast upstroke of the action potential and therefore play a crucial role in transmitting information along nerve fibers from the periphery into the central nervous system. Recently, loss of function mutations in the human VGSC Nav1.7, which is predominantly expressed in nociceptors, has been linked to complete insensitivity to pain[1]. Vice versa, gain-of-function mutations in Nav1.7 lead to inherited autosomal dominant chronic neuropathic pain syndromes, namely erythromelalgia (IEM) or paroxysmal extreme pain disorder (PEPD)[2].

In my talk I will focus on the biophysical changes induced by naturally occurring mutations in Nav1.7. Although IEM and PEPD mutations both provoke a gain-of-function, patients suffer from quite distinct clinical symptoms and therapeutically PEPD seems to be easier to treat than IEM. The use of 3D computer modeling, molecular biological and electrophysiological methods helps us to understand the structural changes that VGSCs need to undergo during gating. The combined knowledge of clinical symptoms and underlying biophysical changes might give a better basis for phenotype and protein structure directed drug design.

1. Cox, J.J., et al., An SCN9A channelopathy causes congenital inability to experience pain. Nature, 2006. 444(7121): p. 894-8.

2. Dib-Hajj, S.D., Y. Yang, and S.G. Waxman, Genetics and molecular pathophysiology of Na(v)1.7-related pain syndromes. Adv Genet, 2008. 63: p. 85-110.

14:00 - 16:15

Friday30 Oct 2009

Gain-of-function mutations in sodium channel Nav1.7: From molecule to pain

Angelika Lampert – Erlangen


16:15 – 16:45: Coffee Break

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Innovative Therapies

Any increase in heart rate increases the cardiac work load per time and as a consequence myocardial oxygen demand. In addition, heart rate is together with stroke volume the crucial parameter contributing to cardiac output thereby exhibiting a fundamental impact on overall cardiovascular physiology. In humans data obtained in the general population and in clinical trials indicate that an increased heart rate is strongly associated not only with a higher cardiovascular mortality and morbidity but also with increased all-cause mortality. Consequently, heart rate lowering by pharmacologic treatment has been a long term goal in cardiovascular medicine. In this regard, drugs that block adrenergic β-receptors, i.e. β-blockers, have been predominantly used as heart rate lowering agents in the past. In the cardiovascular system β-blockers have additional negative inotropic and negative chronotropic actions and they reduce blood pressure as well. These additional cardiovascular mechanisms together with other non-cardiovascular negative effects are unfavorable in some patients and very often limit therefore the use of beta-blockers. In this regard an important observation has been made already 30 years ago when the “funny” pacemaker, i.e. If current, was first described in cardiac myocytes of the sinoatrial node. The term “funny” was used for this current because it demonstrated some unusual features, that is If represents a mixed inward current for both sodium and potassium and is activated on hyperpolarization in the diastolic range of voltages. The funny channels are activated by intracellular cAMP concentrations according to a mechanism mediating regulation of heart rate by the sympathetic and parasympathetic autonomic nervous system. Given its crucial function in pacemaking If became a hot target in the search for drugs aimed specifically at affecting heart rate. Currently, the first and so far only selective heart rate lowering agent available for clinical use is ivabradine. Ivabradine is a specific inhibitor of the pacemaker If current in the sinus node, without altering action potential duration or causing negative inotropy. Clinical studies have already clearly demonstrated the antianginal and antieschemic properties of ivabradine in patients with stable angina pectoris. Moreover, a large clinical trial has recently shown that ivabradine in patients with heart rate above 70 beats per minute prevents coronary events. At present, the availability of ivabradine provides an important and unique opportunity for further experimental and clinical research to test the effects of selective heart rate lowering in cardiovascular diseases such as atherosclerosis, coronary heart disease and heart failure.

16:45 - 18:30

Friday30 Oct 2009

Targeting the If - channel

Reinhold Kreutz – Berlin

Chair: Stephanie Krieg

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Targeting the apoptotic machinery of malignant cells is one attractive concept for the treatment of cancer, which is currently exploited for TRAIL at various stages of preclinical and clinical development. However, the presently published knowledge about TRAIL’s clinical performance points to the need of a further optimization of TRAIL reagents.

In an attempt to improve TRAIL’s tumor selective activity we generated new TRAIL fusion proteins comprising an antibody fragment (scFv) for targeting ErbB2. Further, a variant was designed, where TRAIL is expressed as a single polypeptide chain (scTRAIL). Apoptotic activity of scTRAIL is equivalent to a homotrimeric TRAIL molecule. We studied tumor targeting and apoptosis induction of scTRAIL based fusion proteins in comparison to conventional, homotrimeric TRAIL fusion proteins and nontargeted TRAIL. Interestingly, among the various TRAIL molecules, the tumor antigen targeted scTRAIL fusion protein showed highest apoptotic activity in vitro, with a predominant action via TRAILR2 signaling on Colo205 colon carcinoma cells. Pharmakokinetic studies revealed increased plasma half life of the targeted TRAIL fusion proteins compared to TRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo 205 cells confirm greater response to the tumor targeted ErbB2 specific scTRAIL fusion protein. The underlying mechanisms of this difference in apoptotic and anti tumoral activity will be discussed. Together, in vitro and in vivo data are proof of concept of superior anti tumoral activity of targeted TRAIL molecules, likely via enforced apoptosis induction. Further, we envisage that through targeting of TRAIL, potential side effects should be minimized.

We propose that design concept of single chain TRAIL fusion proteins with tumor targeting capability represents a promising strategy to improve TRAIL’s anti-tumoral action and to minimize potential unwanted actions on normal tissues.

16:45 - 18:30

Friday30 Oct 2009

Death Ligands designed to kill: Engineering of TNF variants for improvement of therapeutic activity

Klaus Pfizenmaier – Stuttgart


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Chromosomal translocations and their encoded fusion genes are hallmarks of leukemias. Leukemic fusion genes are promising candidates for cancer-specific therapeutic approaches because of their exclusive expression in malignant and premalignant tissues. However, their significance for terminally transformed tumour cell is often not well established. We have studied the roles of two leukemic fusion genes, AML1/MTG8 and MLL/AF4, in the maintenance of human leukemia. Using fusion site-specific siRNAs, we have demonstrated that either fusion gene is indispensable for the malignant phenotype both ex and in vivo. Developing an efficient systemic delivery of such leukemia-specific siRNAs may result in new therapeutic approaches with reduced side effects.

16:45 - 18:30

Friday30 Oct 2009

Targeting leukemic fusion genes by RNA interference

Olaf Heidenreich – Newcastle


Evening: HörsaalkinoScrubs Special

20:00

Rudolf-Wöhrl-HörsaalÖstliche Stadtmauerstr. 11

Notes...

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Virology

Primate lentiviruses such as HIV and SIV encode several proteins (Vif, Vpr, Nef, Vpu and Vpx) that are called “accessory” because they can be dispensable for viral growth in vitro. It is evident, however, that these factors are important for viral spread in vivo because they facilitate viral immune evasion and manipulate infected cells to render them permissive for viral replication. Nef is encoded by the genomes of all primate lentiviruses and performs a striking variety of activities, such as downmodulation of CD4 and MHC-I, enhancement of viral infectivity and replication, and modulation of signal transduction pathways. In contrast, vpu is only found in HIV‑1, its chimpanzee precursor SIVcpz and some closely related Cercopithecus viruses, i.e. SIVgsn/mus/mon/den. Vpu counteracts a host restriction factor named tetherin (also called BST-2 or CD317) that “tethers” mature virions at the cell surface and degrades the viral CD4 receptor to promote virus release and replication. Thus, Nef and Vpu activities overlap since both antagonize CD4 and because some SIVs that do not contain a vpu gene use Nef to counteract tetherin. Furthermore, the presence of vpu may facilitate evolutionary changes in Nef function because nef alleles from vpu containing viruses (HIV-1 and its SIV counterparts) have lost their ability to downmodulate TCR-CD3. Thus, the inability of HIV-1 to block T cell activation and apoptosis may contribute to the damaging high levels of chronic immune activation that are observed in AIDS patients but absent in natural SIV-infected primates.

Until recently, data on Vpu function were almost exclusively derived from the HIV-1 NL4-3 molecular clone. In collaborative studies, we analyzed vpu alleles from nearly all primate lentiviruses encoding this accessory gene. We found that Vpus from SIVs infecting Cercopithecus species degrade CD4 and antagonize tetherin. In contrast, SIVcpz uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef due to a deletion in its cytoplasmic domain and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpus from non-pandemic HIV-1 O strains are poor tetherin antagonists and those from the rare group N viruses do not degrade CD4. The finding that only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O may explain why it is almost entirely responsible for the HIV/AIDS pandemic.

Altogether, our current knowledge supports a complex and highly relevant role of Nef and Vpu in HIV and SIV pathogenesis and transmission that clearly warrants further investigation.

9:00 - 11:15

Saturday31 Oct 2009

Role of Nef and Vpu in primate lentiviral pathogenesis and transmission

Frank Kirchhoff – Ulm

Chair: Anna Wiegers

20

Virology

The current swine origin H1N1 influenza A pandemic with a new circulating virus of uncharacterised properties and therefore unknown virulence potential results in huge amounts being spent by governments on vaccine production and stockpiling prophylactic drugs. The benefit of these actions is strongly debated and more knowledge about the virus’ life cycle would be essential to be able to make better informed decisions.

While the natural hosts of the virus are waterfowl, many types of mammal can also be infected and in the case of humans, this can result in pandemics. Usually influenza viruses display a fairly strict species specificity but with the increased emergence of human infections by highly pathogenic avian H5N1 viruses, the need to understand what allows cross species infections and what are the limiting factors is greater than ever. In respect to the current swine flu, it will be important to understand what factors can contribute to high virulence in humans.

While the viral surface proteins are involved in conferring host range specificity, the internal viral polymerase proteins are also known to be involved and PB2 predominantly contributes to host restriction. Together with PA and PB1, PB2 forms the viral RNA-polymerase. In avian viruses PB2 627 is a glutamic acid, while to replicate efficiently in human cells the virus needs a lysine at that position. This is thought to be due to a restrictive factor present in mammalian cell nuclei that inhibits the polymerase complex’s activity.

We used FRAP to investigate the dynamics of individual components of the viral polymerase complex. We found that a full polymerase complex has considerably slower dynamics than its individual components which suggests that the fully formed complex interacts with cellular factors which slow its mobility. Furthermore, in mammalian cells the PB2 627 residue had a major effect on polymerase mobility as a fully human polymerase complex moved faster than a polymerase complex containing an avian PB2. By only changing the 627 position to a “humanising” K, the faster “human” mobility was recovered. At the same time, the avian PB2 resulted in a larger immobile fraction, with the humanised PB2 rescuing to human levels. When carrying out the experiments in avian cells, the identity of residue 627 in an otherwise avian virus PB2 gene did not affect diffusion of the polymerase complex, although it did affect the size of the immobile fraction. We investigated further mutations in the PB2 domain and their potential to contribute to host restriction. These data support the hypothesis that the identity of PB2 627 controls the interaction of the influenza A virus polymerase with host range specific cellular factors.

9:00 - 11:15

Saturday31 Oct 2009

Host specificity of influenza A viruses – investigating the nuclear dynamics of the viral polymerase

Ágnes Föglein – Cambridge

Chair: Anna Wiegers

21

Virology

Human cytomegalovirus (HCMV) viremia and disease are a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). We have recently used the murine model of CMV (MCMV) as a preclinical model to investigate the potential of a novel cell-based strategy to support the humoral antiviral immune response. Memory B-lymphocytes from MCMV immune donor animals adoptively transferred in immunodeficient mice were able to completely prevent a lethal infection. Furthermore, transfer of memory B-cells was also highly effective in protecting from an already ongoing viral infection indicating a therapeutic potential of virus specific memory B-cells. T cells were not involved in the protection. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of post-HSCT immunodeficiency. Currently we develop a novel cell-based therapy on the basis of adoptive transfer of memory B-lymphocytes from the HSCT donor in patients after allogeneic stem cell transplantation. A clinical phase I/II study will be initiated to test the safety and dosing scheme of this novel cell-based strategy.

In parallel, we work on the generation of human monoclonal neutralizing antibodies for therapeutic applications. The main neutralizing antigens for a humoral immune response against HCMV are glycoproteins B, H and N. Affinity optimized antibodies are tested pre-clinically, alone and in combination by cell-based neutralization and spreading assays using a panel of different HCMV strains. So far, we have obtained a set of over hundred human monoclonal antibodies against the glycoprotein G (gB). Only approximately 10 % of these antibodies showed neutralization activity in vitro. The neutralizing monoclonal antibodies recognize a new discontinuous epitope on gB. We used data from the recently established 3D structure of HSV-1 gB for molecular modelling of HCMV gB and based on the model we have identified the region on HCMV gB which is the target of these neutralizing antibodies.

9:00 - 11:15

Saturday31 Oct 2009

Protection from CMV infection in immunodeficient hosts

Thomas Winkler – Erlangen

Chair: Anna Wiegers

22

Immunology

The immediate response to viral infections relies on pattern recognition receptors (PRRs) that sense conserved structural components of invading viruses and subsequently initiate signaling cascades leading to IFN-α/β induction. Retinoic acid-inducible gene-I (RIG-I), consisting of two N-terminal CARDs, a central ATPase motif, and a C-terminal regulatory domain, is a key cytosolic PRR for the recognition of paramyxoviridae, orthomyxoviridae, flaviviridae, and rhabdoviridae. In addition, tripartite motif (TRIM) proteins, containing a RING domain with potential ubiquitin E3 ligase activity, represent a novel class of antiviral molecules involved in innate immunity.

We demonstrate that the interconnection between the sensor RIG-I and a member of the TRIM protein family creates a potent intracellular host defense mechanism, whereas viruses have already evolved a strategy to evade this new class of antiviral regulatory pathway. Specifically, TRIM25 interacts with the N-terminal 1st CARD of RIG-I, and this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the 2nd CARD of RIG-I, resulting in a marked increase in RIG-I downstream signaling activity. Mutational analysis further demonstrated that ubiquitination of RIG-I at Lys-172 is critical for the interaction with its downstream partner MAVS/VISA/IPS-1/Cardif and for RIG-I ability to induce antiviral signal transduction. Gene targeting also showed that TRIM25 is essential for the RIG-I antiviral activity in response to RNA virus infection. Our work further revealed that the non-structural protein 1 (NS1) of human, avian and swine influenza A viruses inhibits the TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising amino acid residues E96/E97 mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and ubiquitination of RIG-I CARDs. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant protein is replication defective in human respiratory cells and loses virulence in mice.

These findings reveal the mechanism by which influenza virus inhibits type I IFN promoter activation during infection.

11:15 - 13:15

Saturday31 Oct 2009

RIG-I- and TRIM25-mediated antiviral innate immunity and viral evasion of Influenza A virus

Michaela Gack – Harvard

Chair: Christian Lehmann

23

Immunology

11:15 - 13:15

Saturday31 Oct 2009

Antibody-based therapies

Ulf Grawunder – 4-Antibody AG, Basel


Notes...

“It is possible, I think, by means of experimentation alone, to determine how far and in what sense we can pursue the investigation of the causes of form.

Thomas Hunt Morgan (1898)

24

Immunology

Although neutrophils are crucial for proper host defence they can also contribute to pathology in a number of inflammatory diseases. Apoptosis of neutrophils and the subsequent removal of these and other apoptotic cells from the site of inflammation are critical events for resolution of inflammation.

Many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells; however the unique micro-environment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. Macrophages show large heterogeneity and comprise various subsets displaying different functional and biochemical properties. The contribution of these distinct populations to the clearance of apoptotic cells and resolution of inflammation is as yet unknown. Furthermore, we have recently reported that immune complexes (IC) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. In disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with IC. We therefore addressed the consequences of IC opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, Mph1), and during resolution of inflammation (type-2 macrophages, Mph2).

Our data demonstrate that IC opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of IC opsonisation. We suggest that IC present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation.

11:15 - 13:15

Saturday31 Oct 2009

Immune complexes: Promoters of apoptotic neutrophil clearance?

Sandra Franz – Edinburgh


13:15 – 14:30: Lunch Break

25

Notes...

Imaging

X-ray computed tomography (CT) has undergone a remarkable development in the past two decades. Significant advances in the existing technologies and new directions in its implementation and application have to be acknowledged.

The state of the art in clinical CT will be reviewed and recent development trends will be analysed briefly; 64-slice high-resolution spiral CT for whole-body imaging constitutes the standard today. It offers whole-body high-resolution imaging within a few seconds scan time. Temporal resolution is sufficient for phase-selective cardiac imaging.

The performance of clinical CT has recently been enhanced significantly by the introduction of dual-source CT scanners. Respective clinical examples shall demonstrate the image quality and the range of applications which are covered routinely.

CT is not only used for anatomic or morphologic imaging. It directly supports molecular imaging in PET/CT combination scanners. However, there are also promising developments aiming at functional and at perfusion imaging. Micro-CT imaging applied in pre-clinical research is a particularly hot topic in Erlangen with its DFG-Forschergruppe 661 funded for “Multimodal Imaging in Pre-Clinical Research”.

14:30 - 16:30

Saturday31 Oct 2009

CT Imaging

Willi A. Kalender – Erlangen

Chair: Martina Hauck “ The future is already here. I t ' s j u s t n o t e v e n l y distributed yet.

William Gibson (1999)

26

Imaging

Stroke is the most important factor for disability in Europe and the US, and by far the most expensive disease. A large fraction of patients are left with a permanent motor, sensory or language deficit affecting the activities of daily living and quality of live. An ischemic lesion affecting cerebral tissue triggers a complex cascade of molecular and systemic mechanisms promoting reorganization of neural functions. There is increasing evidence from studies in animals and humans that both hemispheres contribute to the functional outcome after stroke. Here, functional neuroimaging represents a non-invasive tool to monitor spontaneous recovery of cortical networks.

We use functional magnetic resonance imaging (fMRI) to investigate acute and chronic effects of ischemic lesions on the motor system in human stroke patients, and the impact of neuromodulatory approaches used to manipulate cortical excitability, thereby improving stroke-related impairments. We also compute changes in the interactions of cortical areas, i.e., effective connectivity, by means of differential equations describing non-autonomous systems such as the brain. Connectivity analyses may reveal facilitatory and inhibitory influences and their time-dependent changes during the process of recovery.

The aim is to identify markers which individually predict the potential to respond to a specific therapeutic treatment depending on the individual network pathologies underlying functional impairment.

14:30 - 16:30

Saturday31 Oct 2009

Imaging functional recovery after stroke

Christian Grefkes – Köln

Chair: Martina Hauck

Notes...

27

Imaging

A convenient tool for the observation of dynamic processes in cells is live-cell videomicroscopy. In particular, fluorescent dyes, such as the FM styryl dyes, as well as fluorescent proteins have enabled studies on axonal transport as well as vesicle recycling. As an example of use, a fluorescence video microscopy study on the action of antipsychotic drugs on synaptic vesicle exocytosis is presented, where an inhibitory effect of these drugs could be observed and quantified in real time.

14:30 - 16:30

Saturday31 Oct 2009

Live-cell fluorescence microscopy in neuroscience

Eva Kohler – Erlangen

Chair: Martina Hauck

Evening:Halloween Party

Paisley Park ErlangenNürnberger Str. 15

17:00 – open endFree entry for everyone before 19:00

Notes...

“Of physiology from top to t o e I s i n g , N o t physiognomy alone or brain alone is worthy for the Muse, I say the form complete is worthier far, The Female equally with the Man I sing.

Walt Whitman (1867)

29

29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine Organisation Team

Agata BeraMartin EberhardtStephan Ellmann

Carl-Philipp HacksteinMartina HauckManuela HugleStephanie Krieg

Stephan OttKonstantina Rowald

Anna WiegersFriederike Winter

Stephanie FinzelSimon Kreiser

Christian Lehmann

Cord-Michael BeckerMartin Liepe

Students of the Molecular Medicine degree programme

Members of the SFB 643

Institute of Biochemistry and Molecular Medicine

30

29 Oct – 01 Nov 2009

4th Students' Symposiumof Molecular Medicine Notes

“I did it my way.

Frank Sinatra

symposium of molecular medicine - programme

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