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  HUM-MOLGEN -> Events -> Meetings and Conferences  
 

6th Annual Cytokines & Inflammation

 
  December 13, 2007  
     
 
GTCbio, Orlando, FL
Jan 28-29, 2008


Day 1, Monday, January 28, 2008


What attendees said last year:
“The talks and the attendees were an excellent match, the room was full from the introduction to the closing remarks.” – Scott Durum, NCI, NIH


7:00
Registration & Breakfast


7:55
Chairman’s Opening Remarks


[KEYNOTE PRESENTATION]

8:00
Regulation of T Regulatory Cells

Joost J. Oppenheim, M.D., Chief of the Laboratory of Molecular Immunoregulation, NCI, NIH



(Co-authors: O. M. Z. Howard and X. Chen)
We have identified various means of regulating the level and activity of CD4+CD25+FOXP3+ T regulatory (Treg) cells. They include genetic regulation as illustrated by the difference in the higher levels of functional Tregs seen in Balb/c compared to the C57BL/6 mouse strains. Hormones such as dexamethasone, as expected, augment the level of functional Tregs. A bacterial product pertussis toxin reduces the number and function of Tregs and promotes the Th17 pathway instead. Immunosuppressive drugs can have distinct effects as evidenced by the induction of Tregs by rapamycin, whereas cyclosporine A inhibits the differentiation of Treg cells.

Finally, our studies have revealed a molecular mechanism to clarify the apparently opposing effects of TNF alpha. TNF interacts with TNFR2 initially activating T-effector cells in vitro, this is superseded by considerable expansion of Treg cells both in number and function in the presence of IL-2. Mouse TNFR2-expressing Tregs have more suppressive effects and belong to memory phenotype subset of Tregs with more CTLA-4, GITR, and CD103 expression, but less CD45RB and CD62L. Human Tregs isolated from peripheral blood that are CD4+ CD25+ TNFR2+ cells also express more FOXP3 and are more suppressive. TNFR2 appears to be a marker for the more suppressive subset of mouse and human Tregs. Thus, identification of molecular regulators of Tregs provides a means of targeting immune responses.


Session I - Cytokines Signaling and Regulation


8:45
Interleukin-35: A Novel Cytokine that Mediates Regulatory T Cell Function

Dario Vignali, Ph.D., Associate Member, Department of Immunology, St. Jude Children’s Research Hospital


Regulatory T (TR) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity, limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease (IBD), and regulating homeostatic lymphocyte expansion. However, they also suppress natural immune responses to parasites and viruses as well as anti-tumor immunity induced by therapeutic vaccines. While the manipulation of TR function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr virus-induced gene 3 (Ebi3; IL27β) and interleukin-12 alpha (Il12a; IL12α/p35) are highly expressed by Foxp3+ (forkhead box P3) TR cells but not by resting or activated effector CD4+ T (TE) cells, and that an Ebi3/Il12a heterodimer is constitutively secreted by TR but not TE cells. Both Ebi3 and Il12a mRNA are dramatically upregulated in TR cells co-cultured with TE cells, thereby boosting Ebi3/Il12a production in trans. TR cell-restriction of this cytokine is due to Ebi3 being a downstream target of Foxp3, a transcription factor that is required for TR cell development and function. Ebi3–/– and Il12a–/– TR cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and cure IBD in vivo. As these phenotypic characteristics are distinct from those of other IL-12-family members, this novel Ebi3/Il12a heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naïve T cells, while recombinant IL-35 suppresses T cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that is specifically produced by TR cells and is required for maximal suppressive activity.


9:10
The Pathogenic Role of IL-17 Signaling in Autoimmunity

Xiaoxia Li, Ph.D., Staff, Cleveland Clinic Foundation


Interleukin (IL)-17 is an important proinflammatory cytokine that plays critical functions in immune responses. We recently reported the essential functions of Act1 in IL-17 receptor signaling and IL-17-dependent immune responses. Upon IL-17 stimulation, Act1 is recruited to IL-17 receptor through the SEFIR domain, followed by the recruitment of TAK1 and TRAF6, mediating NFkB activation. IL-17-induced expression of inflammatory genes was abolished in Act1-deficient primary cells. Consequently, Act1 deficiency led to major impact on the development of IL-17-dependent autoimmune inflammatory diseases. While the severity of experimental autoimmune encephalomyelitis (EAE) was greatly reduced in Act1-deficient mice, DSS-induced colitis was significantly attenuated in epithelial-specific Act1-deficient mice. Taken together, these results demonstrate that Act1 is an essential component in IL-17-mediated signaling and required for IL-17-dependent in vivo functions.


9:35
Networking & Refreshment Break


10:05
TH17-Mediated CNS Inflammatory Diseases: Development and Regulation by IL-12 Family Cytokines

Charles E. Egwuagu, Ph.D., Chief, Molecular Immunology Section, Laboratory of Immunology, NEI, NIH



Detection and eventual elimination of pathogens derive from productive interactions between the innate and adaptive immune systems. Interleukin-12 family cytokines play crucial roles in shaping immune responses by influencing cell-fate decisions of differentiating naïve T-cells while cytokines produced by T-cells activate effective defense against the pathogens. However, unregulated secretion of cytokines by T cells is the cause of many autoimmune diseases and recent studies reveal that cytokines produced by cells of the innate system also function to limit adaptive immune responses. IL-17-producing T-cells (TH17 cells) comprise a recently identified TH-cell lineage implicated in tissue inflammation. In humans, TH17 cells increased during active CNS inflammatory disease but decreases following treatment and IL-17-specific antibody treatment reduced disease severity in experimental autoimmune encephalomyelitis and experimental autoimmune uveoretinitis. IL-2, secreted by many T-cell subtypes promotes TH17 expansion while IFN-g, produced by TH1 subtype, inhibits TH17 proliferation by upregulating interleukin-27, an IL-12 family cytokine. These CNS autoimmune diseases provide useful framework for understanding intricate relationships between cytokines secreted by cells of the innate system (IL-12, IL-23 and IL-27) and cytokines produced by cells of the adaptive immune system (IL-2, IL-17 and IFN-g). In this talk I have: (i) Summarized our recent experimental findings that provide new perspectives on how innate and adaptive cytokines converge to orchestrate and regulate host immunity in the eye and possibly, the CNS. (ii) Identified therapeutic targets that can be used to mitigate or prevent development of pathogenic autoimmunity.



10:30
The Innate Immune Response to Virus Infection: Lessons in Evasion from Hepatitis C Virus

John Hiscott, Ph.D., Molecular Oncology Group, Lady Davis Institute, McGill University


The hepatitis C virus (HCV) is an important cause of human chronic liver diseases and is a major public health problem, with more than 170 million people infected worldwide. Clinically, 80% of those individuals exposed to HCV become persistently infected, often resulting in chronic active hepatitis, cirrhosis, and hepatocellular carcinoma. The immune response is rarely effective in eradicating the virus, indicating that HCV has evolved strategies to evade innate immune responses of the host. HCV and many viral infections are detected by the presence of viral nucleic acids, triggering the production of interferons (IFN) and other cytokines that in turn stimulate innate and adaptive immune responses. Although microbial pathogens are usually detected as a consequence of defined pathogen associated molecular patterns (PAMPs) by the Toll-like receptor (TLR) family, viral infection is often detected through the presence of single and double-stranded RNA (dsRNA) by TLR-7 or TLR-3 respectively, as well as CpG containing DNA (TLR-9). Intracellular viral RNA is also detected by two recently characterized RNA helicases, RIG-I and/or Mda5 that function independently of TLR pathways. Both TLR-dependent and -independent recognition of viral infection leads to the activation of the interferon regulatory factors (IRF) -3 and IRF-7 which are essential downstream transcription factors required for the induction of type I IFN; both proteins are targets for phosphorylation by the virus activated kinases TBK1 and IKKe.

Recently the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups: MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy, resulting in its dissociation from the mitochondrial membrane and disruption of signaling to the antiviral immune response. With a novel search program written in python, we also identified an uncharacterized protein - KIAA1271 (K1271) – containing a single CARD-like domain at the N-terminus and a Leu-Val rich C-terminus that is identical to MAVS/IPS-1/VISA/Cardif. Using a combination of biochemical analysis, subcellular fractionation and confocal microscopy, we demonstrated that NS3-4A cleavage of MAVS/K1271 causes relocation from the mitochondrial membrane to the cytosolic fraction of the cytoplasm. Furthermore, virus-induced IKKepsilon kinase, but not TBK1, co-localized strongly with MAVS at the mitochondrial membrane and the localization of both molecules was disrupted by NS3-4A expression. Mutation of the critical cysteine 508 to alanine was sufficient to maintain mitochondrial localization of MAVS/IPS-1/VISA/Cardif in the presence of NS3-4A. The characterization of MAVS/K1271 provides the first link between mitochondrial function and the innate immune response, and outlines the molecular mechanism by which HCV evades the IFN antiviral response.


10:55
Contribution of IL-17 Family Members to Rheumatoid Arthritis: More Targets for Treatment

Pierre Miossec, Professor, Clinical Immunology, University Hospital Edouard Herriot



The role of IL-17 has been established in a growing number of diseases associated with chronic inflammation and matrix destruction. Rheumatoid arthritis (RA) was the first condition where the role of IL-17 was established. Since then progress has identified additional IL-17 family members, has further described the structure of the Il-17 receptor and has demonstrated its regulation by other cytokines.

In synoviocytes, IL-17 was found less active than IL-17A. However when combined with TNF, strong synergy was observed indicating the role of IL-17F in the context of inflammation. Both IL-17RA and IL-RC receptors appear to control IL-17 response of synoviocytes. However dual inhibition of the two receptors appears necessary in order to inhibit the response of synoviocytes when IL-17 is combined with TNF.

RA is associated with Th1 defects as expressed by the reduced expression and production of IFNg in response to the Th1 driving cytokines IL-12 and IL-18. It appears that such defect is the consequence of inflammation and most directly that of IL-17. Indeed exposure of blood cells and isolated T cells to IL-17 reduces IFNg response by selectively inhibiting the expression of the specific chain of the IL-12 receptor. Such effect contributes to the increased risk of tuberculosis reactivation when exposed to TNF inhibitors.

The role of IL-17 in RA has been established and suggests that this cytokine represents a target of interest for treatment.

Key points:
Role of IL-17 in chronic inflammation
Contribution of new IL-17 family members and receptors
Role of IL-17 in RA associated Th1 defects
Basis for IL-17 targeting in inflammation


11:20
Recombinant ST2 Exacerbates Hepatic Fibrosis by Increasing Th2 Response in Injured Liver

Aldo Amatucci, Immunology Department, Biogen-Idec


Excessive scarring or fibrosis is a common feature of a wide spectrum of diseases characterized by an exaggerated T helper cell type 2 (Th2) response. The Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) related protein ST2 is expressed in a membrane bound form selectively by Th2 cells and was shown to be indispensable for some in vivo Th2 responses. ST2 was also found to block TLR signaling. We addressed the impact of the ST2 pathway on fibrogenesis using a mouse model of hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). We showed that cytokine production by intrahepatic lymphocytes from CCl4 injured liver is abrogated in the absence of TLR-4. Interfering with the ST2 pathway using an ST2-Fc fusion protein accelerated and enhanced hepatic fibrosis, paralleled by the increasing ex vivo secretion of Th2 cytokines interleukin (IL)-4, -5, -10 and -13 by intrahepatic lymphocytes of ST2-Fc treated CCl4 gavaged mice. Absence of IL-4/13 signaling in IL-4Ra deficient mice obliterated this ST2-Fc effect on fibrogenesis. Moreover, depletion of CD4+ T cells abrogated both ST2-Fc enhanced Th2 cytokines and accelerated fibrosis. Thus, ST2-Fc caused overproduction of Th2 cytokines by intrahepatic CD4+ T cells possibly by modifying TLR-4 signaling in injured liver. This ST2-Fc driven Th2 response exacerbated CCl4 induced hepatic fibrosis.

Benefits of the talk:
1) Modulation of Th1/Th2 balance in the context of tissue injury.
2) Cytokine response to liver injury dependent on TLR activation.
3) sST2 is not only a decoy receptor for IL-33 but can also inhibit TLR activation.


11:45
IL-21 Increased Potency Design


Kent Bondensgaard, Novo Nordisk




Interleukin-21 (IL-21) is a recently identified type 1 cytokine, which is secreted as a 133-amino acid protein by activated CD4+ T cells. The IL-21 cytokine has been demonstrated to possess potent stimulatory effects on the proliferation, differentiation and activation of several classes of haematopoietic cells including B-cells, T-cells and NK-cells. Lately, IL21 has been shown to be involved in inducing proinflammatory T(H)17 cells.

We have solved the 3D structure of hIL-21 which is dominated by a well-defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule which includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer the helix C segment is presented in a regular, a-helical conformation, while in the other conformer this segments is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding.

IL-21 analogues were designed to stabilize the region around helix C either through the introduction of a segment grafted from hIL-4 or by shortening of the CD loop. These novel hIL-21 analogues were demonstrated to exhibit large increases in potency in cellular assays. Different possible mechanisms for increased potency by cytokine analogues will be discussed


12:10
Lunch Break


Session I - Cytokines Signaling and Regulation (continued)


1:10
IL-22, a Th17 Cytokine, Plays an Important Role in Psoriasis as well as in Mucocutaneous Host Defense


Dimitry M. Danilenko, DVM, Ph.D., Senior Pathologist, Department of Pathology, Genentech, Inc.




IL-22 belongs to the IL-10 family of cytokines, which also includes IL-10, IL-19, IL-20, IL-24 and IL-26. IL-22 is produced by activated CD4+ T cells and regulates proliferation and differentiation of several different epithelia. In a reconstituted human epidermis culture system, IL-22 had pronounced effects on keratinocyte proliferation and differentiation, inducing the psoriasis-associated proteins S100A7 and keratin 16, decreasing the epidermal granular cell layer (hypogranulosis), and persistently activating keratinocyte Stat3 with nuclear localization. Recently, IL-23, a cytokine involved in the development of IL-17-producing T helper cells (Th17 cells), has been implicated in the pathogenesis of psoriasis. IL-22 is preferentially produced by Th17 cells, and can be induced by either IL-23 or IL-6. In a mouse ear inflammation model, IL-22 is the primary mediator of the epidermal hyperplasia (acanthosis) induced by IL-23, and this acanthosis is mediated via activation of Stat3 in keratinocytes. These results suggest that IL-22 produced by Th17 cells may have an important role in the pathogenesis of cutaneous inflammatory diseases, such as psoriasis. In addition, there is evidence that IL-22 also has essential functions in host defense in epithelial tissues. In a murine model of infectious colitis with Citrobacter rodentium, infection of IL-22-/- mice resulted in more severe colitis, increased systemic bacterial burden, and greater mortality. IL-22 induced many anti-microbial genes, such as S100 proteins, RegIIIβ and RegIIIγ, from the colon. Together, the results from these studies demonstrate that IL-22, as an effector cytokine from Th17 cells, mediates the crosstalk between the immune system and epithelial tissues, and plays an important role in both cutaneous inflammatory diseases and mucocutaneous host defense.


1:35
Therapeutic Intervention of Cytokine Signaling in Inflammatory Disease: A Multi-Pronged Approach with Small Molecule Inhibitors


Nandini Kishore, Pfizer




Cytokines are directly implicated in multiple immune processes associated with pathogenesis of rheumatoid arthritis and other inflammatory diseases. Intervention of cytokine signaling with anti-TNF biologics or targeting cytokine receptors with anti-receptor antibodies and soluble receptors has led to favorable outcome in the clinic. Early pre-clinal and clinical data look promising for small molecule inhibitors targeting cytokine signaling in immune and inflammatory cells. Kinases offer a rich plethora of targets for intervention of cytokine signaling at multiple branch points. Diverse pathways targeting kinases in the signaling cascade will be discussed as possible candidates for therapeutic intervention.


2:00
The Role of Cytokines in Cardiac Allograft Vasculopathy after Heart Transplantation


Roel de Weger, Ph.D., Associate Professor, Deartment Pathology, University Medical Centre Utrecht, NI




The only possible therapy for end-stage heart failure is heart transplantation. Despite increasing survival rates of human heart transplant recipients, long-term survival is complicated by loss of the transplants due to Cardiac Allograft Vasculopathy (CAV). The pathogenesis of CAV is multifactorial and mediated by an incompletely understood immunological process. CAV affects the arterial walls from the allograft by a concentric intima proliferation reducing the diameter of the vascular lumen. The luminal narrowing is induced by a mononuclear cell (MNC) infiltrate, composed of lymphocytes, macrophages, smooth muscle cells (SMC) and fibroblasts. We have recently described that the neointima is composed of two distinct layers: a luminal layer (NI-LL) composed of loose connective tissue heavily infiltrated by MNC, and a layer composed of SMC (NI-SMC) directly adjacent to the lamina elastica interna. Clinical and experimental studies demonstrated that recruitment of MNC from the arterial lumen into the neointima and from capillaries into the adventitia is induced by pro-inflammatory cytokines and chemokines. The MNC are important in (I) induction of proliferation and differentiation of SMC and/or fibroblasts, and (II) the deposition of extra-cellular matrix components in the intima. The infiltrating subsets of T-lymphocytes (T helper-1 (Th-1), Th-2, Th-17 and Treg) and macrophages are phenotyped and the production of cytokines and growth factors is analyzed by quantitative PCR and immunohistochemistry. It will be discussed that although the cytokine production of the cells is severely affected by the therapeutic immunosuppression, activated Th1-cells and M2-macrophages are the major MNC cells causing the chronic proliferative response.




2:25
Protease Activated Receptors as Cross Roads between Coagulation and Inflammation


Lars C. Petersen, Ph.D., DSci. Principal Scientist, Haemostasis Biology, Novo Nordisk


Numerous observations indicate that, activation of the coagulation cascade affects the inflammatory process and that inflammation promotes thrombosis. This is illustrated e.g. in animal models of established sepsis showing that treatment with coagulation inhibitors is accompanied by anti-inflammatory effects and by attenuation of organ injury. Coagulation is initiated by exposure of tissue factor (TF) to the circulating blood where it forms a complex with factor VII (VII). TF is normally not present on endothelial cells lining the vessel walls, but TF exposure on these cells can be induced by inflammation; or TF on underlying cells can be exposed upon vessel wall injury. Formation of the TF/VII complex triggers the activation of VII to VIIa and subsequently the activation of coagulation factors X, IX, XI and pro-thrombin. It is well known that the resulting thrombin formation leads to fibrin clot deposition. However, recent studies have revealed that the active proteases, VIIa, Xa and thrombin, are also capable of inducing several types of cellular responses, including signaling, gene transcription, cytokine expression and cell migration with possible inflammatory implications. This is mediated through activation of protease activated receptors (PARs) and the resulting intracellular signaling. Thrombin activates PAR1 and PAR4 whereas VIIa and Xa have been reported to activate either PAR2 or PAR1 depending on the circumstances. Our results on the gene transcription profile induced by TF/VIIa indicating a wound healing type of response are presented, and I discuss 1) possible cross roads between coagulation and inflammation at the cellular level, 2) cellular responses to VIIa stimulation, 3) differential gene-expression induced by VIIa, PAR1 or PAR2, and 4) the importance of TF/VIIa signaling via PAR2 relative to thrombin signaling via PAR1.




2:50
Networking and Refreshment Break


Session II - Inflammation and Cancer


3:20
Th17 Cells and Mucosal Immunity in the Lung

Jay K. Kolls, M.D., Neils K. Jerne Professor of Pediatrics and Immunology, Children’s Hospital of Pittsburgh


Emerging evidence supports the concept that Th17 cells in addition to mediating autoimmunity have critical roles in mucosal immunity against extracellular pathogens. Both IL-23p19 KO and IL-17RKO lack enhanced susceptibility to the intracellaulr pathogens Listeria monocytogenes or Mycobacterium tuberculosis. However both are susceptible to the extracellular pathogens K. pneumoniae. IL-22 and IL-17A are both effector cytokines produced by the Th17 lineage and both cytokines are critical for maintaining local control of the gram negative pulmonary pathogen, Klebsiella pneumoniae. Although both cytokines regulated CXC chemokines and G-CSF production in the lung, only IL-22 increased lung epithelial cell proliferation and increased transepithelial resistance to mechanical injury. These data support the concept that the Th17 cell lineage and their effector molecules evolved to effect host defense against extracellular pathogens at mucosal sites.


3:45
Mechanisms of Crystal Recognition by Inflammasomes

Eicke Latz, MD, Ph.D., Assistant Professor in Medicine, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School


The major task of the immune system is to recognize and respond to conditions that are of danger to the body, such as infections, cancer and traumatic tissue damage. Immune cells express several classes of germline-encoded signaling receptor molecules that can recognize and signal the presence of foreign material (from viruses and bacteria in case of an infection) and altered self molecules that appear under certain disease conditions.
Toll-like receptors are membrane bound immune receptors that recognize a broad range of molecular signatures in the extracellular space and in endo-lysosomal compartments. In the cytoplasm a family of NOD-like receptors (NLR) are expressed that recognize microbial material and can sense altered self molecules that appear under certain disease states. It has recently been shown that crystallization of normally soluble molecules can be interpreted as a danger signal and lead to activation of immune cells via NLRs. For example monosodium urate crystals, which appear in the clinical syndrome known as gout, are potent activators of NLRs. The NLR family members are thought to assemble into multimeric receptor complexes, termed inflammasomes, which control the active state of the inflammatory caspase-1. Active caspase-1 is able to cleave the proforms of the cytokines IL-1b, IL-18 and IL-33 into the active mature cytokines. Thus, inflammasomes are important receptor complexes which control the processing of highly inflammatory cytokines and may play a key role in the development of auto-inflammatory and chronic inflammatory diseases.


4:10
The Alphavbeta6 Integrin: A Mediator of TGF-Beta Activation and Therapeutic Target in Fibrosis and Oncology

Shelia Violette, Director, Discovery Immunology, Biogen-Idec


The avb6 integrin is expressed at low levels in healthy adult tissue and significantly upregulated, mainly on epithelial cells, in fibrotic disease and carcinomas. avb6 binds and activates the latent precursor form of TGF-b and mediates interactions with matrix proteins. TGF-b is central to the initiation and maintenance of fibrosis, a pathological process marked by the replacement of diseased tissue with excess extracellular matrix and ultimately leading to organ scarring and failure. This cytokine is also implicated in promoting tumor progression, and metastases. We evaluated avb6 expression in human disease and in preclinical animal models. avb6 is highly upregulated in lung and kidney disease associated with fibrotic pathology, and in liver disease associated with acute biliary duct injury. It is also highly upregulated in squamous cell carcinomas and metastatic lesions, with expression often predominant at the tumor-stromal interface reflecting a unique mechanism for localized TGF-b activation promoting tumor growth and invasion. We generated potent and selective blocking avb6 antibodies, and tested their ability to inhibit fibrosis and tumor progression in animal models. These antibodies have potent anti-fibrotic activity and inhibit tumor progression in multiple models of disease. A lead clinical candidate antibody has been selected and humanized. Our findings support a role for avb6 in fibrosis and tumor progression and the potential for therapeutic intervention with a function blocking antibody. This approach provides an opportunity to locally inhibit TGF-b and minimize the potential for tumor promoting and pro-inflammatory activity that may be associated with systemic blockade of the TGF-b pathway.

The TGF-b cytokine is a major driver of fibrotic disease and has been implicated in tumor growth and progression. However, chronic systemic inhibition of this target with therapeutics may be limited by the potential for tumor promoting and pro-inflammatory side effects. The avb6 integrin is expressed a low levels in healthy human tissue and highly upregulated in disease. This integrin is also an important mediator of TGF-b activation and thus offers a novel approach for localized inhibition of the TGF-b pathway. We will describe the generation and development of a humanized function blocking avb6 antibody for the treatment of fibrotic disease and for oncology indications.

• Inhibition of a novel target that selectively mediates TGF-b activation in a tissue specific manner.
* Overlapping targets in fibrotic disease and oncology indications.
• Generation of monoclonal antibodies to monitor avb6 expression (diagnostic), and for use in preclinical proof of concept and toxicity studies.
• Development of a humanized monoclonal antibody for therapeutic application in human fibrotic disease and oncology indications.


4:35
Cytokines and Cytokine Modulation in Inflammatory Bowel Disease

Silvia Melgar, Ph.D., Senior Research Scientist, AstraZeneca



Inflammatory bowel disease (IBD) encompasses two major chronic inflammatory diseases, ulcerative colitis (UC) and Crohn´s disease (CD), which affects the gastrointestinal tract in humans. The etiology of IBD is still unknown, but the pathogenesis is likely dependent on the interaction between local immune reactions and environmental factors in genetically susceptible individuals. To this date, there are no specific animal models for either UC or CD. Instead, more than 30 experimental models have been developed, that reflect different features of the intestinal inflammation. Among these, the dextran sulphate sodium (DSS) induced colitis in rodents is widely used, due to the convenient induction of intestinal inflammation (DSS in drinking water ad libitum) and good reproducibility. However, in parallel to the development of new animal models, there has also been a debate regarding the predictability of the animal models for the human diseases. In this talk I (i) will summarize the current human data considering new anti-cytokine therapies (ii) our recent experiments using different conventional therapies such as steroids and biological therapies e.g., anti-cytokine antibodies which has been investigated in vivo and ex vivo models. The results will be discussed in the context of translating information from experimental mouse studies into human efficacy data.

Benefits of this talk:
i) Recent cytokine data from human IBD studies
ii) Animal IBD models and their validation
iii) Relevance of ex vivo to in vivo models
iv) Translation of murine disease in context to human disease


5:00
Poster Session & Networking Reception

Top of the page


Day 2, Tuesday, , January 29, 2008


7:30
Continental Breakfast


7:55
Chairperson’s Review of Day One


Session III – Chemokines


8:00
Chemokines Therapies: Technology to Evolution

Amanda Proudfoot, Ph.D., Principal Scientist, Immunology, Merck Serono Geneva Research Centre



Inappropriate cell recruitment is a hallmark of all autoimmune, allergic and inflammatory diseases. The prevention of inflammation by interfering with cellular recruitment by neutralization of cytokines and adhesion molecules has proven to be successful in the clinic. Chemokines are important potential targets due to the central role they play in the cell recruitment process. Chemokines are unique amongst cytokines as they signal through 7 transmembrane (7TM) receptors, allowing the identification of small molecule inhibitors through high throughput screening. The object of this presentation is to discuss the validity and feasibility of targeting several points of therapeutic intervention offered by the chemokine system, and to assess the state of play within the field to date. Although some trials disappointingly did not achieve their goal, nature has devised strategies to inhibit the chemokine system highlighting the relevancy of these targets.


8:25
Regulation of the Inflammatory Response by the Chemokine Receptor D6

Gerry Graham, University of Glasgow


The chemokine receptor D6 is a highly atypical chemokine biding protein. It is extremely promiscuous and binds all inflammatory CC chemokines but not constitutive CC chemokines. It is not expressed on leukocytes but appears to be expressed by lymphatic endothelial cells in the skin, gut and lung. Previous in vitro work has indicated a role for D6 as a ‘decoy’ receptor which internalises and degrades chemokines with remarkable efficiency. To analyse the roles for D6 in vivo, we have generated D6 null mice and have examined the response of these mice in models of cutaneous inflammation. Specifically we have used the phorbol ester, TPA, to induce cutaneous inflammation. Basically the D6-null mice display an exaggerated response to cutaneous inflammation which is associated with an inability to properly remove chemokines from inflamed sites. In addition, these D6-null mice show aberrant responses in other models of tissue specific inflammation. Thus we have concluded that D6 is essential for the resolution of chemokine-driven inflammatory responses. More recently we have examined the responses of the D6-null mice in murine models of skin tumorigenesis. These studies have shown that D6-null mice are more susceptible to tumorigenesis in a classic 2-stage carcinogenesis model. In addition we have been working with transgenic mice expressing D6 under the control of the keratin 14 promoter. This transgenic over-expression significantly protects mice from cutaneous inflammation and skin tumorigenesis and suggests that D6 may be therapeutically useful if appropriate in vivo expression can be achieved. Finally we have been detailing the cell specific expression patterns of D6 and these data will be presented.


8:50
Inhibition of CCR9 as a Therapeutic Approach: Beyond the Small Intestine


Thomas Schall, President & CEO, ChemoCentryx





CCR9 is a chemokine receptor known to play a pivotal role in migration of T-cells to the mucosal surface of the gut. Ablation of CCR9 or its ligand CCL25/TECK by genetic deletion or neutralizing antibodies impairs the migration of T-cells to the gut in mice. CCL25 is known to be expressed in the human intestinal mucosa , and is up regulated in Crohn’s and Celiac patients.

It is well established that CCR9 is critical for T-cell homing to the small intestine. We have recently found that CCR9 is also critical for development of ulcerative colitis in animal models, indicating a role for this chemokine receptor in cell migration to the large bowel. To extend these observations, we have used immunohistochemical analyses to explore the presence of CCR9 within both small and large bowel tissues collected from healthy individuals and those suffering from inflammatory bowel disease. Using a sensitive immunohistochemical staining protocol, CCR9+ T-cells were detected within both the small and large bowel of healthy donors, and showed a significant increase in numbers in the same tissues collected from patients with Crohn’s Disease and ulcerative colitis respectively. Interestingly, CCL25 was also detected in the mucosa of both small and large bowel. These data are consistent with our previous observations that CCR9 antagonists provide protection in the MDR1a-/- model of murine colitis, and the TNF DARE model of murine Crohn’s disease. They establish expression of CCR9/CCL25 within the large intestines of ulcerative colitis patients, providing scientific rationale for use of CCR9 antagonists (currently in clinical trial for human Crohn’s Disease) to treat patients with Ulcerative Colitis.


9:15
Role of Chemokines in Pain Mechanisms


Catherine Abbadie, Senior Research Fellow, Merck



Many patients suffer from neuropathic pain as a result of injury to the peripheral nervous system (e.g. post-herpetic neuralgia or diabetic neuropathy) or to the central nervous system (e.g. spinal cord injury or stroke). The most distinctive symptom of neuropathic pain is allodynia, whereby normally non-painful stimuli, such as light touch, become painful. Traditionally, inflammatory and neuropathic pain syndromes have been considered distinct entities; however, recent evidence belies this strict dichotomy. Nerve damage can stimulate macrophage infiltration and increase the number of activated T cells. Under these conditions, neuron-inflammatory and immune responses contribute as much to the development and maintenance of pain as the initial damage itself. Recently, studies using animal models have shown that upregulation of chemokines is one of the mechanisms underlying the development and maintenance of chronic pain.

Providing insight into the role of chemokines in pain mechanisms:
• Chemokines in the ‘inflammatory soup’
• Chemokines and acute nociception
• Chemokines and chronic pain
• Chemokines and neuropathic pain: role of CCL2–CCR2
• CCL2 and CCR2 (transgenics, knockout mice, small molecule inhibitors)




9:40
Networking and Refreshment Break


Session IV - Technological Developments in Cytokines Biology


10:10
Visualization and Identification of IL-7 Producing Cells


Scott Durum, Ph.D., Head of Cytokines and Immunity Section, Laboratory of Molecular Immunoregulation, NCI, NIH



IL-7 is required for T cell development and homeostasis, however the actual sites of IL-7 production have never been clearly identified. Using a bacterial artificial chromosome recombineering technique, ECFP was knocked into the IL-7 locus. Because the construct lacked a signal peptide it accumulated inside cells and permitted visualization of IL-7-producing stromal cells. The thoracic thymus, cervical thymus and bone marrow contained such cells. In the thymus, cortical epithelial cells showed extensive IL-7-containing processes that closely contacted thymocytes. Central memory CD8 T cells require IL-7; intravital imaging demonstrated that they home to bone marrow and physically interact with IL-7 producing cells. The results support a paracrine relationship between IL-7 producing cells and the T cells that require IL-7.


10:35
Cytokine Signature of Resident Bone Marrow Myeloid Cell Subsets

M. Lamine Mbow, Senior Research Investigator, Genomics Institute of the Novartis Research Foundation



Studies aimed at determining the immunomodulatory factors that can influence the local immunological milieu are critical in advancing our understanding of the local innate immune response in the bone marrow. We therefore undertook a series of studies to identify and characterize immune cell populations in normal murine bone marrow that have the capacity to respond to various microbial ligands. Our studies led to the identification of previously uncharacterized myeloid immune cell populations that differentially express TLR and chemokine receptors and display unique cytokine signatures in response to TLR agonist stimulation. Specifically we found that the murine bone marrow harbors heterogeneous populations of DEC205+CD11b+Gr-1+ cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205+CD11b+Gr-1low cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-, and IL-12 p70 production following stimulation with TLR7 and TLR9 agonists, R848 and CpG, respectively. In contrast, the DEC205+CD11b+Gr-1high cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205+CD11b+Gr-1high cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.

Benefits of this talk:
1. Description of new bone marrow cell populations involved in the local innate immune response
2. Studies support the concept that discrete myeloid cell populations in the bone marrow play a critical role in innate immunity
3. These studies open the door to new avenues of research to better understand the local innate immune in the bone marrow
4. Opportunity to discuss new concepts regarding the impact of chemokine and innate immune receptors signaling on modulating the local innate immune response in the bone marrow



11:00
IL-33 Signals through ST2 and IL-1R Accessory Protein (AcP) to Amplify both Th1 and Th2 Responses

Dirk Smith, Senior Research Scientist, Inflammation Research, Amgen Inc.


IL-33 is an IL-1 family member recently identified as the ligand for ST2, a member of the IL-1 receptor family. IL-33 has been show to induce intracellular signals similar to those utilized by IL-1 and other members of the IL-1 family. IL-1 signaling depends on a heterdimeric receptor comprised of a primary binding subunit (IL-1R) and the IL-1R Accessory Protein (AcP), a second subunit required for signaling. We have found that IL-33 binding to ST2 recruits AcP and that the presence of AcP augments binding affinity and is necessary to elicit a cellular response. Additional control of IL-33 activity may be provided by the soluble form of AcP, which enhances the ability of soluble ST2 to neutralize IL-33 in vitro. Stable ST2 expression is found on polarized Th2 T cells as well as immature and mature mast cells. Indeed, IL-33 potently activates Th2 cytokine production from human Th2 cells and also drives the maturation and activation of human mast cells. In cooperation with other cytokines, IL-33 can also act on other immune cells to amplify Th1 as well as Th2 type responses. Based on our findings, we propose that IL-33 represents a Th2-oriented cytokine that is also capable of amplifying non-polarized inflammation depending on the physiologic context.


11:25
Novel Interferon Molecules Generated By Gene Shuffling: From Molecular Breeding to Clinical Evaluation

Julian Symons, D. Phil., Head, HCV Biology, Roche


The use of pegylated interferon alpha (IFN alpha) remains the cornerstone of treatment for patients with chronic hepatitis C (CHC). However, currently approved treatment regimens only provide an approximate 50% sustained virological response in those individuals infected with genotype 1 HCV. R7025 (Maxy Alpha) is a novel human pegylated IFN alpha molecule generated using DNA Shuffling technology and is currently in clinical development for the treatment of CHC. In vitro, R7025 demonstrated up to 50-fold greater antiviral activity when compared to PEG-IFN alpha-2a against a number of viruses. In addition, the molecule exhibited greater immunomodulatory activity as shown by its ability to differentiate naïve human T cells into IFN gamma secreting Th1 cells, activate natural killer cells and cause the maturation of immature dendritic cells. Interestingly, in contrast to the enhanced antiviral and immunomodulatory activity, R7025 demonstrated similar activity to PEG-IFN alpha-2a in a Daudi cell antiproliferative assay. This unusual ratio of antiviral/immunomodulatory: antiproliferative activity has recently been mapped to a novel pseudogene motif in a putative IFNAR1 binding region of the molecule (Brideau-Andersen et al, Proc. Natl. Acad. Sci. (USA) 104, 8269, 2007). To determine the in vivo pharmacokinetic (PK) and pharmacodynamic (PD) properties of the molecule, R7025 was administered s.c. to cynomolgus monkeys at doses of 0.03 and 0.3 mg/kg and serum concentrations were determined using an enzyme-linked immunosorbent assay. The time of maximum concentration (Cmax) of R7025 was achieved between 33 and 48 hours and a sustained serum concentration was observed for one week, similar to that observed for PEG-IFN alpha-2a and consistent with a once weekly dosing interval. The pharmacodynamic activity of R7025 was measured by the induction of mRNA from IFN-sensitive genes in cellular RNA derived from whole blood and by serum levels of 2’,5’-oligoadenylate synthetase (2’,5’-OAS) and neopterin. Enhanced gene expression was observed at 8 hours after R7025 administration and mRNA levels for each gene remained elevated over the course of the week. Increased serum 2’,5’-OAS activity and neopterin levels were observed between 4 and 24 hours post administration and both remained above baseline levels for at least 10 days. In conclusion, R7025 demonstrates greatly enhanced in vitro properties ideally suited to the treatment of CHC and a unique ratio of antiviral/immunomodulatory to antiproliferative activity. Further, in vivo, the molecule exhibits PK/PD properties consistent with at least once weekly dosing intervals.


11:50
Luncheon


12:50
Oral Presentations from Outstanding Abstracts


Please submit your abstract by December 28, 2007


Session V - Targeting of Cytokine Receptors


1:20
Blocking IL-13 Signaling with Soluble IL-13 Receptors: A Potential Therapeutic Strategy

Ray Donnelly, Ph.D., Senior Investigator, Division of Therapeutic, Proteins, FDA Center for Drug Evaluation & Research


IL-13 plays a central role in the pathogenesis of many allergic and atopic diseases. The functional receptor complex for IL-13 is a heterodimer composed of the ligand-binding chain, IL-13R-alpha 1, and the IL-4R-alpha chain. IL-13 binds initially to the IL-13Ra1 chain, and then rapidly recruits the IL-4Ra chain to assemble the active receptor complex. Ligand-mediated assembly of the IL-13 receptor complex induces the activation of STAT6 and subsequent expression of STAT6-responsive genes such as arginase-1 and eotaxin. In view of IL-13’s central role in allergic disease, there is a strong rationale for developing IL-13 antagonists. One approach to blocking IL-13 activity is to use the soluble IL-13 receptor-alpha 2 chain (IL-13Ra2). IL-13Ra2 has a higher affinity for IL-13 than the membrane-associated IL-13 receptor, IL-13Ra1. We found that sIL-13Ra2 can competitively inhibit the activation of STAT6 by IL-13 in several cell types, including macrophages and fibroblasts. Soluble IL-13Ra2 was more effective than either soluble IL-13Ra1 or soluble IL-4R protein at blocking IL-13 signaling. These findings indicate that soluble IL-13Ra2 or an IL-13Ra2-Fc fusion protein might provide a very effective means by which to block IL-13 activity.



1:45
Targeting IgE vs. Th2 Effector Function in Models for Type I Hypersensitivity and Airway Hyperresponsiveness

Henry Hess, Head of Immuno-pharmacology, Biogen-Idec


2:10
Regulation of Tight Junction Functions by Agonist and Antagonist Peptides

Sefik S. Alkan, Executive VP, Discovery and Preclinical Development, Alba Therapeutics


Tight junctions (TJ) are intercellular elements that control paracellular permeability and cellular polarity. It is now understood that TJs are highly dynamic structures that are regulated in response to exogenous stimuli such as antigen exposure. Endogenous stimuli such as TNF, IFN-g, IL-1, IL-4, IL-13 are also known to induce TJ disassembly while TGF-b, IL-10, IL-17 increase barrier function. Barrier leak is associated with pathological conditions such as celiac disease, IBD, MS and Type1 diabetes. Alba’s barrier function platform consist of a series of peptides derived from zonula occludens toxin (Zot), a protein secreted by Vibrio cholerae that transiently and reversibly opens epithelial TJs. Our pilot “agonist”, AT-1002, is a 6-mer, Zot derived peptide that induces leak in tissues such as intestinal mucosa, nasal epithelia, vascular endothelia and the blood brain barrier. Some agonists demonstrate profound mucosal adjuvant effect, while others may be useful for trans-epithelial and endothelial drug delivery. Our platform also includes “antagonists” such as AT-1001, an 8-mer peptide which inhibits AT-1002 mediated tight junction disassembly. AT-1001 treatment also inhibits re-distribution of TJ protein ZO-1 and MAP kinase activation caused by AT-1002 in Caco2 cells. The net result of these events is inhibition of stimulus induced tight junction opening. In addition, AT-1001 also inhibits ZO-1, F-actin and p-myosin light chain redistribution induced by gliadin (the causative agent of Celiac Disease) in IEC6 cells. Alba is currently developing oral AT-1001 for the treatment of Celiac Disease and exploring its use in Type 1 diabetes and Inflammatory Bowel Disease.


2:35
Targeting Cytokines in Asthma

Nestor A. Molfino, M.D., MSc, FCCP, Senior Director, Clinical Development, Inflammation and Autoimmunity, Medimmune-AZ Biologics Unit


Asthma is a condition or group of conditions characterized by airway inflammation, variable airflow obstruction and airway hyperresponsiveness. While the cause of asthma is unknown and gene environment interactions are likely, a number of pro-inflammatory cytokines have been associated with the asthma phenotype. The most widely studied cytokines include those derived from Th2 T-cells, namely IL4, IL5, and IL13. In the last few years there have been a number of studies in asthmatics trying to block these cytokines or its receptors with mixed results.
In the current presentation, human data with new monoclonal antibodies targeting cytokines or its receptors will be presented and discussed.


3:00
Potent, Orally Active CXCR2-CXCR1 Antagonists as Anti-inflammatory Therapy

Jianhua Chao, Schering Plough


3:25
Conference Concludes

 
 
Organized by: Nina Tran
Invited Speakers: Visit our web to see invited speakers!
 
Deadline for Abstracts: December 28, 2007
 
Registration: Register 2, the 3rd goes FREE - Register Now! 10% off if you register by December 28, 2007!
E-mail: nina.tran@gtcbio.com
 
   
 
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