NIHRDataInsights/HRCSData huggingface dataset:
Title: Regulators of WIP function and podosome activity
Funder: Medical Research Council

Abstract: Dendritic cells (DC) and macrophages elaborate podosomes, specialised integrin and f-actin rich structures that serve as labile adhesion sites to the extracellular matrix. The actin nucleation promoting protein WASP (Wiskott-Aldrich Syndrome Protein) acting through the Arp2/3 complex is the major regulator of actin filament formation in podosomes since DC and macrophages from WASP-null humans and WASP knockout mice fail to make podosomes. Such leukocytes exhibit defects in cell migration and chemotaxis in vitro and in vivo which are thought to underlie the immune dysfunction seen in WAS patients. In 80% of studied WAS patients, the syndrome arises from missense mutations in the WASP-binding site of the WASP-interacting protein WIP. Within cells WASP does not exist as a single entity but is complexed with WIP (WASP-Interacting Protein) and we have shown that WIP is required to protect WASP from degradation by endogenous calpains and to translocate WASP to sites of podosome formation. At some stage, WASP has to be released from WIP in order to facilitate its degradation and thus the turnover of podosomes. We propose here that the WIP/WASP complex regulates the formation and turnover of podosomes in a process influenced by WIP phosphorylation. Earlier findings suggested that a serine phosphorylation of WIP within the WASP-binding domain regulates its association with WASP . Despite repeated attempts, we failed to reproduce this finding in our current work (data not shown). We have subsequently learned that the earlier reports have since been modified by the author. So to date, there is no known link between the phosphorylation of WIP and the release of WASP from a complex. In this study we aim to elucidate: a) the spatio-temporal kinetics of actin, WASP and WIP within podosomes following expression of WIP phosphomutant isoforms b) the role of WIP phosphorylation in the regulation of macrophage/DC podosome function and cell invasion through matrix barriers c) the endogenous kinase(s) that phosphorylate WIP and their upstream activation. To this end, we shall use a human model myeloid cell system together with DC generated from Wild Type and WIP-null mice. We have developed highly efficient and non-perturbing lentivirus delivery of WIP, WASP and actin cDNAs to cell cultures and high resolution optical imaging of protein interaction kinetics during the formation and turnover of podosomes during cell migration on and into basement membrane derived matrix and across an endothelium.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of T-bet in Foxp3+ T regulatory cell-mediated protection from autoimmune inflammation
Funder: Medical Research Council

Abstract: T regulatory (Treg) cells expressing the Foxp3 transcription factor are an essential component of immune tolerance. Treg function in multiple ways to prevent the development of autoimmune inflammation and can also induce the natural resolution of inflammation once it is established. This latter property makes new therapeutic approaches aimed at improving Treg function particularly appealing. Using murine experimental autoimmune encephalomyelitis (EAE) as a model of organ-specific (CNS) type 1 autoimmune inflammation, we have previously described that Treg accumulate in the CNS in response to inflammation and are absolutely essential to its natural resolution. These CNS Treg are remarkably suppressive (as assessed by ex vivo functional assays). We have also established therapeutic models in which we transfer myelin-responsive TCR transgenic Treg to protect against, or to cure ongoing EAE. These therapeutic studies revealed that Treg can readily express T-bet, a transcription factor primarily associated with T helper (Th)1 cells, a CD4+ population known to be pathogenic in EAE. Moreover, we find that the CNS Treg that trigger natural EAE resolution strongly express T-bet, whereas their counterparts from the peripheral lymphoid organs of the same mice are T-bet-. Our central hypothesis is that the induction of T-bet expression in Treg at sites of type 1 inflammation has two essential roles:- a) enabling efficient Treg-mediated suppression of Th1 effector function and b) inhibiting the potentially pro-inflammatory action of the Treg themselves. Furthermore, we propose that integrating T-bet expression as a component of Treg functional profile will provide maximal therapeutic potency. To test this hypothesis, our research aims are to:- 1) determine whether T-bet expression within CNS Treg is required specifically for their ability to suppress Th1-driven pathology during the natural resolution of CNS inflammation. 2) understand the impact of T-bet expression on the Treg transcriptional profile. 3) profile the functionality of T-bet-sufficient versus T-bet-deficient Treg extracted from the inflamed CNS. 4) assess whether T-bet expression is required for therapeutic Treg function in EAE. 5) explore whether we can enforce T-bet expression in Treg in vitro and in vivo. We shall interrogate our hypothesis by use of a unique collection of GM mice in which specifically Treg can or cannot express T-bet (i.e. the pathogenic T cell populations will be T-bet-sufficient). This project will therefore provide important new information on Treg biology and may open new avenues to harnessing their therapeutic potential.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Allergenicity and allergen dominance: structural requirements for IgE-dependent recognition by B cells & effector cells
Funder: Medical Research Council

Abstract: IgE is the antibody isotype involved in mediating allergic reactions. Our structural and functional studies on B-cell switching to IgE expression, regulation of IgE synthesis, and the mechanism of IgE/receptor interactions (which has spawned an anti-IgE small-molecule drug discovery programme), together with the development of a novel cloning/expression system to produce IgE molecules from human B-cells and plasma cells from allergic subjects, places us uniquely to investigate a fundamental question in allergy, i.e. what makes a protein allergenic? We shall test our hypothesis that the disposition of epitopes on the allergen structure, and their IgE-binding affinity, determine whether they are recognized by the IgE-B-cell receptor (to cause B-cell proliferation and IgE synthesis) and by receptor-bound IgE on mast cells and basophils (to cause immediate hypersensitivity reactions); antigens that are not allergens may fail at one or both of these stages. We further propose that the bent structure of IgE places isotype-specific constraints upon these recognition steps. We shall study peanut allergy as a paradigm, not only for its medical importance, but also because certain peanut allergens are known to be dominant - IgE antibodies to these correlate with clinical reactivity - while others are not. Specifically we shall: 1) Clone and express peanut allergen-specific IgEs from single B-cells and plasma cells from allergic subjects; these will have naturally ?matched? H- and L-chains, which we believe is critical for understanding pathogenicity in allergic disease. 2) Determine the structures of allergen/IgE/receptor complexes to understand the constraints of the Fab regions in IgE. 3) Employ TIRF microscopy with lipid bilayers to study interactions of allergens (and combinations of allergens) with membrane-bound and receptor-bound IgE using the peanut allergen-specific antibodies. 4) Investigate the bias in VH gene usage in the peanut allergen-specific IgEs, explore the role of known B-cell superantigens (SAg) with SAg-specific IgEs from human tissue, and discover whether dominant allergens behave as ?superallergens?. 5) Determine the structural basis of ?cytokinergic? IgE and its pathogenic potential in allergic disease by comparing IgEs with matched and unmatched VH/VL domains from allergic patients. 6) Correlate the structural studies with functional assays of IgE-BCR signalling and mast cell/basophil activation, using peanut allergens with combinations of the allergen-specific IgEs. Answers to the fundamental questions addressed here in peanut allergy will underpin immunotherapeutic and potentially disease-modifying ?cures? for other allergies, and may lead to the development of clinically relevant diagnostic tools to profile patient reactivity to dangerous allergens.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: The role of Caveolin-1 in regulating primary T cell responses
Funder: Medical Research Council

Abstract: The question of how T cell activation is controlled is fundamental to understanding how adaptive immune responses are regulated. T cells are activated by engaging cell surface receptors which link to multiple intracellular signalling pathways. Intracellular signalling needs to be co-ordinated in space and time so that proliferation and differentiation of T cells are both initiated and terminated appropriately. Failure to do so can lead to uncontrolled T cell expansion and potentially lethal inflammatory sequelae. We report, for the first time, expression in T cells of a molecule, Caveolin 1 (Cav1), known to be important for co-ordination of signal transduction pathways in many cell types. Cav1 is involved in regulation of inflammation and anchorage dependent growth control in endothelial and epithelial cells through its interaction with key signal transduction proteins such as: G protein a subunits, eNOS, EGF-R, PKCa, Integrins and Src-family kinases. We present preliminary data showing that Cav1 is involved in T cell receptor signalling and is phosphorylated in responce to T cell receptor engagement. We propose to further characterise its role in regulating T cell responses. These studies will tell us whether the function of Cav1 and phospho-Cav1 in T cells is the same as that described for adherent cells, which will enhance our knowledge about both Cav1 function and T cell biology. Alternatively there may be aspects of Cav1 function that are unique to T cells which will provide novel insights into the regulation of T cell activation and signalling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Mechanisms and immune consequences of Neutrophil trafficking via lymphatics
Funder: Medical Research Council

Abstract: Emerging evidence indicates that neutrophils - the key phagocytes of the innate immune system can also modify and even initiate adaptive immune responses either by cross-talk with antigen presenting cells or by directly functioning as antigen presenting cells themselves. These properties are critical in generating protective immune responses to important intracellular pathogens such as mycobacteria and Toxoplasma and protozoal parasites such as Leishmania where neutrophils induce Th1 responses that promote effective killing by macrophages. The capacity of neutrophils to modify such responses requires their exit from peripheral tissues and trafficking to the lymph nodes via afferent lymphatics. Just recently such trafficking has been observed in vivo using fluorescence microscopic imaging. However, the molecular mechanisms orchestrating the entry of neutrophils to lymphatics including the adhesion receptors chemokines and proteinases involved have yet to be elucidated. Moreover, the interactions of neutrophils with resident lymph node populations, their influence on ongoing immune responses and their fate within the lymph nodes are also obscure. This grant proposal seeks to investigate each of these areas using a combination of in vitro transmigration assays with primary lymphatic endothelial cells and function blocking antibodies together with in vivo assays measuring lymph node trafficking of fluorescent BCG treated neutrophils in appropriate transgenic and gene knockout animals by flow cytometry and confocal microscopy. In addition we will assess the cytokine profile of neutrophils having trafficked to the lymph nodes and investigate whether the presence of such neutrophils can influence or override the polarity of neighbouring responses in the same location. This is a straightforward proposal designed to understand the basic biology of a potential rate-limiting step connecting innate and adaptive immunity. The results of this research could well be exploited for the optimization of neutrophil-based vaccination strategies or for reshaping protective immune responses.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Modulating the local immune environment via gene transfer to suppress intraocular inflammation
Funder: Medical Research Council

Abstract: Uveitis is a recurrent autoimmune condition and a major cause of blindness in children and the working age population. If inadequately treated it can cause irreversible sight loss and current treatment options largely require systemic immunosuppression. Whilst improved treatments using systemic immunosuppressants and more recently biological therapies have been developed, there remain significant safety issues and side effects. Prolonged use of corticosteroids can induce cataract and glaucoma, as well as an increased risk of cardiovascular disease, bone loss and gastric ulceration. Multiple episodes of disease relapse also substantially increase the long-term cost of healthcare. Viral gene therapy vectors encoding anti-inflammatory cytokines have been used to treat other autoimmune diseases such as rheumatoid arthritis. Results from this lab have shown that the technique can attenuate the mouse model of experimental autoimmune uveitis (EAU) and endotoxin-induced uveitis (EIU). Given our increased understanding of cytokine biology and receptor-ligand interactions involved in immune mediated damage in these models, it is now timely to investigate the translational potential of immunomodulatory gene therapy as an agent for regulating the local immune tissue environment. AAV2/8 vectors efficiently transduce both photoreceptors and RPE cells, with high levels of transgene expression after just a few days. We will use AAV2/8 vectors carrying mouse cDNAs driven by CMV promoters to express different immunomodulatory molecules, including but not limited to, IL-1ra, IL-4, IL-10, CD200, TGF?, FasL and sTNFR (p55 [R1]and p75 [R2]) linked to eGFP by an IRES sequence, so that vector expression can be observed directly. These vectors have been generated in the lab, and most have been tested in vitro for biological function. Preliminary in vivo work showed a partial rescue of EAU and EIU. Further development of individual vectors and the use of combinations of vectors that target different pro-inflammatory pathways should provide a more complete, synergistic treatment of the disease and better preservation of visual function. The AAV2/8 vector will be delivered by intravitreal, intracameral or subretinal injection and the therapeutic effect on established disease compared to untreated EAU controls. The prevention of disease onset will also be examined. Immunohistochemistry and multi-colour flow cytometry will be used to quantify and assess the phenotype of infiltrating cells within the retina. In vivo fundus imaging, including non-invasive Topical Endoscopic Fundal Imaging developed in this lab will determine the overall disease effect upon retinal morphology and vascular permeability. Electroretinography will be carried out to quantify the preservation of retinal function.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Influence of interferon & Toll like receptors on endothelial hormones: relevance to pneumonitis & pulmonary hypertension
Funder: Medical Research Council

Abstract: Pulmonary vascular inflammation can occur with viral infection or interferon (IFN) therapy, which is used to treat hepatitis C and multiple sclerosis. Pulmonary hypertension (PH) is linked to infection with HIV and human herpes virus-8 and respiratory viruses are implicated in pneumonitis and acute lung injury. Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogen, which is associated with pulmonary vascular inflammation and, indelibly, with PH. Our pilot data shows that ET-1 is released from human pulmonary vascular smooth muscle cells when stimulated by the virally induced type I and type II IFNs. We have also shown in pilot experiments that human pulmonary vascular cells release ET-1 when activated with viral toll like receptor (TLR)8 ligands, but not when stimulated with bacterial TLR4 ligands. Our main aim is therefore to understand how IFN signalling regulates ET-1 and other vasoactive genes such as prostacyclin and nitric oxide (NO) in pulmonary vascular cells from patients with and without PH. Our other aims will be to determine the effect of viral mimics and of infection of cells with prototype viruses, using protocols firmly established in Professor Johnston‘s group, on IFN release, signalling and vasoactive gene expression in human pulmonary vascular cells. We will use cell culture and cell based assays. Cultures of human pulmonary artery smooth muscle cells and lung microvascular endothelial cells will be maintained according to protocols established over many years in Professor Mitchell‘s group. Bone morphogenetic protein receptor type 2 (BMPR2) ‘mutant‘ vascular smooth muscle cells from patients with PH will be cultured according to protocols of Professor Nicholas Morrell who is a collaborator. Cell cultures will be characterised by morphology and expression of CD31 (endothelial cells) and ?-actin (smooth muscle cells). ET-1 and prostaglandin release will be measured using ELISA. Cell proliferation will be measured by counting cells, measuring DNA levels and, if required, FACS analysis of proliferation/apoptosis markers. Gene expression will be measured using quantitative PCR. Protein expression will be measured using western blotting and imaged in cells using en face confocal microscopy. This study has scientific and medical importance. Data obtained will lead to a better understanding of viral and IFN-induced vascular inflammation. If our hypothesis is correct this project will provide the essential pre-clinical data required for a clinical study in which patients at risk of serious life-threatening reactions to virus or IFN therapy may be identified by measuring levels of vasoactive hormones such as ET-1.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: MRC Centre for Immune Regulation
Funder: Medical Research Council

Abstract: The mission of the Centre is to gain a better understanding of the regulation of the immune response with a view to developing strategies to augment beneficial responses and limit harmful responses leading to disease. The distinctive feature of research in the Centre, that unifies its component programmes, is its focus on microenvironmental factors controlling lymphocyte responses. Elucidation of these basic mechanisms is targeted to selected diseases with a particular focus on immune factors in auto-immune and inflammatory disease in joints, liver and kidney, and on chronic bacterial and viral infections in man. This is achieved by incorporation into the Centre of groups working on basic mechanisms alongside those investigating disease specific processes. In support of its mission, the Centre has the key aims of providing a well found research environment with state of the art technologies and a training environment in which young scientists and clinicians can gain experience in a wide range of cellular, molecular and in-vivo techniques in immunology. Existing strengths in the Centre focus on four areas of activity, mechanisms in lymphocyte development; induction and regulation of immunity in secondary lymphoid tissues; mechanisms in chronic inflammation; and cellular immunity to viral infection and tumours. To complement these, 3 new initiatives will be developed to add expertise in chemokine biology to the Centre s basic science programmes, to develop the capacity of the Centre to study the impact of ageing on the immune response and to develop the translation of Centre discoveries to therapeutic benefit.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM
Title: Over two years for the development of gene therapy to treat X-linked lymphoproliferative disease (XLP)
Funder: Action Medical Research

Abstract: Background_x000D_ X-linked lymphoproliferative disease (XLP) is a rare, inherited condition affecting an estimated 300 to 500 children worldwide. The altered gene responsible is linked to the X chromosome so, although girls can carry the disease, only boys will develop symptoms Boys with XLP become sick in childhood or early adolescence. Symptoms vary, but the most common effect of XLP is that the immune system reacts abnormally to certain viral infections with serious, sometimes life-threatening, complications. In addition, about a third of patients develop lymphoma, a cancer of the immune cells. At the moment, bone marrow transplant can be offered as a treatment. However, to get the best results, it is necessary to find a good match for the patient and carry out the transplant before they have any symptoms _x000D_The research project_x000D_ The researchers have evidence that inserting a normal copy of the affected gene into the patient's own bone marrow stem cells can improve XLP. This avoids the time-consuming and often difficult process of finding a suitable bone marrow donor. They now need to make this treatment safer and more effective by making sure that the 'new' gene is only active in the kinds of cells that it would be in a healthy person. Another way of using gene therapy is to insert a normal copy of the gene directly into the patient's T cells (white blood cells produced by the bone marrow that help to fight infection). This could alleviate most of the symptoms experienced by patients with XLP and may be safer than modification of bone marrow stem cells. The team will focus on this new approach, developing techniques for the gene therapy of T cells in the laboratory, and making sure it is safe and effective. Not only will this work offer a potentially curative option for children with XLP, but the techniques developed could be applied to many other childhood diseases, including some other primary immunodeficiencies.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFLAMMATORY AND IMMUNE SYSTEM