6A_02_S

Molecular genetics and biology of small heat shock proteins causing inherited peripheral neuropathy

Joy Irobi¹, Ines Dierick¹, Berlinda Vanloo², Jan Gettemans², Ludo Van Den Bosch³, Wim Robberecht³, Jean-Pierre Timmermans⁵, Peter De Jonghe^1,4 & Vincent Timmerman¹

1. Department of Molecular Genetics, VIB, University of Antwerp, Antwerpen, Belgium
2. Department of Medical Protein Research, VIB, Ghent University, Gent, Belgium
3. Laboratory of Neurobiology, Department of Experimental Neurology, University of Leuven, Leuven, Belgium
4. Division of Neurology, University Hospital Antwerp, Antwerpen, Belgium.
5. Laboratory of Cell Biology & Histology, Department of Veterinary Sciences, University of Antwerp, Antwerpen, Belgium

Distal hereditary motor neuropathies (distal HMN) are characterized by a selective degeneration of the axons of motor neurons, while the sensory neurons are spared. The biological process of this selective degeneration of motor neurons is still unknown. In 2 distal HMN type II pedigrees linked to chromosome 12q24.3, we identified the same mutation (K141N) in the small heat shock 22kDa protein 8 (HSPB8). A second mutation (K141E) was found in 2 smaller families. Co-immunoprecipitation showed an increased binding of both HSPB8 mutants to the interacting partner heat shock protein27 (HSPB1). We previously reported a Russian family with autosomal dominant axonal Charcot-Marie-Tooth disease (CMT) and assigned the locus (CMT2F) to chromosome 7q11-q21. This locus contained HSPB1 as one of the candidate genes. Mutation analysis of HSPB1 revealed a (S135F) missense mutation segregating in the CMT2F family. Screening for HSPB1 mutations in a large cohort of CMT2/distal HMN patients identified additional mutations. Expression of mutant HSPB8 in COS-1 and N2a cells promoted formation of intracellular aggregates and a reduction of neuronal cell survival. In vitro chaperone activity assay showed a reduction on the cytoprotective function of mutant proteins. Early passages of the primary fibroblast cultures from the distal HMN patient’s skin biopsy showed the formation of aggregate/aggresome complex, which sequestered several molecules including mitochondria. Measurement of mitochondria transmembrane potentials in these primary fibroblast cells evidenced that patients but not controls persons exhibited a depolarized mitochondrial potential.

6A_04_S

Defective chaperone networks

Péter Csermely, Tamás Korcsmáros, István A. Kovács and Máté S. Szalay

Department of Medical Chemistry, Semmelweis University, Puskin street 9., H-1088 Budapest, Hungary; Email: csermely@puskin.sote.hu

Molecular chaperones are not only fascinating molecular machines, but have a number of functions, which can be understood only by considering the emergent properties of protein-protein interaction, signalling and organelle networks – and that of chaperones as special constituents of cellular networks. Moreover, chaperones themselves are networks of amino acid side chains offering vulnerable points for damage. Why are chaperones special in the context of cellular networks? Chaperones (1) have weak links, i.e. low affinity, transient interactions with most of their partners; (2) connect hubs, i.e. act as ‘masterminds’ of the cell being close to several centre proteins with a lot of neighbours; (3) are in the overlaps of network modules, which confers them a special regulatory role. Chaperones may be inhibited by (1) mutations; (2) their damage e.g. after oxidation; (3) their overload, i.e. a growth in the need of chaperones and/or a decrease of their availability or (4) by pharmacological inhibitors. Inhibitory modes (1) through (3) may occur in various diseases and during the aging process. Chaperone inhibitors are efficient multi-target drugs in several diseases such as cancer. Defective chaperones may uncouple or quarantine modules of cellular networks, which increase protection and efficiency of the cell during stress. Moreover, after stress chaperones are essential to re-build inter-modular contacts by their low affinity, ‘quasi-random’ sampling of the potential interaction partners in different cellular modules. This opens the way to the chaperone-regulated disassembly, re-assembly, adaptation and modular evolution of cellular networks, and helps us to design novel therapeutic and anti-aging strategies.

6A_05_S

Protein Aggregation Mechanisms: New lessons from human alphaB-crystallin chaperonapathy in mice

Ivor J. Benjamin

University of Utah, Division of Cardiology, Department of Internal Medicine, Salt Lake City, USA, ivor.benjamin@hsc.utah.edu

Protein aggregation skeletal and cardiac diseases are caused by mutations in ?B-crystallin (CryAB, HSPB5) or desmin and exhibit characteristic hallmarks of protein misfolding and large cytoplasmic aggregates. The mechanisms of protein misfolding diseases remain poorly understood but defining their pathogenesis might uncover new pathways as potential targets for therapeutic interventions. Our recent genetic studies in mice have shown that selective hR120GCryAB expression in the heart induces a novel toxic gain-of-function mechanism mimicking reductive stress. Reductive stress refers to an abnormal increase in the amounts of reducing equivalents (e.g., glutathione, NADPH), which has been demonstrated in lower eukaryotes but is uncommon in the mammals and/or in disease states. This work has broad implications for the treatment of chaperonopathies and neurodegenerative disorders in which small MW Hsps participate as key players and not innocent bystanders in disease pathogenesis involving novel mechanisms

6B_02_S

aB-Crystallin: A New Player in Cancer

Vincent Cryns (Invited Speaker)

v-cryns@northwestern.edu

aB-crystallin, a member of the small heat shock protein family, is induced by cellular stress and functions to limit stress-induced damage by suppressing protein aggregation. We have demonstrated that aB-crystallin inhibits apoptosis induced many stimuli, at least in part, by disrupting activation of the cell death protease caspase-3. We have also shown that aB-crystallin is commonly expressed in poor prognosis basal-like breast cancer and likely contributes to the aggressive behavior of these tumors. This presentation will focus on new insights into the mechanisms by which aB-crystallin inhibits apoptosis and promotes tumor progression and its integral role in the cellular stress response.

6B_03_S

Immunological role of membrane-bound and exported heat shock protein 70 (Hsp70)

Gabriele Multhoff¹, Mathias Gehrmann¹, Jürgen Radons²

2Inst. of Medical Biochemistry and Molecular Biology, University of Greifswald, Germany

Stress or heat shock proteins (HSPs) are remarkably conserved in all living organisms. Their synthesis is induced in response to a variety of physiological and environmental insults. In the cytosol HSPs play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, preventing protein aggregation, transport of proteins, and supporting antigen processing and presentation. On the plasma membrane and in the extracellular milieu they act as danger signals for the adaptive and innate immune system. Either they act as carriers for immunogenic peptides, induce cytokine release or provide recognition sites for activated natural killer (NK) cells. Here we will discuss the problem why Hsp70, the major stress-inducible, cytosolic member of the HSP70 family is selectively found in the plasma membrane of tumor cells but not on normal cells and elucidate the immunological consequences (1,2).

References

6B_04_S

Hsp72 and Hsp27 regulate the p53 pathway and suppress the senescence program in cancer cells

Yaglom J., Gabai V.L., O’Callaghan C., Sherman M.Y.

Boston University School of Medicine

Many tumors have high levels of the major heat shock proteins Hsp72 and/or Hsp27, which correlates with aggressiveness of tumors, resistance to chemotherapy and poor prognosis. Originally, it was suggested that these Hsps facilitate tumorigenesis because they can suppress apoptosis of cancer cells caused by activation of oncogenes, like myc, or by the adverse factors of tumor microenvironment. Here we demonstrate that both Hsp72 and Hsp27 can suppress the p53 pathway and prevent senescence, another major break on cancer development. For example, over-expression of Hsp27 inhibited activation of the p53 pathway by doxorubicin, nutlin-3 or TGF-b, and suppressed development of senescence in response to these stimuli. In contrast, specific depletion of Hsp27 or Hsp72 in a variety of cancer cell lines led to inhibition of Hdm2, activation of p53 and induction of p21. As a result, about 30-40% of cell population became growth arrested and developed features of cell senescence, while the rest of the population also having activated p53 continued to divide slowly, but became sensitive to radiation, doxorubicin and other drugs. These data indicate that high levels of Hsp72 and/or Hsp27 allow cancer cells to avoid activation of an intrinsic senescence program by suppressing p53. At least in certain cancer lines, the intrinsic senescence program was associated with activation of oncogenes. In fact, in MCF-7 cells, which have constitutively active mutant of PI3 kinase (PIK3CA oncogene), depletion of Hsp72 or Hsp27 led to activation of p53 and senescence. However, inhibition of PIK3CA that down-regulates PIP3 prevented activation of p53 and development of senescence upon depletion of Hsps. Similar results were seen with down-regulation of PIP3 in cell lines transformed by PTEN oncogenic mutations. These data indicate that overexpression of Hsps plays a crucial role in supporting division of cells at early stage of transformation upon activation of oncogenes that control PIP3 levels.

6B_05_S

Hsp70 interacts with the plasma membrane phospholipid phosphatidyl serine

Mathias Gehrmann¹, Daniela Schilling¹, Claudia Steinem², A. Graham Pockley^1,3, Gabriele Multhoff¹

¹Technische Universität München and GSF - Institute for Pathology, Munich, Germany, ²University of Göttingen, Göttingen, Germany and ³University of Sheffield, Sheffield, UK

We have previously demonstrated a tumour-specific, plasma membrane localisation of Hsp70 and shown that membrane bound Hsp70 acts as a target recognition structure for NK cell-mediated cytolysis. The membrane association of Hsp70 remains poorly understood and this study used artificial unilamellar liposomes having different phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidylglycerol (PG) compositions to investigate the interaction of Hsp70 with plasma membrane phospholipids. For the assay, Hsp70 (5µg) was incubated with liposomes (10µl) for 30 min at room temperature, after which the mixture was centrifuged for 2 h at 200,000g. The presence of protein in the pelleted fraction, which is indicative of protein/lipid interactions, was determined by Western blot analysis. Hsp70, but not Hsp60 or Hsp90, bound to unilamellar PC/PS (8/2) liposomes under physiological conditions (pH 7.4), even in the absence of CaCl2 or MgCl2. The amount of Hsp70 in the pellet increased as the amount of PS in the liposomes increased (PC/PS, 9/1, 8/2, 7/3, 2/8, 1/9). Hsp70 did not bind to PC/PG (8/2) liposomes, thereby confirming that the interactions are not charge-related. Hsp70 bound to PS-containing liposomes was recognised by commercially available anti-Hsp70 antibodies, whereas membrane-bound Hsp70 is only detectable using the cmHsp70.1 antibody (multimmune GmbH, Munich). If PS is indeed a natural binding partner of Hsp70 in vivo, then this might have important physiological implications. It might also be that the plasma membrane PS content of tumour sublines exhibiting distinct membrane Hsp70 expression patterns differs.

6B_06_S

Extracellular mtHsp70 and immune resistance

Zvi Fishelson, Oren Moskovich and David Pilzer

Tel Aviv University, Dept. Cell & Developmental Biology, Sackler School of Medicine, Tel Aviv, Israel, lifish@post.tau.ac.il

Mitochondrial hsp70 is involved in mitochondrial import/export of proteins, protection from stress and apoptosis, senescence and cancer. The level of mtHsp70 is elevated in many tumor types. Our findings (Pilzer and Fishelson, Int. Immunol. 17: 1239, 2005) suggested a role for mtHsp70 in protection of cancer cells from immune death. Following complement attack, the complement membrane attack complexes (MAC) and mtHsp70 were both found on membrane vesicles released from the cell surface. Vesicle release and accumulation of extracellular mtHsp70 could be inhibited by exogenous application of anti-mtHsp70 antibodies. Silencing of mtHsp70 with siRNA reduced MAC elimination by vesiculation and enhanced sensitivity to MAC-induced cell death. MKT-077 is a cationic rhodacyanine dye that is highly toxic selectively to cancer cells. MKT-077 binds to mtHsp70 and dissociates it from p53, one of its client proteins. Our results demonstrated that MKT-077 similarly prevented binding of mtHsp70 to purified complement C9, the main MAC component. Treatment of human erythroleukemia K562 cells with MKT-077 sensitized them to MAC-mediated lysis but not to lysis induced by another pore-former, streptolysin O. Pre-treatment with MKT-077 also reduced the extent of MAC-mtHsp70 vesiculation after sublytic complement attack and, thus, enhanced the amount of MAC remaining on the cells. Experiments with human colorectal carcinoma HCT116 p53+/+ and p53-/- cells excluded the involvement of p53 in the effect of MKT-077 on complement-mediated lysis. Our results suggest that during an immune attack, mtHsp70 binds to the MAC at the plasma membrane plane and plays a role in its clearance from the cell surface by vesiculation and thus, supports cell resistance to complement-mediated lysis.

6B_07_S

Antagonism between major stress protein Hsp70 and Myc oncoproteins in the execution of apoptotic program

Irina Guzhova¹, Elena Afanasyeva¹, Elena Komarova¹, Lars-Gunnar Larsson², Boris Margulis¹

¹ Institute of Cytology RAS, St Petersburg, Russia
² Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.
guzhova@mail.cytspb.rssi.ru

Two proteins contribute to the targeting of a cancer cell population towards apoptosis or survival, Myc oncogene known mainly as proapoptotic factor and Hsp70 stress protein possessing the ubiquitous protective activity. Based on our data indicating that Hsp70 can have especially high protective activity in heat shock-treated U-937-v-Myc cells we suggested that the key function of the former is the abrogation of apoptosis specifically mediated by Myc. To check this we used two models, in one of whose over-expression of Myc oncogene was continuous (v-Myc in U-937 cells) and in another Myc expression was controlled by estradiol (c-Myc in Rat1MycER cells). It was found that the stable expression of Hsp70 rendered U-937-v-Myc cells more resistant than the original U-937 cell clones when both having been induced to apoptosis with etoposide and camptothecin. Using anti-Hsp70 siRNA we also demonstrated that resistance of U-937-v-Myc cells to the cytotoxic effect of above anti-cancer drugs was fully related to Hsp70 level, whereas in U-937 cells such effect of Hsp70 depletion was not observed. In order to prove the data we employed another model system, Rat1MycERcells, with inducible expression of Hsp70 and of c-Myc. It was found that apoptosis induced by camptothecin and sodium butyrate and mediated by active MycER was efficiently and dose-dependently suppressed by Hsp70. Taken together, our results led us to conclusion that Hsp70 might act as a complement to Myc-driven oncogenesis.

6C_01_S

Divergent Outcomes of Hsp90 Inhibition – Problem or Opportunity ?

Francis Burrows

Biogen IDEC, San Diego, USA

Hsp90 is a ubiquitous molecular chaperone implicated in the pathophysiology of a range of diseases and so is a target of high interest for therapeutic intervention. Most drug targets eventually become resistant, e.g. by mutation, overexpression or anergy, but Hsp90 is uncommon in that it directly controls both cytotoxic and cytoprotective pathways through it’s key role in regulating mitogenic and survival signaling as well as antiapoptotic stress responses. Recent work has indicated that the HSF-1-dependent heat shock response (HSR) decreases the cytotoxic potency of Hsp90 inhibitors in cancer cells in vitro by several mechanisms, but, since normal cells are thought to mount a more robust HSR than their transformed counterparts, it is unclear what impact inhibition of the HSR might have on the therapeutic index of these drugs in vivo. On the other hand, conditions characterized by excessive cell death, such as neurodegenerative diseases, stroke and peripheral neuropathy, could potentially be treated by pharmacological induction of the HSR in neurons. Biogen IDEC has identified potent Hsp90 modulators that induce client protein degradation and the HSR with equal potency, which are in clinical development for cancer. In addition, we are also exploring the potential of compounds that selectively induce client protein degradation or favour cytoprotective pathways for use in Alzheimer’s Disease and Huntington’s Disease in addition to oncology.

6C_02_S

Acetylation as a dynamic regulator of Hsp90 function: Implications for further development of pharmacologic Hsp90 inhibitors

L. Neckers

Urologic Oncology Branch, National Cancer Institute, Bethesda, MD, USA

Heat shock protein 90 (Hsp90) chaperones a key subset of cellular signaling proteins and is necessary for malignant transformation. However, many aspects of Hsp90 regulation remain unresolved. Hsp90 is subject to an array of post-translational modifications which affect its function, including acetylation. In the last few years, several investigators have reported that histone deacetylase (HDAC) inhibitors and knock-down of HDAC6 induce Hsp90 acetylation and inhibit its activity. While pharmacologic inhibition and RNA knockdown of HDACs have been very useful in identifying reversible acetylation as a potential regulator of Hsp90 activity, it is unclear how this process functions at the molecular level. Use of HDACi and/or HDAC knockdown techniques allow study of only the hyperacetylated (but not hypoacetylated) chaperone, and the importance of the acetylation state of individual residues of Hsp90 in the context of hyperacetylation of the proteome cannot be queried. Furthermore, contributory effects due to histone hyperacetylation cannot be discounted. Direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. These sites are now being identified. K294 (Hsp90alpha) was the first amino acid in Hsp90 to be formally identified as an acetylation site and this residue is highly conserved in eukaryotic Hsp90. Conservative mutational analysis of K294 revealed its acetylation status to be a strong determinant of client protein and co-chaperone binding to Hsp90 in mammalian cells. Interestingly, although acetylation status of K294 affects Hsp90 ATPase activity, ATP binding is not altered. In yeast, human Hsp90 mutants that cannot be acetylated at K294 have reduced ability to support viability, while an acetylation-mimicking mutation of K294 possesses the opposite property. Further, acetylation status of K294 may determine Hsp90 sensitivity to N-terminal pharmacologic inhibitors. These data suggest that controlled acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.

6C_04_S

Drugging the cancer chaperone: Preclinical discovery and clinical development of Hsp90 inhibitors

Paul Workman

Cancer Research UK Centre for Cancer Therapeutics, The Institute for Cancer Research, Haddow Laboratories, Sutton, Surrey SM2 5NG

We are in a very exciting era of cancer drug discovery in which effective mechanism-based therapies are being designed and developed that act on the oncogenic proteins and pathways that are hijacked by pathological genetic and epigenetic changes to bring about the initiation of cancer and subsequent malignant progression. Therapeutic selectivity for tumor versus normal cells is achieved by taking advantage of various ‘dependencies’ that develop during the induction of cancers. Two major forms of cancer dependency are related to the molecular chaperone and heat shock protein 90 (Hsp90). Firstly, many mutant and overexpressed oncoproteins that are involved in oncogene dependence or addiction require Hsp90 for their stability and function. Secondly, cancer cells also require Hsp90 and other stress response proteins to help protect them against the adverse environmental conditions present in solid tumors. Moreover, since Hsp90 inhibition causes combinatorial degradation of many cancer-causing proteins, Hsp90 inhibitors are able to attack all of the hallmark phenotypic traits of cancer cells and should have a reduced liability to the development of resistance compared to more conventional drugs. In this presentation, I will provide an update on the preclinical discovery and clinical development of Hsp90 inhibitors. In particular, I will describe what we have learned from the clinical experience with the first-in-class Hsp90 inhibitor 17-AAG. This is a derivative of the natural product geldanamycin which has been a pathfinder Hsp90 drug, demonstrating evidence of target inhibition and therapeutic activity in various cancers, including melanoma, breast, prostate and multiple myeloma. I will also describe our discovery of the new synthetic small molecule class of Hsp90 inhibitors based on the resorcinylic pyrazole and isoxazole scaffold and discuss their optimization by structure-based design in collaboration with Vernalis Limited. Finally, I will describe our recent work identifying genes and proteins that are involved in sensitivity to Hsp90 inhibitors and that have potential to act as biomarkers for clinical use.

6C_05_S

Alcohol exposure regulates HSF-1 and heat shock proteins 70 and 90 in murine macrophages: implication in TNFa production

P. Mandrekar, D. Catalano, V. Jeliazkova and G. Szabo

Dept. of Medicine, Univ. Mass. Med. Ctr, Worcester, MA, USA. pranoti.mandrekar@umassmed.edu 

Alcohol use affects innate immune responses, particularly TNFa , and results in alcoholic liver disease. Heat shock proteins, mediators of the stress responses influence TNFa production. Here we hypothesized that alcohol exposure regulates TNFa production and NFk B activation via modulation of the heat shock transcription factor (HSF), hsp70 and hsp90. Murine RAW 264.7 macrophages were exposed to 25mM, 50mm and 75mM concentrations of alcohol for 24h, 48h and 72h followed by lipopolysaccharide (LPS) for 15 minutes (for HSF and NFk B binding) or 16h (for hsp70, hsp90) to study the effects of alcohol. At the end of the stimulation, nuclear extracts prepared and subjected to HSF and NFk B binding assays whereas hsp70 and hsp90 in whole cell lysates were determined by Western blotting. Our findings demonstrate that HSF binding was increased after alcohol exposure. Supershift analysis showed the presence of HSF-1 proteins and not HSF-2. Interestingly, hsp70 was increased by short-term alcohol but prolonged alcohol exposure decreased hsp70. Conversely, hsp90 levels were decreased after short-term alcohol treatment and increased by prolonged alcohol treatment in macrophages. Immunoprecipitation experiments of hsp90, a chaperone for IKK, showed decreased hsp90-IKKb complexes after short-term alcohol whereas prolonged alcohol revealed presence of hsp90-IKKb and increased IKK kinase activity. Geldanamycin, an hsp90 inhibitor, blocked alcohol-induced increase in TNFa production suggesting an important role for hsp90 in alcohol-induced inflammation. Collectively, our results suggest that alcohol exposure regulates HSF-1 binding, hsp70 and hsp90 and thus could contribute to elevated TNFa and alcohol-induced liver injury.

6C_06_S

HSP90 and ErbB2 in the Cardiac Response to Doxorubicin Injury

Kathleen Gabrielson, Djahida Bedja, Scott Pin, Allison Tsao, Lucio Gama, Bibo Yuan, Nicole Muratore, Yi Xu and Nazareno Paolocci

A major drawback to chemotherapeutic agent doxorubicin is cardiac toxicity. To understand the signal transduction pathways activated in doxorubicin cardiac toxicity, and the potent synergic effect seen when doxorubicin is combined with anti-ErbB2 (trastuzumab), we developed an in vivo rat model that exhibits progressive dose-dependent cardiac damage and loss of cardiac function after doxorubicin treatment. The hearts of these animals respond to doxorubicin damage by increasing levels of ErbB2 and the ErbB family ligand, NRG1ß, and by activating the downstream Akt signaling pathway. These increases in ErbB2 protein levels are not due to increased ErbB2 mRNA, however, suggesting post-transcriptional mechanisms for regulating this protein in the heart. Accordingly, levels of HSP90, a known ErbB2 protein stabilizer and chaperone, are increased by doxorubicin treatment and co-immunoprecipitation reveals binding of HSP90 to ErbB2. Isolated cardiomyocytes are more susceptible to doxorubicin after treatment with HSP90 inhibitor, 17AAG, suggesting that the HSP90 is protective during doxorubicin treatment. Thus, our results provide one plausible mechanism for the susceptibility of the heart to anti-ErbB2 therapy post-doxorubicin therapy in subclinical and clinical conditions. Additionally, these results have lead us to further investigate the biology of HSP90 inhibition in the heart under various conditions. These isolated heart studies will also be included in this presentation.

6D_01_S

Stress proteins are inducers of anti-inflammatory regulatory T cells

Willem van Eden

Dept of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Yalelaan 1, 3584 CL Utrecht, The Netherlands. Email: w.vaneden@uu.nl

Especially since the (re-)discovery of T cell subpopulations with specialized regulatory activities, mechanisms of anti-inflammatory T cell regulation are studied very actively and are expected to lead to the development of novel immunotherapeutic approaches, especially in chronic inflammatory diseases. HSP are possible targets for regulatory T cells due to their enhanced expression in inflamed (stressed) tissues and the evidence that HSP induce anti-inflammatory immuno-regulatory T cell responses.

Initial evidence for an immuno-regulatory role of heat shock proteins (HSP) in chronic inflammation was obtained through analysis of T cell responses in the rat model of adjuvant arthritis and the findings that HSP immunisations protected against the induction of various forms of autoimmune arthritis in rat and mouse models. Since then, immune reactivity to HSP was found to result from inflammation in various disease models and human inflammatory conditions, such as RA, diabetes type 1 and atherosclerosis.

Now, also in the light of a growing interest in T cell regulation, it is of interest to further explore the mechanisms through which HSP can be utilised to trigger immuno-regulatory pathways, capable of suppressing such a wide and diversified spectrum of inflammatory diseases.

6D_02_S

Heat shock proteins as immunomodulators

Salvo Albani

The focus in clinical research in rheumatoid arthritis is increasingly shifting toward early aggressive intervention to achieve remission or a state of low disease and to prevent the development of erosions and functional limitations. These objectives have been achieved in general by continuing therapies that are costly and have long-term side effects. The question remains on whether we can induce lasting immune tolerance and, if so, what mechanisms should we target? Are the drugs currently available sufficient to meet this goal and are we using them properly? The fact that DMARD withdrawal leads to relapses of active diseases in many patients indicates that complementary approaches aimed at maintaining tolerance are needed. A translational itinerary from idea to end of Phase II trial epitope specific T cell immune therapy will be discussed as a potential complementary therapeutic tool to currently existing therapies for RA. This approach relies on a mechanism of modulation of autoimmune inflammation based on immune recognition of heat shock protein derived epitopes.

6D_03_S

What makes arterial endothelial cells a target for the autoimmune attack in the earliest stages of atherosclerosis?

Georg Wick, M.D., Professor and Chairman

Division of Experimental Pathophysiology and Immunology, Biocenter, Innsbruck Medical University Fritz-Pregl-Str. 3/IV, 6020 Innsbruck, Austria

The autoimmune hypothesis of atherogenesis postulates that preexisting cellular and humoral immunity to either microbial heat shock protein 60 (HSP60) or bona fide autoimmunity to biochemically altered autologous HSP60 leads to an attack on stressed arterial endothelial cells (ECs). We have shown in various animal models with spontaneously occurring autoimmune diseases that two essential sets of genes have to be present for an autoimmune disease to develop, i.e. genes that code for autoimmune reactivity of the immune system and genes that are responsible for target organ susceptibility. In the case of atherosclerosis, the arterial ECs express HSP60 that is also transported to the cell surface after being subjected to classical atherosclerosis risk-factors. We have shown the HSP60-inducing effect of most of these risk-factors, including mechanical stress (hypertension), oxygen radicals, oxidized low-density lipoproteins (oxLDL), proinflammatory cytokines (TNF?), and cigarette smoke constituents. Exposure to these classical atherosclerosis risk-factors entail the simultaneous expression of HSP60 and various adhesion molecules (ICAM-1, VCAM-1, P-selectin). Due to their lifelong mechanical pre-stress by the arterial blood pressure, arterial ECs have a lower threshold for the HSP60 inducing effect of atherosclerosis risk-factors as compared to venous ECs. However, when veins are subjected to arterial blood flow conditions, e.g. after arterial-venous bypass operations, HSP60 expression and intimal infiltration with mononuclear cells with subsequent restenosis occurs similar to the pathogenetic events in classical atherosclerosis.

This work was funded by the European Union MOLSTROKE (Molecular basis of vascular events leading to thrombotic stroke) project (LSHM-CT-2004-005206) and the European Vascular Genomics Network (EVGN) (LSHM-CT-2003-503254)

6D_05_S

Heat Shock Modulates Inflammatory Activation of Human Intestinal Microvascular Endothelial Cells (HIMEC)

Parvaneh Rafiee, Mary F Otterson, David G Binion

Medical College of Wisconsin, Milwaukee, WI

Background and Aims: The heat shock response is an evolutionarily conserved mechanism for the maintenance of cellular homeostasis following sublethal noxious stimuli, where heat shock proteins (HSPs) induced by stress, chaperone intracellular proteins that might otherwise be denatured. HSPs are now appreciated to modulate signaling cascades during periods of repeated stress, including chronic inflammation. Although the microvascular endothelium plays a critical role in chronic intestinal inflammation in IBD, little is known about the role of HSPs in the inflammatory activation of HIMEC, the gut specific microvascular population.

Methods: HIMECs from small and large intestine between passages 8 to12 were used. HIMECs were activated with thermal stress as well as TNF-a and LPS. HSPs and iNOS expression were characterized using RT-PCR and Western blot analysis. HIMEC activation was assessed using whole cell ELISA for detection of cell adhesion molecules, and leukocyte binding under low shear stress flow adhesion.

Results: HIMECs exposed to thermal stress (42C, 1h) with recovery times of 1, 6 and 20h demonstrated induction of HSP family members including HSP32 (HO-1), HSP60, HSP70, HSP90. TNF-a /LPS activation of HIMEC maintained at 37C also demonstrated induced expression of HSP60 and HSP70 at the mRNA and protein levels. To investigate the effect of HSP induction on inflammatory activation, HIMEC preconditioned with thermal stress were assessed with and without TNF-a /LPS activation. Heat shock alone induced significant increases in ICAM 1. TNF-a /LPS activation following heat shock, resulted in the highest levels of ICAM 1 and E selectin expression detected in these cells, which corresponded to significantly increased leukocyte adhesion. Heat shock with and without TNF-a /LPS failed to induce increased iNOS expression, which normally functions to downregulate inflammatory activation in HIMEC.

Conclusions: Heat shock significantly enhanced TNF-a /LPS induced activation of HIMEC. The mechanisms involved increased pro-inflammatory ICAM 1 and E selectin expression, which was not accompanied by an increase in the downregulatory expression of iNOS. Further defining the heat shock response may yield insight into mechanisms which enhance inflammatory activation of HIMEC in the setting of chronic inflammation in IBD.

6D_06_S

A novel role of HSP70 as a modulatory agent for dendritic cells phenotype and function

P Stocki, U Holtick, NJ Morris, AM Dickinson

Haematological Sciences, Newcastle University, UK; corresponding author: pawel.stocki@ncl.ac.uk

Heat shock protein 70 (HSP70) is a chaperone proteins which was reported to be implicated in an activation of innate immunity. However, recent data strongly opposes the proposed function of HSP70 as a proinflammatory cytokine highlighting the problem of a bacterial contamination in a purified recombinant human HSP70 (rhHSP70). As it was shown, endotoxin free rhHSP70 is incapable to launch immune activation raising the rhHSP70-endotoxin debate and suggesting that the real role of HSP70 in an immune system, if any, hasn’t been reviled yet. To eliminate the problem of endotoxin contamination we purified HSP70 from two different leukemic cell lines which were growing in the endotoxin free environment. In addition, the purification products were tested for LPS content giving negative result. We examined the HSP70 role on monocyte derived dendritic cells (DC) presenting its modulatory effect on a DC activation state. 24h incubation of DC with HSP70 was effective in down regulating surface expression of HLA-DR as well as CD80/86/83. As a control we used an endotoxin-contaminated rhHSP70 (erhHSP70) which in contrast to HSP70 was highly potent DC maturation molecule. We further examined the functional effect of HSP70 treated DC on proliferation of allogeneic T cells, where HSP70 was responsible for its down regulation. The modulatory effect of HSP70 on DC was also investigated on DC cytokine level after CD40L activation, presenting decreased level of IL-4/6/10 and TNF? confirming the observed phenomenon. Although our results which clearly show that HSP70 has an immunological potential as an anti inflammatory agent are contradicting most of the published data they are supported by the most recent publication on the field of HSP70 conscious about possible endotoxin contamination.

6E_01_S

Psychosocial factors in the metabolic syndrome

Eric Brunner

e.brunner@ucl.ac.uk

The cluster of cardiovascular risk factors labeled the metabolic syndrome is linked with low social status. Prevalence studies show a stepwise relationship - the lower the social position, the greater the probability the syndrome will be present. Systematic differences in diet and physical activity contribute to social patterning of the syndrome. In addition, psychosocial factors including chronic work stress are linked with its development. The Whitehall II study, set up in 1985 to study the psychosocial, behavioral and biological causes of health inequalities in an office-based cohort of 10,308 adults, obtained repeat measures of job stress using a standard self-report questionnaire (Karasek-Theorell job strain questionnaire). Components of the metabolic syndrome (waist circumference, BP, fasting glucose, serum HDL-cholesterol and triglycerides) were measured after 14 years of follow-up. A dose-response relation was found between exposure to job stress and risk of ATPIII metabolic syndrome which remained after adjustment for age and socioeconomic status (OR=2.25 (95% CI 1.3-3.9)). A second prospective study showed a similar dose-response relation with general (BMI) obesity and central obesity. These findings add to other evidence that psychosocial stressors from everyday life are linked to coronary risk. Cross-sectional studies link the metabolic syndrome to adverse neuroendocrine and autonomic activity (increased urinary cortisol and noradrenaline metabolite outputs, and decreased heart rate variability). The metabolic syndrome is a valuable research concept for studying population health and social-biological translation.

6E_02_S

Neuroendocrine mechanisms in the metabolic syndrome

Jan W Eriksson

E-mail address: jan.eriksson@medic.gu.se , jan.eriksson@astrazeneca.com

Abstract

Psychosocial stress is now considered to be an important risk factor for metabolic syndrome and type 2 diabetes, and development of insulin resistance can be a common denominator. Insulin resistance can be described as an impaired insulin action in target tissues, i.e. muscle, fat and liver. In muscle, insulin-stimulated transmembrane glucose transport and the first step in intracellar glucose metabolism, i.e. glucose-6-phosphorylation, appear to be rate-limiting defects. In adipose tissue, insulin resistance is manifested as impaired glucose uptake but also as an impaired suppression of lipolysis and, in addition, there may potentially be dysregulated production and secretion of adipokines and other adipose-derived biomolecules. In the liver, there is attenuated insulin action with respect to glucose uptake and storage as well as suppression of glucose production.

Type 2 diabetes is in most cases caused by a combination of beta cell dysfunction and insulin resistance. Physical inactivity, adiposity due to overeating, stress and smoking are risk factors that interact with susceptibility genes in the development of the disease. The metabolic syndrome is often used to define a cluster of risk markers that predict cardiovascular disease but also type 2 diabetes.

There is no consensus about a unifying underlying mechanism in insulin resistance. However, it is now generally accepted that central adiposity plays a key role in the metabolic syndrome and it is linked to insulin resistance and risk for type 2 diabetes as well as cardiovascular disease. Dysregulated neuroendocrine signalling in adipose tissue can contribute to the development of insulin resistance and metabolic syndrome, and such pathways can mediate the metabolic effect of stress responses that are evoked in the CNS. An overview of the field will be given and recent results will be presented. Topics will include the links between neuroendocrine dysregulation and visceral fat accumulation, fat cell metabolism, alterations in fat cell recruitment, differentiation and growth as well as adipokine production.

Recent data will be presented with focus on the the interplay between different neuroendocrine sysems - the cortisol axis, sex hormones, the autonomic nervous system, catecholamines and the renin-angiotensin system. A hypothesis will be presented about a common ‘final pathway’ that might merge the insulin-antagonistic actions of hormones, inflammation, glucose and lipids, namely the generation of excess reactive oxygen species (ROS). Potential pharmacological interventions targeting the different signalling pathways, including oxidative stress, will also be discussed.

6E_03_S

Role of stress response genes, including HO-1, in Type 1 diabetes and diabetic complications

Prof. Dr Nader G. Abraham

Director of Stem Cells and Gene Therapy,New York Medical College, Valhalla, New York 10595

Hyperglycemic episodes, which cause vascular complications as a result of oxidative stress and complicate even well controlled cases of diabetes, are closely associated with the development of vascular disease. Several antioxidants and stress response genes have been shown to partially protect the vascular system. However, the continuous generation of O₂^- via heme-dependent enzymes, including NADPH oxidase and mitochondrial cytochrome, has shown that such a strategy is difficult to achieve and problematic in diabetes. Heme oxygenase (HO-1) is the sole enzyme that degrades heme and subsequently regulates NADPH oxidase and the cytochrome oxidase system and O₂^- formation via increases in bilirubin generation and ferritin synthesis, which are anti-oxidants, and CO, which is anti-apoptotic. In an effort to overcome this impasse, we examined molecular (gene transfer) and pharmacological (cobalt protoporphyrin, CoPP) approaches to increased HO-1/HO-2 expression in providing cardiovascular protection in Type I diabetic animal models. Upregulation of HO-1 expression attenuates hyperglycemia-mediated increases in vascular dysfunction, circulating endothelial cells and fragmentation, decreases O₂^-, heme levels and iNOS, but increases EC-SOD. Translocation of HO-1 to the mitochondrial compartments enhances cytochrome oxidase and anti-apoptotic molecules, and decreases cytochrome C release, suggesting that HO-1 may regulate mitochondrial pro-apoptotic and anti-apoptotic proteins. Using the loss-of-function, HO-2(-/-) and gain-of-function strategy, we will present data to document that HO-2 siRNA treatment decreased basal levels of EC-SOD and phosphorylated Akt proteins (serine-473 and threonine-308), although no change in Akt protein expression was observed. HO-2 siRNA was also associated with an increase in 3-nitrotyrosine (3-NT) and apoptotic signaling kinase-1 (ASK-1). Additionally, we will present data to show that an increase in HO-1/HO-2 levels, i.e., CO and bilirubin, has a salutary effect, modulating the vascular phenotype, as reflected by the increases in anti-apoptotic proteins, thus rendering endothelial cells resistant to oxidant stress hence preventing vascular dysfunction and the development of Type 1 diabetes.

6E_04_S

Type 2 diabetes, hyperglycemia, and chronic oxidative stress

R. Paul Robertson

The pathogenesis of type 2 diabetes, also known as adult-onset diabetes, is usually attributed to a combination of pancreatic islet beta cell dysfunction and resistance to the action of insulin in important targets, such as liver, muscle, and fat tissue. The cause of beta cell failure is polygenic in nature, whereas the cause of insulin resistance is at least partially explained by associated obesity. It is important to note, however, that many type 2 diabetic people are not obese, and that the majority of obese individuals do not develop type 2 diabetes. Thus, it appears that type 2 diabetes is primarily a genetic disease that can be made worse, but is not caused, by excessive body fat.

A major issue in the field of type 2 diabetes research is why this disease is often characterized by a continual and inexorable decline in glucose control despite optimal drug treatment. This decline in beta cell function is associated with chronically elevated blood glucose levels and has led to the notion of beta cell exhaustion because of continual stimulation by glucose, on the one hand, and the possibility that high concentrations of glucose are chemically toxic to the beta cell on the other. There is an intrinsic paradox at play in these considerations. Since glucose is considered to be a physiologic compound and supportive of beta cell function at many levels from insulin gene transcription through insulin secretion, shouldn’t high glucose levels stimulate insulin synthesis and increase insulin stores rather than lead to exhaustion of the beta cell?

One concept that has emerged is that of glucose toxicity, i.e. chronically high glucose levels form metabolites that can be harmful. This has led to the idea that glucose toxicity of the beta cell might be attributable to formation of excess levels of reactive oxygen species. Glucose excess can promote increased generation of ROS through several metabolic pathways. One can envision that the normal route of glycolysis and oxidative phosphorylation might become oversaturated with glucose. This in turn might lead to shunting excess traffic of glucose molecules along any of several alternative routes, including methylglyoxal formation and glycation; enediol and ?-ketoaldehyde formaton (glucoxidation); diacylglycerol formation and protein kinase C activation; glucosamine formation and hexosamine metabolism; and sorbitol metabolism (1). ROS are normally generated along all these pathways, including oxidative phosphorylation. One need only to imagine a flooding of all these pathways by glucose as a mechanism for excessively high concentrations of ROS in many tissues, including pancreatic beta cells.

This pathophysiologic construction suggests that ROS, which like glucose function as positive chemical mediators in physiologic processes, become negative forces when they are present in excess concentrations. Both ROS and glucose are seen as having good and evil sides, depending on whether their levels are normal or excessive. This general consideration has led to the term glucose toxicity with a major mechanism of action being chronic oxidative stress.

Experimental evidence for chronic oxidative stress as a mechanism for glucose toxicity of the beta cell

The concept of glucose toxicity was first proposed by Unger and Grundy in 1985 (2) and the first biochemical and molecular evidence to support this hypothesis at the level of the beta cell was reported in the early 1990s. In studies that used the beta cell line HIT-T15, serial observations were made over many passages that demonstrated chronic exposure of beta cells led to decreased insulin gene expression, insulin stores, and glucose-induced insulin secretion (3). This was followed by identification of the molecular defects for decreased insulin mRNA levels. Protein levels of two critically important transcription factors, PDX-1 and MafA, were low to non-detectable after prolonged culturing of HIT-T15 cells in media containing supraphysiologic concentrations of glucose (4, 5). It was determined that the molecular mechanism for decreased PDX-1 levels was post-transcriptional, whereas as the mechanism for decreased MafA is post-translational (6). The transcriptional machinery needed for insulin gene expression was not abnormal (4), so major attention was focused on the insulin promoter region through a series of studies involving mutation of the PDX-1 and MafA DNA binding sites and reconstitution of glucotoxic beta cells with these two proteins (4-6). Mutation of the insulin promoter binding sites for either protein in non-glucose toxic beta cells led to marked decreased in promoter activity. Reconstitution of glucotoxic beta cells by transient transfection of either PDX-1 or MafA lead to improved promoter activity. Most recently, it was shown that adenoviral reconstitution of HIT-T15 cells with both PDX-1 and MafA fully restored insulin promoter reporter activity and partially normalized levels of insulin mRNA (6).

As these experiments were evolving, it became evident from the concepts put forward by Wolff and colleagues (7) that the mechanism of glucose toxicity might involve generation of ROS, specifically from glucose autoxidation. This line of thinking was instrumental in early work assessing whether the defects in insulin gene expression and abnormal insulin secretion associated with exposure to high glucose concentrations could be ameliorated by antioxidants. This led to reports of preventive effects in vivo and in vitro, animal models of type 2 diabetes and beta cell lines. Treatment of db/db mice and Zucker Diabetic Fatty rats with NAC preserved insulin gene expression and beta cell function (8, 9). Inclusion of NAC or aminoguanidine in media containing supraphysiologic concentrations of glucose protected HIT-T15 cells against the loss of PDX-1 and insulin gene expression (10).

A central role for glutathione peroxidase in the beta cell

The status of antioxidant enzyme expression in pancreatic beta cells is of major importance to the thesis that chronic oxidative stress may cause beta cell dysfunction. The pancreatic islet is unusual in that in contains the lowest complement of antioxidant enzymes of any other tissue. Early reports indicated that isolated islets from rodents contain very low levels of SOD-1, SOD-2, catalase, and glutathione peroxidase mRNA, protein, and activity levels (11, 12). Similar observations have been made using human isolated islets. This raises the question whether the islet is designed so that low levels of ROS are purposely encouraged to facilitate processes such as gene transcription and exocytosis. Whatever the explanation, these low levels of antioxidant enzymes become a handicap when excess levels of ROS are formed in the beta cell or surrounding tissue.

This paradoxical situation wherein low levels of ROS might be good for the beta cell, but higher levels might not be, led to a series of experiments in which it was documented that rodent and human islets exposed to high concentrations of glucose formed greater ROS concentrations that islets exposed to normal glucose concentrations (10.) This in turn led to experiments in which the potential protective effects of glutathione peroxidase overexpression was tested in islets exposed to r high concentrations of glucose. The reason glutathione peroxidase was chosen is that it metabolizes both hydrogen peroxide and lipid peroxides. SOD-1 and SOD-2 were not chosen because their catabolization of superoxide forms hydrogen peroxide, an ROS. Catalase was not chosen because it catabolizes hydrogen peroxide, but not lipid peroxides. Glutathione peroxide overexpression increased activity of the enzyme 6-fold, roughly equivalent to the activity present in liver, which was sufficient to prevent the deleterious effects of ribose on insulin gene expression, insulin content, and glucose-induced insulin secretion (10).

These experiments have set the stage in our lab for assessing the potential benefits of transgenic experiments in which glutathione peroxidase is overexpressed in the beta cells of animal models of type 2 diabetes. It is being ascertained whether overexpression of this enzyme in beta cells in db/db mice will protect them from beta cell deterioration as they begin to develop hyperglycemia and, if so, whether this will result in attenuation of beta cell failure, as was seen in the case of NAC and aminoguanidine treatment of Zucker Diabetic Fatty rats. If this proves to be the case, then the scene will be set to develop glutathione peroxdase mimetics for use in pre-clinical and clinical trials. This approach will determine whether such drugs will provide a novel, ancillary layer of protection to patients being treated with conventional anti-diabetic drugs, which usually do not completely normalize blood glucose concentrations in patients with diabetes.

References

1. Robertson, R.P. (2004) Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J. Biol Chem 279, 42351-42354.

2. Unger, R.H. and Grundy, S. (1985) Hyperglycaemia as an inducer as well as a consequence of impaired islet cell function and insulin resistance: implications for the management of diabetes. Diabetologia 28, 119-121.

3. Robertson, R.P., Zhang, H.J., Pyzdrowski, K.L. and Walseth, T.F. (1992) Preservation of insulin mRNA levels and insulin secretion in HIT cells by avoidance of chronic exposure to high glucose concentrations. J. Clin. Invest. 90, 320-325.

4. Olson, L.K., Redmon, J.B., Towle, H.C. and Robertson, R.P. (1993) Chronic exposure of HIT cells to high glucose concentrations paradoxically decreases insulin gene transcription and alters binding of insulin gene regulatory protein J. Clin. Invest. 92, 514-519.

5. Sharma, A., Olson, L.K., Robertson, R.P. and Stein, R. (1995) The reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to high glucose concentration is associated with the loss of RIPE3b1 and STF-1 transcription factor expression. Mol. Endocrinol. 9, 1127-1134.

6. Harmon, J.S., Stein, R. and Robertson, R.P. (2005) Oxidative stress-mediated, post-translational loss of MafA protein as a contributing mechanism to loss of insulin gene expression in glucotoxic beta cells. J. Biol. Chem. 280, 11107-11113.

7. Wolff, S.P. and Dean, R.T. (1987) Glucose autoxidation and protein modification. The potential role of 'autoxidative glycosylation' in diabetes. Biochem. J. 245, 243-250.

8. Tanaka, Y., Gleason, C.E., Tran, P.O., Harmon, J.S. and Robertson, R.P. (1999) Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc. Natl. Acad. Sci. USA 96, 10857-10862.

9. Kaneto, H. et al. (1999) Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity. Diabetes 48, 2398-2406.

10. Tanaka, Y., Tran, P.O., Harmon, J. and Robertson, R.P. (2002) A role for glutathione peroxidase in protecting pancreatic beta cells against oxidative stress in a model of glucose toxicity. Proc. Natl. Acad. Sci. USA 99, 12363-12368.

11. [Grankvist K, Marklund SL, Taljedal IB. (1981) CuZn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse. Biochem J 199, 393-398.

12. Tiedge M, Lortz S, Drinkgern J, Lenzen S. (1997) Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells. Diabetes 46, 1733-1742.

Legends

Figure 1. Plasma glucose levels in Zucker Diabetic Fatty (ZDF) rats, a genetic model of type 2 diabetes, who were treated with placebo, n-acetylcysteine, or aminoguanidine beginning at 6 weeks of age. Both drugs are antioxidants and both drugs ameliorated the degree of hyperglycemia developed by the animals. Zucker lean controls (ZLC) that do not develop hyperglycemia are shown for comparison. Taken from ref. 8.

Figure 2. Molecular mechanisms of actions leading to defective insulin gene expression in glucotoxic beta cells. In this model, insulin gene transcription is intrinsically normal, but gene expression of two critical transcription factors, PDX-1 and MafA, is not. Normally, PDX-1 binds to the insulin promoter at three sites, A1, A3, and A5. MafA binds to C1 only. During the development of glucose toxicity, PDX-1 fails to become expressed because of a post-transcriptional defect, and MafA fails to become expressed because of a post-translational defect. Consequently, insulin gene expression at the mRNA level decreases, as does insulin stores and glucose-induced insulin secretion.

Taken from ref.1.

Figure 3. Preventive effects of adenoviral infection of GPx cDNA in isolated islets against the oxidative effects of ribose. Ribose in non-infected cells decreases insulin gene expression and glucose-induced insulin secretion from rat islets in vitro (two lower panels). Adenoviral infection of islets with GPx cDNA increase intrinsic GPx activity in islets 6-fold (top panel) and prevents the adverse effects of ribose on islets (bottom panels). Infection with virus not containing GPx (Ad) has no effects on islets under control conditions or on the adverse effects of ribose on islet function (bottom panels). Taken from Ref. 10

6E_05_S

HSP72 protects against obesity-induced insulin resistance

Mark A. Febbraio

Baker Heart Research Institute, Melbourne, Australia. Email: mark.febbraio@baker.edu.au

Patients with type 2 diabetes have reduced gene expression of Heat Shock Protein (HSP) 72 which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of ?B kinase and tumor necrosis factor-? can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. We first show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next utilized heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high fat feeding.

6E_06_S

Heart Rate Variability and Glycated Hemoglobin

Nanna Hurwitz Eller

Department of Occupational Medicine, Hilleroed Hospital, 3400 Hilleroed, Denmark.

Introduction: By the means of spectral analysis of heart rate, the activity in the autonomous nervous system can be expressed as Heart Rate Variability, HRV. Low Total Power, TP (total variance in HRV, ms²) and low High Frequency power, HF (0,18-0,4Hz), the latter known to mirror vagal tone, is associated with higher cardiovascular morbidity.

Aim: To analyse associations between TP, HF and HbA1c.

Methods: The study included seventy-four healthy non-diabetic participants (50 women, 24 men, mean age 49 years). Levels of HbA1c were obtained in 1998 and 2002. In 2002 ECG during the 45 minutes clinical examination were analysed and spectral analysis carried out. LogTP, logHF and logHF /TP were used as dependent variables. The analyses were carried out by means of general linear models, repeated measurements (9 levels of 5 minutes), separately for each gender using HbA1c in 1998, 2002 and expressed as change between 1998 and 2002 as the independent variable, one at a time. Results were adjusted for age and time of examination.

Results: HbA1c in 1998 was only weakly associated with HRV in 2002. HbA1c in 2002 and expressed as change (2002-1998) were significantly associated with HRV, i.e. high HbA1c and high change were associated with low HRV, most pronounced in men.

Conclusions: Increase in HbA1c and actual level of HbA1c was strongly associated with HRV, in non-diabetic women and men. This indicates that the increased cardiovascular risk accompanying insulin resistance may be caused by low HRV.

6F_01_S

Leukocyte reprogramming after endotoxin encounter

Jean-Marc Cavaillon

Unit Cytokines & Inflammation, Institut Pasteur, Paris. jmcavail@pasteur.fr

Stressfull situation associated with sepsis and non-infectious systemic inflammatory response syndrome (SIRS) such as trauma, surgery, haemorrhage, or ischemia/reperfusion, is associated with a profound alteration of immune status that could explain the increased sensisitivity of the patients to nosocomial infections. Endotoxin (lipopolysaccharide, LPS) is often found in plasma of sepsis patients, and endotoxin translocation from the gut frequently occurs in SIRS patients. During sepsis and SIRS, pro-inflammatory cytokines and inflammatory mediators contribute in synergy to tissue injury, organ dysfunction, and possibly to lethality. To dampen this overzealous process, a counter regulatory loop exists, involving anti-inflammatory cytokines and neuromediators that can also alter the immune status. In sepsis and SIRS patients, a reduced ex vivo pro-inflammatory cytokine production, particularly tumor necrosis factor (TNF), in response to LPS and other Toll-like receptor (TLR) agonists has been reported. However, cells remain responsive to whole bacteria, and their capacity to produce anti-inflammatory cytokine is maintained or even enhanced. Thus the terms "leukocyte reprogramming" well define the process. Modification of intracellular signaling are presently deciphered in patients' leukocytes and some aspects recall what is known about "endotoxin tolerance". The reduced capacity of circulating monocytes to produce TNF and other cytokines can be mimicked in vitro by a pre-treatment of monocytes or macrophages with lipolysaccharide (LPS). This is not a specific phenomenon and it can be induced by other agents or events. Cross-tolerance between LPS, TLR2, 4, 5, or 9 specific ligands, and TNF has been reported. It is possible that cross-tolerance is induced by microbial and endogenous (alarmins) signals of danger.

6F_02_S

Acyloxyacyl hydrolase shortens the duration of post-infection immunosuppression

Mingfang Lu and Robert S. Munford

University of Texas Southwestern Medical Center, Dallas, Texas, USA 75390-9113 Mingfang.Lu@utsouthwestern.edu Robert.munford@utsouthwestern.edu

Serious tissue infections may be followed by a period during which the host animal is much more susceptible to infection with the same or other microbes. One experimental model for this clinical phenomenon is endotoxin tolerance or reprogramming. For several days after receiving a low parenteral dose of Gram-negative bacteria or their cell wall lipopolysaccharide (LPS, endotoxin), many of an animals’ reactions to LPS and other microbial agonists are “reprogrammed” so that proinflammatory responses are reduced while anti-inflammatory ones are maintained or increased. Whereas the adaptive function of this stress reaction may be to prevent harmful inflammation-induced damage to the host, it is often also immunosuppressive. We found that deacylation of LPS by a host lipase, acyloxyacyl hydrolase (AOAH), is required to terminate LPS-induced immunosuppression in mice. Whereas wildtype mice recover within 10 days after receiving 10 µg LPS intraperitoneally, mice that lack AOAH remain immunosuppressed for at least one month. The peritoneal cells in Aoah^-/- mice retain intact LPS for much of this time, their proinflammatory responses to LPS (a TLR4 agonist) and Micrococcus luteus (a TLR2 agonist) are blunted, and their ability to kill Gram-negative bacteria is impaired. By extinguishing the LPS signal, AOAH seems to help the body “re-load” its defensive armamentarium to fight another infection.

6F_03_S

Reprogramming of Macrophages by Endotoxin Tolerance

Schade, F. Ulrich, Butterbach, Katja, Plitzko, Daniela

ulrich.schade@uni-due.de

The term "Endotoxin Tolerance" (ET) has been introduced for a state of low responsiveness of experimental animals or humans treated with a low dose of lipopolysaccharide (LPS, endotoxin). ET has attracted a lot of attention since it was shown that cross tolerance exists to other bacterial products such as lipoteichoic acid or lipopeptides. Furthermore, it turned out that ET protected against bacterial infections - Gram-negative, as well as Gram-positive - and ischemia-reperfusion injury. It, therefore, is of potential clinical interest to get insight into the cellular and molecular basis of ET. To this end, we studied different cellular components of the peritoneal cell populations and Kupffer cells of endotoxin tolerant and normal mice with regard to gene expression, intracellular signal transduction and regulation of cytokine production. Corresponding results will be presented. HSP 70 is among the genes that are significantly upregulated in peritoneal cells of ET mice compared to normal mice.

6F_04_S

Immuno-neuro-endocrine adaptation in critical illness

I.Vermes, A.Beishuizen and A.B. J.Groeneveld

Medical Spectrum Twente, Enschede & Free University Medical Centre Amsterdam,The Netherlands (i.vermes@home.nl)

Critical illness is a severe stress stimulus that disturbs the milieu interior and induces homeostatic responses specific to the stimulus and generalized responses when the disturbances are prolonged and severe. The immune-neuroendocrine response during critical illness is a dynamic process, differing between the acute and chronic phases. This acute phase lasts typically from about a few hours to several days, depending on the severity of the illness and is characterized by an appropriate hormonal reaction: the mobilization of fuel stores of the organism, together with apparent restrains on their utilization. During the acute phase of critical illness the secretory activity of the pituitary gland is stimulated whereas anabolic target organ hormones are inactivated. Due to the development of intensive care medicine patients who would previously have died during the acute phase nowadays survive and enter the chronic or prolonged phase of the critical illness. This prolonged phase lasts for days and is characterized by an inappropriate hormonal response, resulting in a chronic increase in metabolic rate and a breakdown of body tissue. The secretory activity of the pituitary gland is uniformly inhibited in relation to reduced levels of target organ hormones. These hormonal changes suggest an imbalance between immunosuppressive and immunostimulatory hormones which might be the cause of the increased susceptibility to infectious complications during the chronic phase of severe illness. The suppressed pituitary activity allows the respective target organ hormones to decrease, resulting in a restored balance between the catabolic and anabolic hormonal responses. This recovery phase is characterized by restored sensitivity of the pituitary gland to reduced feedback control. The distinction of acute and prolonged critical illness as different entities with regard to the immuno-neuroendocrine adaptation may be helpful in further understanding of the pathogenesis of critical illness and the targeting of therapeutic intervention.

6F_05_S

In vivo imaging of the effect of bacterial endotoxins on arterial endothelial cells: molecular imaging of heat shock protein 60 expression

Marius C. Wick¹, Christina Mayerl², Georg Dobrozemsky³, Roland Haubner³, Hermann Dietrich⁴, Werner Jaschke¹, and Georg Wick²

1Department of Radiology, ²Division for Experimental Pathophysiology and Immunology, Biocenter, ³Department of Nuclear Medicine, ⁴Central Animal Laboratory Facilities,Innsbruck Medical University, Austria

Background. Bacterial endotoxins (LPS) are known to act as stress factors for endothelial cells.

Aims. As a proof of principle, the present project aimed at developing a novel radiotracer-based non-invasive molecular imaging method for in vivo visualization of early aortal HSP60-expression in a normocholesterolemic rabbit model after induction of endothelial stress.

Material and Methods. In 14 rabbits, endothelial stress was induced by i.v. injection of bacterial endotoxin (lipopolysaccharide, LPS at 10m g/kg) while 8 animals were untreated controls. Non-invasive in vivo molecular imaging was performed 24 hours after intravenous injection of I-124 radiolabelled monoclonal anti-HSP60 antibodies using computertomography (CT) and positron-emission-tomography (PET). In vitro correlation of in vivo imaging was done by en face immunohistochemistry and autoradiography of the aorta.

Results. Compared to control animals, quantitative analyses of in vivo non-invasive molecular PET-CT images of rabbit aortae revealed a significantly increased endothelial binding of I-124 anti-HSP60 antibodies upon application of LPS as an endothelial stressor, especially at sites of aortal branching. This was confirmed by in vitro anti-HSP60 immunohistochemistry and autoradiography data.

Conclusion. Our first results showed, as a proof of principle, that HSP60-expression in normocholesterolemic rabbits is significantly increased after the induction of endothelial stress and non-invasive in vivo molecular imaging of early aortal HSP60-expression using radiotracer labelled anti-HSP60 antibodies is possible.

Acknowledgements: This work was supported by the Propter Homines Foundation, Vaduz, FL.

6F_06_S

Protective role of N-procalcitonin in endotoxic shock

Eva Tavares, Rosario Maldonado, Inmaculada Capilla, Cesar Sevillano and Francisco J. Minano

Corresponding author: jminano@us.es

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. Procalcitonin (PCT), a 14-kDa propeptide of calcitonin, is a circulating biomarker of bacterial infection but PCT itself has no known activity. Circulating levels of PCT and its free aminopetide N-procalcitonin (N-PCT), have been found dramatically increased in septic patients, and these rises are correlated with severity and mortality. Importantly, in sepsis, the levels of N-PCT may be even higher than the PCT values. In vitro studies suggest that N-PCT may function as a factor suppressing the propagation of inflammation through the inhibition of several processes involved during a response to a bacterial stimulus.These findings together with N-PCT's sequence conservation during evolution, suggest that N-PCT has a critical, and as yet undefined, pathophysiological function. In this study, we assessed the role of N-PCT as a mediator of sepsis in endotoxin-challenged rats. Intraperitoneal administration of an lethal dose (LD₁₀₀) of E. coli endotoxin to normal rats induced a substantial increase in PCT, IL-1b and TNF-a in plasma. The administration of recombinant human N-PCT intraperitoneally significantly protected rats from endotoxin-induced mortality from 100% to 15%, and resulted in a decrease in PCT, IL-1b and TNF-a levels. By contrast, N-PCT did not modify IL-10 levels. These results confirm a critical part for PCT in the pathogenesis of endotoxic shock and indicate that N-PCT is a protective peptide expressed in murine endotoxemia, and does so by down-regulating the systemic production of proinflammatory cytokines in endotoxin-challenged animals.

This work was supported by grants from the Andalusian Government (Consejeria de Salud, 053/05 and 0364/06), Spanish Ministry of Health (FIS 06/1394) and the Valme Foundation.

6G_01_S

Stress and prions: lessons from the yeast model

Yury O. Chernoff

School of Biology and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Altlanta, Georgia 30332-0230, USA; yury.chernoff@biology.gatech.edu

Amyloids are fiber-like ordered aggregates generated via intermolecular cross-ß interactions. In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the transmissible protein isoforms (prions) that cause infectious neurodegenerative diseases in mammals (including humans) and manifest themselves as non-Mendelian heritable elements in yeast and other fungi. Propagation of the prion state in yeast is controlled by the concerted action of chaperone proteins. A crucial role in this process is played by the chaperone protein Hsp104, which promotes breakage of amyloids into smaller oligomeric seeds initiating new rounds of prion proliferation. Prion formation and propagation are also influenced by other stress-related chaperones (Hsp70 and Hsp40), and by alterations of the protein trafficking and degradation networks. Therefore, prion propagation employs enzymatic machinery which is normally supposed to protect cells from environmental stresses. Some stresses induce prion formation or loss. It is possible that prions arise as by-products of the reversible assembly of highly ordered complexes, protecting certain proteins from destruction during unfavorable conditions.

6G_02_S

Intracellular Fate of Misfolded Proteins Associated with Retinal Degeneration

Shalesh Kaushal, Syed M. Noorwez

University of Florida, Departments of Ophthalmology and Cell Biology, Gainesville, Florida, USA, skaushal@ufl.edu

Misfolded proteins are found in many inherited diseases, which collectively are called protein conformational disorders. These misfolded proteins, typically containing one or more mutations, are retained intracellularly in the endoplasmic reticulum or in cytoplasmic aggregates. Our previous studies clearly demonstrated that the clinically common P23H opsin mutant associated with autosomal dominant retinitis pigmentosa is misfolded and retained in the cell. Pharmacological induction of the heat shock response has emerged as an attractive strategy for modulating the cellular folding environment and treating protein conformational diseases. The induced ensemble of heat shock proteins act as molecular chaperones in protein folding and protect against the formation of misfolded protein aggregates. Using a tetracycline-inducible HEK293 cell lines, we have studied the effect of the heat shock response on P23H opsin and folded rhodopsin levels in the cell. The known chemical inducers of the heat shock response, geldanamycin (GA) and celastrol, increase the total opsin levels by 1.8- and 1.6-fold (n=3), respectively. Further, GA and celastrol reproducibly increase folded rhodopsin levels by 1.7- and 1.4-fold (n=3). The level of hsp70 in the treated cells was elevated by 4 fold by GA and 1.2-fold with celastrol. Both treatments appear to induce the heat shock response and stabilize the folded form of opsin i.e. the form capable of binding retinal. Further, the extent of hsp70 increase correlates with te yields of folded P23H opsin. Thus, heat shock induction clearly alters the cellular stability and fate of P23H opsin. Both compounds are attractive candidates for treating P23H opsin associated retinal degeneration.

6G_03_S

Misfolded Proteins in Aging and Neurodegenerative Disease

Cindy Voisine

Misfolded and damaged proteins challenge homeostasis and the capacity of molecular chaperones and clearance machineries to restore the health of the cell. Whereas acute exposures to heat shock and other environment stress leads to the induction of cytoprotective responses, the chronic expression of misfolded and mutant proteins can result in irreversible toxicity as occurs in human neurodegenerative disease. We are interested in understanding how an organism and specific populations of cells detect and respond to diverse types of proteotoxic stress. We have established C. elegans models to identify the genes that regulate protein homeostasis in response to chronic expression of aggregation-prone proteins associated with neurodegenerative disease. These studies have identified an important molecular link between the insulin-signaling pathway that regulates longevity and HSF-1, the master regulator of protein folding quality control and the heat shock response, revealing that longevity is closely linked to the cellular folding capacity. Using forward genetics, genome-wide RNAi screens and a candidate gene approach, we have identified a functionally diverse set of genes that sort into six networks involved in RNA metabolism, protein synthesis, protein folding, protein degradation, protein trafficking, and mitochondrial function and energy metabolism that influence protein homeostasis. Many of these genes are also important for the regulation of heat shock gene expression which suggests that genetic modifiers for protein quality control defines a protein-folding proteome that also functions to sense an imbalance in protein homeostasis and regulation of chaperone expression. Using our C. elegans models, we are characterizing key Hsp70 chaperone networks that coordinately function to protect cells during aging and in response to misfolded disease proteins.

6G_04_S

Chaperome Systems and Protein Misfolding Stress in Trafficking Disease

William E. Balch_1,2,6, Atanas Koulov1, Darren Hutt1, Bogdan Tanasa1, Wendy Kellner1, Helen Plutner1, Paul Lapointe1, Scott Stagg1, Clint S. Potter1,5, Bridget Carragher1,5, Jeffery W. Kelly3,4 and John R. Yates III1

The Scripps Research Institute, Departments of Cell1 and Molecular Biology2,and Chemistry3, The Skaggs Institute for Chemical Biology4, The National Resource for Automated Molecular Microscopy5, and The Institute for Childhood and Neglected Diseases6, 10550 North Torrey Pines Road, La Jolla, CA 92037.

Protein folding in the eukaryotic cell is highly sensitive to the local environment of the cytoplasm or membrane trafficking compartments, and requires the assistance of multiple chaperones that define the folding buffer of the cell- the chaperome (Wang et al. (2006) Cell, 127:803). Different cell types exploit the variable composition of the chaperome environment to maintain protein folding homeostasis- reflecting the kinetic and thermodynamic properties of the protein fold. Perturbation of this relationship, as occurs in misfolding diseases such as cystic fibrosis (CFTR), Gaucher's (glucocerebrosidase), childhood emphysema (alpha-1-antitrypsin), type II diabetes and numerous amyloid diseases (Alzheimers (APP), Parkinsons (alpha-synuclein)), results in an imbalance between the protein fold and the folding environment leading to disruption of normal physiology. It is becoming increasingly apparent that folding pathways in aging and disease can be altered by the cell through stress sensitive pathways to maintain or reestablish the proper chaperome environment to maintain functionality. Emerging mechanisms by which these activities work provides insight into the pathways that can be manipulated through biological and chemical approaches to adjust the flow of folded and functional protein through the exocytic and endocytic pathways.

6G_05_S

A reverse genetic overexpression screen reveals non-canonical chaperones as potent suppressors of polyglutamine proteins

Harm H. Kampinga, J. Hageman, M. Rujano, M. Vos

1Department of Radiation and Stress Cell Biology, University Medical Center Groningen, Univ. of Groningen, The Netherlands; E-mail: h.h.kampinga@med.umcg.nl

As they are able to bind misfolded proteins, Heat Shock Proteins (HSP) may prevent accumulation of toxic poly-Q protein aggregates and as such inhibit disease-associated pathogenesis. Several in vitro reports have pointed to the classical Hsp70 chaperone machines and their cofactors as suppressors of polyglutamine-related protein aggregation. Yet, the (limited) in vivo work with mouse models has yielded disappointing results from minor delays in disease progression to no effect at all. It has become clear recently that the many different Hsp subfamily members may have different and substrate specific chaperone-like functions. To seek for chaperones that may be particularly effective in dealing with polyglutamine proteins, we conducted a reverse genetic overexpression screen of the human chaperonome. We identified a number of non-canonical Hsp40 family members as well as a new member of the small Hsp family as superior inhibitors of polyglutamine aggregation. Unlike the canonical DnaJB1 (that has mild suppressive activity in vitro), the suppression by the non-canonical Hsp40’s was not annihilated by mutating the HPD motif in their J-domain, normally required for interaction with Hsp70 family members. At suboptimal concentrations of the non-canonical Hsp40’s, overexpression of none of the Hsp70 family members was able to further reduce the polyglutamine aggregation. All these data suggest that they act independently of the Hsp70 machine. The action of the small Hsp was independent on its N-termimal domain and as such seems to be executed by the crystallin domain.

6G_06_S

The Hsp60-(p.Val98Ile) variant protein associated with autosomal dominant spastic paraplegia SPG13 displays subtle effects when co-expressed with wild type Hsp60

Peter Bross¹, Marit N. Nielsen¹, Jane H. Christensen¹, Jakob Hansen¹,Niels Gregersen¹, Debbie Ang², Costa Georgopoulos²

Peter.Bross@ki.au.dk

We have earlier reported that a mutation (c.292G>A/p.Val98Ile) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin is associated with dominantly inherited hereditary spastic paraplegia SPG13 . To mimic heterozygosity for the Hsp60-(p.Val98Ile) mutation and to assess a potential dominant negative effect of co-expression of a wild type and the mutant variant of Hsp60 on the function of the 7-meric chaperonin complex, we used a flexible bacterial model system. These cells lack the endogenous chaperonin genes and are maintained alive by expression of a plasmid-encoded Hsp60/Hsp10 operon. The cells harbour a second plasmid with a Hsp60/Hsp10 operon encoding a mutant Hsp60. The two operons can be turned on and off separately. We compared the behaviour of bacterial cells co-expressing either wild type Hsp60 and Hsp60-(p.Val98Ile) or wild type Hsp60 and an artificially constructed Hsp60 ATPase mutant. Induction of co-expression of the Hsp60 ATPase mutant severely inhibited bacterial growth, whereas co-expression of the Hsp60-(p.Val98Ile) mutant variant had only subtle yet specific effects. Additional experiments indicate that both mutant Hsp60 variants form heterologous complexes with wild type subunits. By varying temperature and relative amounts of the wild type and Hsp60-(p.Val98Ile) mutant variant, we have found conditions under which the mutant exerts a clear effect. We propose that the major in vivo consequences of heterozygosity for the Hsp60-(p.Val98Ile) variation are due to subtle qualitative and quantitative effects remaining to be discovered.

6H_01_S

The neural bases of negative affect and stress responses in schizophrenia

Ute Habel

Department of Psychiatry and Psychotherapy, RWTH Aachen University, uhabel@ukaachen.de

Stress has a major impact on the course and psychopathology of schizophrenia, having significant influences especially on emotion but also cognitive processes. In schizophrenia, emotion dysfunctions are a hallmark with a special role of negative affect and reduced capacities to manage stressful situations and stimulations. Data acquired from different samples of healthy subjects and schizophrenia patients will be presented to clarify the neural basis of dysfunctional emotion processes applying fMRI. Emotion and cognition are mostly investigated as different entities, while practically both functions are inseparable and are interacting with each other continuously. However, interactions between emotion and cognition have not been investigated in greater detail in patients. Similarly, in healthy persons there are only a few and rather contradictory studies exploring the general effects of emotion on cognitive function. We developed and validated tasks investigating the interplay between emotions and cognition. Stress was induced in patients and comparison subjects by means of negative olfactory stimulation while subjects had to perform a combined 0-back/2-back task. A control condition with neutral room air stimulation was also applied. According to subjective ratings mood induction was successful. The impairing effect of the negative mood induction was visible during working memory performance (2-back) only, in which the mean number of correct target reactions was significantly lower during negative olfactory stimulation as compared to the neutral condition. The neuroimaging data reveal a complex dysfunctional interaction in patients, where especially frontal and cingular regions show aberrant activation patterns. Hence, regions of major importance in emotion regulation and integration are affected.

6H_02_S

Stress, dopamine, brain tissue volumes and vulnerability to psychosis

Machteld Marcelis, E. Cavalier, John Suckling, Paul Hofman, Philippe Delespaul, Jim van Os

Dept of Psychiatry & Neuropsychology, POBox 616, 6200 MD, Maastricht University, The Netherlands m.marcelis@sp.unimaas.nl

The present study investigated whether individuals with schizophrenia, and those with an elevated genetic risk for schizophrenia, display an altered plasma HVA-response to metabolic stress. Besides, associations between the metabolic stress response and brain tissue volumes were examined. Patients with psychosis (n=50), non-psychotic first-degree relatives of patients with psychosis (n=51) and controls (n=50) underwent, in randomised order, double-blind administration of placebo and the glucose analog 2-deoxy-D-glucose (2DG), which induces a mild, transient clinical state of glucoprivation. Plasma HVA was assessed twice before the start of the 2DG/placebo infusion (baseline), as well as four times post infusion. MRI cerebral tissue volumes were derived from automated segmentation procedures. During the stress condition, significant increases in plasma HVA were found. The increase in plasma HVA level during the stress condition was significantly stronger in patients than in controls, whereas this was not the case in relatives v. controls. The HVA level increase in the stress condition was stronger in patients with lower grey and white matter volumes. In conclusion, patients with psychosis, but not their non-psychotic relatives, show an altered dopaminergic/noradrenergic mediated stress response, possibly reflecting acquired sensitization of catecholamine systems by repeated environmental stressors or repeated stimulation with agonistic drugs. HVA level increases were stronger in patients with lower grey and white matter volumes, supporting the hypothesis that alterations in cortico-subcortical connections affect psychosis susceptibility through an altered stress response.

6H_03_S

Structural Brain Changes Underlying Vulnerability to Schizophrenia

Michio Suzuki^1,2, Tsutomu Takahashi^1,2, Shi-Yu Zhou^1,2,3, Yasuhiro Kawasaki^1,2, Masayoshi Kurachi^1,2

1Department of Neuropsychiatry, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan, ²CREST, JST, Tokyo, Japan, ³Department of Psychiatry and Medical Psychology, Dalian Medical University, Dalian, China

Morphologic changes in schizophrenia are thought to represent a complex and dynamic process in which multiple brain regions are differentially affected. Common neurobiological abnormalities among the schizophrenia-spectrum may be essential for the pathogenesis of schizophrenia, but some additional pathological changes may also be required for the development of full-blown schizophrenia. Clarifying the neurobiological similarities and differences between established schizophrenia and schizotypal (personality) disorder, a schizophrenia-spectrum disorder without manifestation of overt and sustained psychosis, would potentially discriminate the pathophysiological mechanisms underlying the vulnerability to schizophrenia from those associated with overt psychosis. Detailed volumetric comparisons using magnetic resonance imaging (MRI) revealed differential morphologic alterations in the brain between the patients with schizotypal disorder and those with schizophrenia. Volume reductions in the amygdala, hippocampus, superior temporal gyrus, and anterior parietal cortex common to both patient groups may represent the vulnerability to schizophrenia. Volume loss of the prefrontal cortex, posterior parietal cortex, cingulate, insula, and fusiform cortex preferentially observed in schizophrenia may be critical for overt manifestation of psychosis. On the other hand, preserved volume in these regions might have relevance to the protection factors from overt psychosis in schizotypal disorder.

6H_04_S

Early emotional stress by analysis of stress line in molar and susceptibility to schizophrenia

Kunio Yui ^{1, 2)}, Koichi Watanabe³⁾, Masayoshi Kobayashi³⁾, Koichi Nishijima⁴⁾

4) Department of Psychiatry, Jichi Medical School

[Introduction]: It is well known that early life events or urbanicity, that interacts with multiple genes, induces persistent sensitization to stress possibly through an imbalance in interactions between dopaminergic and glutamatergic systems. This stress sensitization may be critical in the development or relapse of schizophrenia. Laboratory method for estimating early stress is therefore needed. Ameloblast activity in human molar’s enamel is slowed during 1-2 days extreme stress, and the segment of enamel rods is smaller and often misshapen, making a particular dark line seen by microscopy, which we called Pathological Stress Line (PSL). We studied the nature of stress sensitization by analysis of PSL. According to animal and clinical studies, severe emotional stress induce changes of mineral density in bone. Thus, to clarify the type of stress related to PSL, we examine mineral changes in PSL portions.

[Methods]: We examined PSL in third molar in 35 chronic paranoid schizophrenics (25 males and 10 females, 41.9±13.5 yeas old)and 32 normal controls (5 males and 27 female, 28.3±9.1 years old). Changes of density in potassium (P), calcium (Ca) and magnesium (Mg) in PSL portion were examined by Scanning Microscope and Electron Probe Microanalyser (EPMA). Since the rate of enamel elongate is well known, PSL was assessed due to its length and definition at half yearly between 9 to 13 years old: 0, none; 1, slight; 2, mild; 3, moderate; 4, severe. Mineral changes were assessed in PSL portions rated as 2-4 due to extent of changes of mineral density in one area (1.5 X 1.5 mm): 0, none; 1, 1/4; 2, 1/3; 3, 1/2, 4, 2/3.

[Results]: PSL scores in the 35 schizophrenics were significantly higher than the 32 normal controls (4.8±5.1 vs. 2.0±2.3, P<0.01). The 35 schizophrenics exhibited PSL indicative of stressors experienced at age of 10.5-11.5 years old. Scores in P and Ca were significantly decreased and those of Mg were significantly increased in PSL portions.

[Conclusion]: The present findings suggest that stress sensitization may be induced at age of 10.5-11.5 years, which is comparable to previous studies indicating that stress exposed at age of 10-13 years may be related to the development of adulthood schizophrenia. Decreased density of P and Ca and increased density of Mg have been reported to be caused by severe emotional stress. The findings on EPMA suggest that severe emotional stress at age of 10.5-11.5 years may induce stress sensitization.

6H_05_S

Psychosis in correlation with chronic stress and vasopressin

Dóra Zelena

Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Szigony 43, Hungary, zelena@koki.hu

The role of stress in the development and maintenance of symptoms in major psychiatric disorders such as schizophrenia is well-established. The biological component of stress is mediated largely through the endocrine system, predominantly by the hypothalamo-pituitary-adrenal (HPA) axis. The two main hypothalamic secretagogues of the axis are CRH and vasopressin with more important role of the latter during chronic stress. Vasopressinergic dysfunction in schizophrenics was also indicated and the naturally vasopressin mutant Brattleboro rats was suggested to be a good model for schizophrenia. Therefore we addressed here the question if the lack of vasopressin in Brattleboro rats will lead to disturbances in chronic stress-induced HPA axis changes parallel with the development of schizophrenia. The classical somatic chronic stress symptoms (body weight reduction, thymus involution, adrenal gland hypertrophy) and also the signs of HPA axis hyperactivity (resting corticosterone elevation and POMC mRNA elevation in the anterior lobe of the pituitary) were present in all three studied situation (repeated restraint, streptozotocin-induced diabetes mellitus, repeated morphine injections) with similar extent in control and vasopressin deficient rats disclosing the involvement of V1b receptors in the process. On the other hand vasopressin as a neurotransmitter may act on other brain regions and there are many compensatory mechanisms in Brattleboro rats (e.g. oxytocin, CRH). Moreover both the adrenomedullary system and the sympathetic nerves are more active in vasopressin deficient rats. So our conclusion is that vasopressin may act on the development of schizophrenia through influencing other neurotransmitters in brain and not the HPA axis. Chronic stress may exacerbate the schizophrenia not through HPA axis changes but e.g. glucocorticoid toxicity in hippocampus.

6H_06_S

Nutritional metabolic stress and epigenetic mechanism of schizophrenia

Sahebarao Mahadik

Stress, both environmental (nutrition, toxins, hypoxia) and emotional is ubiquitous and can contribute to epigenetic molecular mechanisms for a wide variety of non-communicable diseases from birth to aging. Maternal as well as fetal nutritional stress (deficiency of micronutrients or oxidative cell damage due to very high caloric intake) has been shown to imprint the risk for adolescent and adult non-communicable disorders such as brain disorders, diabetes and cardiovascular disease. Among these, maternal folic acid and vitamin B12 deficiency has been considered to contribute to the altered gene expression by altered one carbon metabolism and thereby methylation of proteins, DNA and phospholipids, particularly containing omega-3 fatty acids. Role of maternal nutrition has been considered in schizophrenia but evidence is based on epidemiological data. We studied the plasma levels of folic acid, vitamin B12, cortisol and homocysteine, one of the most important factor in oxidative stress-mediated cellular dysfunction, particularly neural cell function during brain development. The drug-naive patients at the early onset of psychosis (N=28) and matched healthy controls (N=34) were enrolled in a study. All the study protocols were properly approved by the Institutional Ethical Committee and each study subject signed the consent to participate in the study. Compared to normal controls, patients had significantly (p<0.05) lower levels of folic acid (5.40 ± 3.99 vs 3.46 ± 1.71 ng/ml) as well as vitamin B12 (236.32 ± 132.66 vs 185.52 ± 73.39 pg/ml). These changes were associated with the significantly increased (p=0.05) plasma levels of homocysteine in patients (15.99 ± 10.09 vs 11.65 ± 3.88 m moles/l). It has already been shown that altered methylation of histones, DNA and phospholipids as well as the increased morbidity to diabetes and cardiovascular disease in schizophrenia. Our data have important implications to prevention of these metabolic stress mediated fetal risks for adult disorders including schizophrenia.

6I_01_S

Stress and the gut: role of corticotropin releasing factor (CRF) signaling pathways

Y. Taché

Center for Neurovisceral Sciences, Department of Medicine, University of California, Los Angeles, USA

The characterization and distribution of corticotropin-releasing factor (CRF) family of peptides, CRF, urocortin 1, urocortin 2 and urocortin 3, and the two G-protein coupled receptors, CRF₁ and CRF₂, as well as the development of selective CRF₁ and CRF₂ receptor antagonists provided novel means to understand mechanisms involve in the stress response. The activation of brain CRF₁ signaling pathway plays a primary role in the endocrine (activation of pituitary adrenal axis), behavioral (anxiety, depression), autonomic (sympathetic activation), and decrease immune responses to stress. Combined anatomical, pharmacologic and molecular approaches support a role of CRF receptor activation in both the brain and the gut as part of key mechanisms involved in stress-related alterations of gut propulsive function. Inhibition of gastric emptying and stimulation of colonic motor function are the commonly encountered patterns resulting from exposure to various stressors in experimental animals and humans. Activation of brain and peripheral CRF₂ receptors mediates stress-related inhibition gastric motor function while that of CRF₁ receptors are involved in the stimulation colonic secretory and motor functions. Clinical investigations also support the notion that stress contributes to visceral hypersensitivity of the gut observed in patients with irritable bowel syndrome (IBS) as established by their lowered threshold of pain to colorectal distension (CRD). Experimental models have been developed that recapture clinical features of IBS with regard to stress-related hyperalgesia to CRD, gender differences, comorbidity with anxiety/depression and altered bowel habit. Data obtained using pharmacologic approaches in rodents subjected to stress indicate that the activation of CRF₁ receptor contributes to the development of hyperalgesia to CRD while CRF₂ receptors display opposite effects. The mechanisms through which CRF₁ antagonists alleviate colonic responses involved the prevention of stress-related activation of sacral parasympathetic ouflow, colonic enteric cholinergic neurons and mast cells. The pre-clinical and clinical phase I data support that targeting of CRF₁ receptors may open new therapeutic venues for stress-related functional gastrointestinal disorders. Supported by NIHDDK and VA Merit grants.

6I_02_S

Effect of capsaicin on the esophageal motility of patients with gastro-esophageal reflux disease (GERD)

Agnes Király

3rd Dept. of Medicine, University of Pécs, Hungary. e-mail: agnes.kiraly@aok.pte.hu

Capsaicin-sensitive afferents play a role in the regulation of esophageal motility. Vanilloid receptor is activated by acidic pH which triggers pain and motor responses leading to esophageal emptying . The aim was to investigate the effect of topical capsaicin (Tabasco) suspension on esophageal sensation and motility in healthy controls (N=10), patients with non-erosive GERD (NERD) (N=10), erosive esophagitis (ERD) (N=10) and with Barrett’s metaplasia (BM) (N=10). Visual analog scale was used to determine sensation, the esophageal body and lower esophageal sphincter (LES) response were analyzed. Topical 1.5 x 10 ^-4 M capsaicin significantly increased the amplitudes (68 +/- 3 to 88.5 +/- 4.7 ; 66+/-2 to 80+/-3; 70+/-1 to 96+/-4 mm Hg, P < 0.05) and propagation velocity (2.7 +/- 0.3 to 4.3 +/- 0.3 ; 3.1+/-0.3 to 4.2+/-0.1; 2.5+/-0.1 to 3.7+/-0.2 cm/s, P < 0.05) of esophageal pressure waves and LES pressure (15 +/- 1.4 to 27 +/- 2.6; 13+/-1 to 20+/-1; 14+/-1 to 29+/-3 mm Hg, P < 0.05) in controls, NERD and ERD patients respectively. None of the above responses were present in BM patients. Capsaicin enhanced esophageal bolus transit (3.3+/-0.1 to 2.7+/-0.1; 3.2+/- 0.1 to 2.5 +/- 0.1 s, P < 0.05) in controls and NERD patients. Unchanged bolus clearance was found in ERD and BM patients (4.0+/-0.1 vs. 4.1+/-0.1; 4.2+/- 0.2 vs. 3.9 +/-0.1 s NS). Significantly increased perception to capsaicin was found in NERD (66+/-7) and ERD (78+/-9) patients vs. to healthy controls (47+/-3) mm. Esophageal capsaicin induced a profound increase in emptying which could improve clearance of the esophagus. Chronic acid exposure produces allodynia in NERD and ERD patients which is related to their symptom development. Capsaicin-mediated esophageal clearance is disturbed in ERD and BM patients.

6I_03_S

Stress at the cellular level – novel roles of heat shock proteins in gastric mucosal protection and healing.

A. S. Tarnawski and H. Gergely

VALBHS & Univ of California, Irvine, CA, USA. atarnawski@yahoo.com

Cells respond to stress by activating heat shock protein (HSP) response, which constitutes an universal defense system, essential for maintaining cell and tissue homeostasis by stabilization of denatured proteins. In gastric mucosa, the surface epithelial cells are directly exposed to temperatures ranging from 4° C to 75° C, therefore HSP activation is very relevant. We examined expression of constitutive and inducible HSP70 (c and iHSP70) in gastric mucosa of rats at baseline and following treatment with cytoprotective drugs: sucralfate, rebamipide, and talcid. In normal gastric mucosa cHSP70 is expressed mainly in the surface epithelium. Cytoprotective drugs enhanced expression of cHSP70 in surface epithelial and progenitor cells induced iHSP70 in the same areas and significantly reduced ethanol-induced gastric mucosal injury. Ethanol-induced gastric injury is significantly increased in mice with KO gene for heat shock factor 1 (HSF-1) a transcription factor for HSP genes. This is due to increased apoptosis in response to ethanol injury in HSF-1 null mice. This study provides evidence that HSPs after their HSF-1-dependent upregulation protect gastric mucosa against injury. Recent data indicate that HSPs are involved in healing of experimental gastric ulcers. HSP32 are elevated during early stage of healing; HSP47 are increased in granulation tissue and HSP70 is increased in the epithelial cell of the ulcer margin and progenitor cells where it co localized to EGF, IGF-1 and Cox2. Conclusions: 1) Gastric mucosa expressed HSP at baseline and their expresses is activated by cytoprotective drugs and in response to injury. 2) HSPs also play important roles in ulcer healing by local interactions with growth factors and by protection of regenerating cells.

6I_04_S

Increased expression of transcription factor Egr-1 in stress-induced gastric ulceration

T. Beregova¹, V. Kucharskyij¹, O. Drobinska¹, S. Szabo²

Revutskogo str., 44, apt.86, Kyiv, Ukraine 02140 tberegova@univ.kiev.ua

The transcription factor early growth response-1 (Egr-1) is activated by many environmental signals. Recently it was demonstrated the enhanced duodenal Egr-1 transcription activity and followed increased expression of target genes such as bFGF, PDGF and VEGF in cysteamine-induced duodenal ulceration in rats. However, the involvement of Egr-1 expression in stress-induced gastric ulceration was not determined. The aim of our study was to investigate the effects of stress on Egr-1 expression in rat gastric mucosa (GM) at the different time point. The rats were immobilized for 6, 12 and 24 hr. Egr-1 expression was determined by immunoblotting. It was established that 6 hr of stress exposure induced only single punctated hemorragies in GM. 12 hr of stress produced the multiply punctated hemorragies. 24 hr of stress exposure developed ulcers, errosions and massive hemorragies in rat stomach. The gastric injuries were 23,01±4,07 mm². Egr-1 expression was barely detectable in GM of untreated rats. 6 hours of stress increased the gastric Egr-1 expression by 8 fold (p<0.05) compared to control. At 12 hr of stress exposure Egr-1 expression in injured GM was enhanced only 2 fold in comparison to control. Egr-1 expression dropped to the basal level after 24 hr of stress exposure. Thus, the highest level of Egr-1 expression was observed in rat GM 6 hr after stress exposure in comparison with untreated rats and rats after 12 and 24 hr of stress exposure. So stress-induced gastric ulceration leads to increased Egr-1 expression in rat GM. The results highlight the role of Egr-1 in experimental ulceration.

6I_05_S

New molecular mechanisms of duodenal ulceration.

S. Szabo, T. Khomenko, X.M. Deng, G. Tolstanova , L Chen, K. Osapay, Zs. Sandor