Publications by year
In Press
Gold L, Williams D, Groenendyk J, Michalak M, Eggleton P (In Press). Unfolding the complexities of ER chaperones in health and disease: Report on the 11th International Calreticulin Workshop.
Cell Stress and ChaperonesAbstract:
Unfolding the complexities of ER chaperones in health and disease: Report on the 11th International Calreticulin Workshop
The 11th International Calreticulin workshop was held May 15–18, 2015 at New York University School of Medicine-Langone Medical Center, New York. The meeting highlighted many of the new discoveries in the past two years involving the important role of molecular chaperones in physiological and pathological processes. Crucial to the understanding of these disease processes was the role of chaperones in maintaining quality control of protein processing in the endoplasmic reticulum, the importance of Ca2regulation acting through its action in stress-related diseases, and the trafficking of glycoproteins to the cell surface. Central to maintaining healthy cell physiology involves correct ER-associated protein degradation of specific misfolded proteins and information on different mechanisms involved in the degradation of misfolded proteins was revealed. This was a landmark meeting for the chaperone field in terms of new insights into their roles in physiology including the unfolded protein response, innate/adaptive immunity, tissue repair, the functions of calreticulin/chaperones from the cell surface and extracellular environment and in diseases including from neurodegenerative disorders, prion disease, autoimmunity, fibrosis-related disease, the host immune response to cancer and hematologic diseases associated with calreticulin mutations. The 12th calreticulin workshop is planned for the spring of 2017 in Delphi, Greece.
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2019
Wiersma VR, Clarke A, Pouwels SD, Perry E, Abdullah TM, Kelly C, Soyza AD, Hutchinson D, Eggleton P, Bremer E, et al (2019). Galectin-9 is a Possible Promoter of Immunopathology in Rheumatoid Arthritis by Activation of Peptidyl Arginine Deiminase 4 (PAD-4) in Granulocytes.
Int J Mol Sci,
20(16).
Abstract:
Galectin-9 is a Possible Promoter of Immunopathology in Rheumatoid Arthritis by Activation of Peptidyl Arginine Deiminase 4 (PAD-4) in Granulocytes.
The aetiology of rheumatoid arthritis (RA) is unknown, but citrullination of proteins is thought to be an initiating event. In addition, it is increasingly evident that the lung can be a potential site for the generation of autoimmune triggers before the development of joint disease. Here, we identified that serum levels of galectin-9 (Gal-9), a pleiotropic immunomodulatory protein, are elevated in RA patients, and are even further increased in patients with comorbid bronchiectasis, a lung disease caused by chronic inflammation. The serum concentrations of Gal-9 correlate with C-reactive protein levels and DAS-28 score. Gal-9 activated polymorphonuclear leukocytes (granulocytes) in vitro, which was characterized by increased cytokine secretion, migration, and survival. Further, granulocytes treated with Gal-9 upregulated expression of peptidyl arginine deiminase 4 (PAD-4), a key enzyme required for RA-associated citrullination of proteins. Correspondingly, treatment with Gal-9 triggered citrullination of intracellular granulocyte proteins that are known contributors to RA pathogenesis (i.e. myeloperoxidase, alpha-enolase, MMP-9, lactoferrin). In conclusion, this study identifies for the first time an immunomodulatory protein, Gal-9, that triggers activation of granulocytes leading to increased PAD-4 expression and generation of citrullinated autoantigens. This pathway may represent a potentially important mechanism for development of RA.
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2018
Jung J, Eggleton P, Robinson A, Wang J, Gutowski N, Holley J, Newcombe J, Dudek E, Paul AM, Zochodne D, et al (2018). Calnexin is necessary for T cell transmigration into the central nervous system.
JCI Insight,
3(5).
Abstract:
Calnexin is necessary for T cell transmigration into the central nervous system.
In multiple sclerosis (MS), a demyelinating inflammatory disease of the CNS, and its animal model (experimental autoimmune encephalomyelitis; EAE), circulating immune cells gain access to the CNS across the blood-brain barrier to cause inflammation, myelin destruction, and neuronal damage. Here, we discovered that calnexin, an ER chaperone, is highly abundant in human brain endothelial cells of MS patients. Conversely, mice lacking calnexin exhibited resistance to EAE induction, no evidence of immune cell infiltration into the CNS, and no induction of inflammation markers within the CNS. Furthermore, calnexin deficiency in mice did not alter the development or function of the immune system. Instead, the loss of calnexin led to a defect in brain endothelial cell function that resulted in reduced T cell trafficking across the blood-brain barrier. These findings identify calnexin in brain endothelial cells as a potentially novel target for developing strategies aimed at managing or preventing the pathogenic cascade that drives neuroinflammation and destruction of the myelin sheath in MS.
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2017
Hutchinson D, Clarke A, Heesom K, Murphy D, Eggleton P (2017). Carbamylation/citrullination of IgG Fc in bronchiectasis, established RA with bronchiectasis and RA smokers: a potential risk factor for disease.
ERJ Open Research,
3(3), 00018-2017.
Abstract:
Carbamylation/citrullination of IgG Fc in bronchiectasis, established RA with bronchiectasis and RA smokers: a potential risk factor for disease
Bronchiectasis (BR) and smoking are risk factors for rheumatoid arthritis (RA) development. The mechanisms by which smoking and BR trigger RA are unknown, but are associated with concurrent rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (anti-CCP) positivity. Anti-carbamylated protein antibodies (anti-CarP) have also been observed in BR patients and can be induced by smoking. Given that RF only has one antigen, immunoglobulin G (IgG) we have suggested that post-translational modifications to the Fc region of the heavy chain of IgG (IgGH) are a potential explanation for the clustering of the RA-associated autoantibodies in RA.Protein analysis was undertaken on 22 individuals. Four of the individuals had a diagnosis of BR at the time of protein analysis and subsequently developed RA up to 18â€
months following blood sampling. Four smoking RA patients and 4 patients with both BR and RA and 10 healthy controls were also studied.We identified modified arginines (Arg) frequently in the variable region and CH3 domains of IgG in patients and control subjects alike, but only observed carbamylated Lys and/or citrullinated Arg modifications in the RF binding site of the IgG CH2 domain of 5/12 (41.7%) patients investigated (1 BR, 2 RA and 2 BRRA), but in no control subjects (0/10, 0%) p=0.02.This is the first report of citrullination and carbamylation at the RF binding site of IgG in RA. These results point towards the concept of a universal antigen in RA, an antigen that is post-translationally modified at the Fc region of IgGH.
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Hutchinson D, Eggleton P (2017). Could Autophagy Induced by Misfolded Mutant α1 -Antitrypsin Z in Synovitis Explain the Association of α1 -Antitrypsin Z with Increased Anti-Citrullinated Protein Antibody Production in Rheumatoid Arthritis? Comment on the Article by McCarthy et al.
Arthritis Rheumatol,
69(12), 2403-2404.
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Clarke A, Perry E, Kelly C, De Soyza A, Heesom K, Gold LI, Ollier W, Hutchinson D, Eggleton P (2017). Heightened autoantibody immune response to citrullinated calreticulin in bronchiectasis: Implications for rheumatoid arthritis.
Int J Biochem Cell Biol,
89, 199-206.
Abstract:
Heightened autoantibody immune response to citrullinated calreticulin in bronchiectasis: Implications for rheumatoid arthritis.
Calreticulin (CRT) and citrullinated (citCRT) are implicated in rheumatoid arthritis (RA) pathology. citCRT binds to RA shared epitopes (SE) on HLA-DR molecules with high affinity and triggers pro-inflammatory events in adjacent cells. The aim of the study was to detect the presence of citCRT prior to developing RA and evaluate if citCT is a target for autoantibodies in RA cohorts with and without lung disease. Antibodies were assessed by ELISA against native CRT, citCRT and general protein citrullination, in sera from 50 RA patients without lung disease, 122 bronchiectasis (BR) patients, 52 bronchiectasis patients with RA (BRRA), 87 asthma patients and 77 healthy controls (HC). Serum citCRT was detected by immunoblotting and mass spectrometry. Genomic DNA was genotyped for HLA-DRB1 alleles. Patients were assessed for DAS28, rheumatoid factor, and anti-cyclic citrullinated peptide antibodies. Extracellular citCRT was detected in BR patients sera prior to them developing RA. A citCRT SE binding peptide GEWKPR261citQIDNPDYK was identified. Anti-CRT antibodies were observed in 18% of BR patients with or without RA. Anti-citCRT antibodies were observed in ∼35% of BR or RA patients, increasing to 58% in BRRA patients. In the RA alone patients 7/20 (35%) who were negative for RF and anti-CCP were anti-CRT antibody positive and had higher DAS28 scores than triple negative RA alone patients. Three of the four BR patients who developed RA over 18 months were anti-citCRT+ve SE+ve. The detection of citCRT in BR and development of anti-citCRT in BR patients suggests citCRT antigens are early targets of antigenicity in these patients, especially in SE+ve patients prior to the onset of RA.
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Eggleton P, Smerdon GR, Holley JE, Gutowski NJ (2017). Manipulation of Oxygen and Endoplasmic Reticulum Stress Factors as Possible Interventions for Treatment of Multiple Sclerosis: Evidence for and Against.
Adv Exp Med Biol,
958, 11-27.
Abstract:
Manipulation of Oxygen and Endoplasmic Reticulum Stress Factors as Possible Interventions for Treatment of Multiple Sclerosis: Evidence for and Against.
Multiple sclerosis (MS) is normally considered a chronic inflammatory disease of the central nervous system (CNS), where T-cells breaching the blood brain barrier react against proteins of the axonal myelin sheaths, leading to focal plaques and demyelination in the brain and spinal cord. Many current therapies are immunosuppressive in nature and are designed to target the immune system at an early stage of the disease. But there is no cure and MS may evolve into a neurodegenerative disease, where immunomodulatory treatments appear less effective. Neurodegeneration is influenced by oxidative and endoplasmic reticulum (ER) mediated stress which can be induced independently of immune processes. Since 1970, MS patients have been self-managing their long term symptoms using hyperbaric oxygen and reporting improvement in their symptoms, especially bladder control. In contrast, the majority of clinical trial evidence does not support the views of patients. Therefore does oxygen under pressure affect brain tissue by modulating oxidative or ER stress at the cellular level resulting in CNS tissue repair or deterioration? This chapter reviews our understanding and the role of oxidative and ER stress in the context of employing hyperoxia treatments to treat MS and evaluate its effects on neural cells.
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Eggleton P, Smerdon GR, Holley JE, Gutowski NJ (2017). Manipulation of oxygen and endoplasmic reticulum stress factors as possible interventions for the treatment of multiple sclerosis: Evidence for and against. In Asea AA, Geraci F, Kaur P (Eds.)
Multiple sclerosis: Bench to bedside global perspectives on a silent killer, Switzerland: Springer, 11-28.
Abstract:
Manipulation of oxygen and endoplasmic reticulum stress factors as possible interventions for the treatment of multiple sclerosis: Evidence for and against.
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Asea AAA, Geraci F, Kaur P (2017).
Multiple Sclerosis: Bench to Bedside Global Perspectives on a Silent Killer., Springer.
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Multiple Sclerosis: Bench to Bedside Global Perspectives on a Silent Killer
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Haile Y, Deng X, Ortiz-Sandoval C, Tahbaz N, Janowicz A, Lu J-Q, Kerr BJ, Gutowski NJ, Holley JE, Eggleton P, et al (2017). Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.
J Neuroinflammation,
14(1).
Abstract:
Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.
BACKGROUND: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. METHODS: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. RESULTS: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. CONCLUSIONS: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.
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2016
Hutchinson D, Murphy D, Clarke A, Eggleton P (2016). Are Rheumatoid Factor, Anti-Citrullinated Protein Antibodies, and Anti-Carbamylated Protein Antibodies Linked by Posttranslational Modification of IgG? Comment on the Article by Koppejan et al.
Arthritis Rheumatol,
68(11), 2825-2826.
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Eggleton P, Bremer E, Dudek E, Michalak M (2016). Calreticulin, a therapeutic target?.
Expert Opin Ther Targets,
20(9), 1137-1147.
Abstract:
Calreticulin, a therapeutic target?
INTRODUCTION: Calreticulin is an endoplasmic reticulum (ER) resident protein critical for maintaining Ca(2+) homeostasis and glycoprotein folding in the ER. The protein has also been identified on the cell surface of apoptotic and necrotic cells and implicated to play a role in immunogenic cell death and other extracellular functions. The molecular events that promote cell surface association of calreticulin are not clear. Under cell stress conditions (environmental, drug induced, hypoxia), calreticulin may be upregulated as it attempts to regulate cell survival, death or repair. The initial signaling mechanisms involved in these processes may be regulated by the unfolded protein response (UPR) and genome damage response (GDR) pathways. AREA COVERED: Here, the phenomenon of cell surface calreticulin and its extracellular functions are discussed, with a major emphasis on the process of immunogenic cell death. The evidence of how cell surface calreticulin may act as a damage associated molecular pattern molecule is evaluated, in addition to how these properties of the protein can be exploited for therapeutic vaccine development, cancer treatment and mediating other inflammatory processes. In addition, clarification of calreticulin functions from its intracellular, cell surface, and extracellular locations are provided. EXPERT OPINION: While the protein folding and immune-stimulatory properties of calreticulin can be exploited to develop therapies, the molecular pathways involved remain to be elucidated. Nevertheless, exploiting the multifaceted properties of calreticulin may in the future provide a means to treat a number of diseases.
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Eggleton P (eds)(2016).
Endoplasmic reticulum and its role in tumor immunity. Lausanne · Switzerland, Frontiers Media SA.
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Endoplasmic reticulum and its role in tumor immunity
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2015
Quirke A-M, Perry E, Cartwright A, Kelly C, De Soyza A, Eggleton P, Hutchinson D, Venables PJ (2015). Bronchiectasis: a model for chronic bacterial infection inducing autoimmunity in rheumatoid arthritis.
Arthritis and Rheumatology,
67(9), 2335-2342.
Abstract:
Bronchiectasis: a model for chronic bacterial infection inducing autoimmunity in rheumatoid arthritis.
Objective: Bronchiectasis (BR) is a risk factor for rheumatoid arthritis (RA). Here we examine the potential of BR in generating rheumatoid factors (RFs) and anti-citrullinated peptide antibodies (ACPA) in patients with BR alone and in patients with BR and RA (BRRA).
Methods: We studied 122 patients with BR alone, 50 BRRA, 50 RA without lung disease, with 87 asthma and 79 healthy subjects as controls. RFs were measured by an automated analyzer, and ACPA using CCP2. Fine specificities to citrullinated α-enolase (CEP-1), citrullinated vimentin (cVim) and fibrinogen (cFib) with their arginine control peptides (REP-1, Vim and Fib) measured by ELISA.
Results: in the BR patients 39% were ever smokers compared to 42% of the controls. Serum samples from BR patients had an increased frequency of RF (25%; p< 0.05) and 5% to CCP2, 7% to CEP-1, 7% to cVIM (all p=ns) and 12% cFib (p
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de Bruyn M, Wiersma V, Samplonius DF, Klip HG, Helfrich W, Nijman HW, Eggleton P, Bremer E (2015). CD20+ T cells have a predominantly Tc1 Effector Memory phenotype and are expanded in the ascitis of patients with ovarian cancer.
Oncoimmunology,
4Abstract:
CD20+ T cells have a predominantly Tc1 Effector Memory phenotype and are expanded in the ascitis of patients with ovarian cancer
Recent studies have highlighted a small subset of T cells that express the B cell marker CD20. In healthy volunteers this subset was INF-y producing, but in rheumatoid arthritis patients this subset was characterized by IL-17 production. However, the origin and relevance of this population of T cells remains unclear. Here, we identified that CD20 is transferred to T cells after B cell/T cell interaction.
After isolation of CD20+ T cells from the peripheral blood of healthy volunteers, the expression of CD20 remains stable for up to 48h of ex vivo culture. This CD20+ T cell population had a predominant (CD8+) effector memory T cell phenotype (~60-70%). Further, CD20+ T cells were found to almost exclusively produce IFN-y (~70% vs. ~20% in the CD20- T cell population). Compared to healthy
controls, this effector memory and IFN-y producing phenotype was retained in T cells from peripheral
blood or ascitic fluid of ovarian cancer patients. However, the percentage of CD20+ T cells was strongly increased in the ascitic fluid of OC patients (from ~6% in the peripheral blood to 23% in ascitic fluid). Taken together, the data presented here indicate that CD20 can be transferred to T cells upon T cell/B cell interaction. Further, CD20+ T cells are of memory and IFN-y producing phenotype and are strongly increased in ascitic fluid.
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Eggleton P, Michalak M, Bremer E (2015). Editorial: Endoplasmic Reticulum and its Role in Tumor Immunity.
Front Oncol,
5 Author URL.
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Perry E, Eggleton P, De Soyza A, Hutchinson D, Kelly C (2015). Increased disease activity, severity and autoantibody positivity in rheumatoid arthritis patients with co-existent bronchiectasis.
International Journal of Rheumatic Diseases,
18Abstract:
Increased disease activity, severity and autoantibody positivity in rheumatoid arthritis patients with co-existent bronchiectasis.
Aim: Patients with rheumatoid arthritis (RA) and co-existent Bronchiectasis (BRRA) have a 5-fold increased mortality compared to rheumatoid arthritis alone. Yet previous studies have found no difference in clinical and serological markers of RA disease severity between BRRA patients and RA alone. RA disease activity measures such as DAS28-CRP and anti-cyclic citrullinated peptide antibodies (anti-CCP) however have not been studied, so we assessed these parameters in patients with BRRA and RA alone.
Methods: BRRA patients (n = 53) had HRCT proven bronchiectasis without any interstitial lung disease and ≥2 respiratory infections/year. RA alone patients (n = 50) had no clinical or radiological evidence of lung disease. DAS28-CRP, rheumatoid factor (IgM) and anti-CCP were measured in all patients, together with detailed clinical and radiology records.
Results: in BRRA, BR predated RA in 58% of patients. BRRA patients had higher DAS28 scores (3.51 vs. 2.59), higher levels of anti-CCP (89 vs. 46%) and RF (79 vs. 52%) (p = 0.003) compared to RA alone. Where hand and foot radiology findings were recorded, 29/37 BRRA (78%) and 13/30 (43%) RA alone had evidence of erosive change (p = 0.003). There were no significant differences between groups in smoking history or DMARD/biologic therapy.
Conclusions: Increased levels of RA disease activity, severity and RA autoantibodies are demonstrated in patients with RA and co-existent bronchiectasis compared to patients with RA alone, despite lower tobacco exposure. This study demonstrates that BRRA is a more severe systemic disease than RA alone.
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Wiersma VR, Michalak M, Abdullah TM, Bremer E, Eggleton P (2015). Mechanisms of translocation of ER chaperones to the cell surface and immunomodulatory roles in cancer and autoimmunity.
Frontiers in Oncology,
5(7).
Abstract:
Mechanisms of translocation of ER chaperones to the cell surface and immunomodulatory roles in cancer and autoimmunity
Endoplasmic reticulum (ER) chaperones (e.g. calreticulin, heat shock proteins and isomerases) perform a multitude of functions within the ER. However, many of these chaperones can translocate to the cytosol and eventually the surface of cells, particularly during ER stress induced by e.g. drugs, UV irradiation and microbial stimuli. Once on the cell surface or in the extracellular space, the ER chaperones can take on immunogenic characteristics, as mostly described in the context of cancer, appearing as damage-associated molecular patterns recognized by the immune system. How ER chaperones relocate to the cell surface and interact with other intracellular proteins appears to influence whether a tumor cell is targeted for cell death. The relocation of ER proteins to the cell surface can be exploited to target cancer cells for elimination by immune mechanism. Here we evaluate the evidence for the different mechanisms of ER protein translocation and binding to the cell surface and how ER protein translocation can act as a signal for cancer cells to undergo killing by immunogenic cell death and other cell death pathways. The release of chaperones can also exacerbate underlying autoimmune conditions, such as rheumatoid arthritis and multiple sclerosis, and the immunomodulatory role of extracellular chaperones as potential cancer immunotherapies requires cautious monitoring, particularly in cancer patients with underlying autoimmune disease.
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Eggleton P, Bishop A, Smerdon G (2015). Safety and efficacy of hyperbaric oxygen therapy in chronic wound management: current evidence.
Chronic Wound Care Management and Research,
2Abstract:
Safety and efficacy of hyperbaric oxygen therapy in chronic wound management: current evidence
The breathing of pure oxygen under pressure to treat tissue damage has been employed for almost 45 years and has been investigated through prospective, retrospective and randomized control trials. The physiological effects of oxygen treatment on wound tissue are profound and include the activation of immune cells, changes in cytokine production, and modulation of inflammatory and bactericidal mediators. HBO also influences the biochemistry of whole cells, altering cell proliferation, angiogenesis, clotting and tissue regeneration. The precise effects of HBO on individual cell types and tissues are only beginning to be revealed in both animal and human studies. Many independent studies using HBO adjunctively with standard wound care have observed improved healing, most particularly for diabetic foot ulcers, and can result in a significant reduction in major amputations. Side effects occur infrequently, but myopia, ear barotrauma and rarely oxygen toxicity have been reported. As antibiotics become less available, and clinician time and complex dressings become more expensive, the use of HBO as a means of treating a variety of wound types may become an increasingly appropriate option for treatment.
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Wiersma VR, de Bruyn M, Wei Y, van Ginkel RJ, Hirashima M, Niki T, Nishi N, Zhou J, Pouwels SD, Samplonius DF, et al (2015). The epithelial polarity regulator galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux.
Autophagy,
11, 1373-1388.
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The epithelial polarity regulator galectin-9 induces fatal frustrated autophagy in KRAS mutant colon carcinoma that depends on elevated basal autophagic flux.
Oncogenic mutation of KRAS in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant Galectin-9 (rGal-9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rGal-9 rapidly internalizes via clathrin- and PKC/CRAF/MEK-dependent endocytosis and accumulates in the lysosomal compartment. Treatment with rGal-9 is accompanied by activation of frustrated autophagy in KRASmut CRC, but not in BRAFmut CRC. In KRASmut CRC, rGal-9 acts as a lysosomal inhibitor that inhibits autophagosome/lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rGal-9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rGal-9 has potent antitumor activity towards refractory KRASmut CRC cells that may be exploitable for therapeutic use.
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de Bruyn M, Wiersma VR, Helfrich W, Eggleton P, Bremer E (2015). The ever-expanding immunomodulatory role of calreticulin in cancer immunity.
Frontiers in Oncology,
5Abstract:
The ever-expanding immunomodulatory role of calreticulin in cancer immunity.
Calreticulin is a pleiotropic molecule that normally resides in the lumen of the endoplasmic reticulum (ER). Here it has various functions, ranging from regulation of calcium homeostasis to ensuring proper protein folding. More recently, calreticulin gained especial interest for its extracellular functions, where it has direct immunomodulatory activity. In this respect, calreticulin activates dendritic cells (DCs) and macrophages. In addition, certain anti-cancer therapies induce the translocation of calreticulin from the ER to the cell surface of dying cancer cells, where calreticulin dictates the immunogenicity of these cells. Interestingly, treatment with Tumor Necrosis Factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) also induces membrane calreticulin exposure on cancer cells. As shown here, calreticulin directly interacts with TRAIL and its receptor (TRAILR)-signaling complex, as well as with other TNF family members. of note, TRAIL is a well known immunomodulatory molecule, and is expressed on the surface of natural killer T-cells (NK T-cells). Therefore, calreticulin may have an as yet unrecognized wide(r) impact on immunity, with the TNF ligand family modulating virtually all aspects of the immune response.
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2014
Wiersma VR, He Y, Samplonius DF, van Ginkel RJ, Gerssen J, Eggleton P, Zhou J, Bremer E, Helfrich W (2014). A CD47-blocking TRAIL fusion protein with dual pro-phagocytic and pro-apoptotic anticancer activity. British Journal of Haematology, 164(2), 304-307.
Quirke AM, Perry E, Kelly C, De-Soyza A, Eggleton P, Hutchinson D, Venables P (2014). A6.3 Patients with bronchiectasis, with or without rheumatoid arthritis, have an elevated anti-citrullinated peptide antibodies (ACPA) response.
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A6.3 Patients with bronchiectasis, with or without rheumatoid arthritis, have an elevated anti-citrullinated peptide antibodies (ACPA) response.
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Eggleton P, Ukoumunne OC, Cottrell I, Khan A, Maqsood S, Thornes J, Perry E, Isenberg D (2014). Autoantibodies against C1q as a diagnostic measure of lupus nephritis:systematic review and meta-analysis.
Journal of clinical and cellular Immunology,
5(2).
Abstract:
Autoantibodies against C1q as a diagnostic measure of lupus nephritis:systematic review and meta-analysis
Objectives: to evaluate the diagnostic accuracy of C1q autoantibodies in identifying lupus nephritis (LN) in patients with systemic lupus erythematosus (SLE).
Data Sources and methods: Citation indexes were searched and 370 articles published from 1977 to 2013 were evaluated. The 31 selected studies included in the meta-analysis were cross-sectional in design. Among the 31 studies, 28 compared anti-C1q antibodies in 2769 SLE patients including those with (n = 1442) and without a history of LN (n = 1327). Nine studies examined anti-C1q in 517 SLE patients with active (n = 249) and inactive LN (n = 268). Hierarchical summary receiver operating characteristic (HSROC) random effects models were fitted to pool estimates of accuracy across the studies.
Results: Anti-C1q antibodies discriminated between patients with and without a history of LN, with a median specificity of 73.5%. The HSROC model estimated the corresponding sensitivity to be 70.4%. A hypothetical patient with a 55% prior probability of having a history of LN as opposed to no history (the median prevalence across 28 eligible studies) would have a post-test probability of 76.4% following a positive test result (positive predictive value) or 33.0% following a negative test result (negative predictive value). For differentiating active from inactive LN the median specificity of anti-C1q antibodies was 80%, with a corresponding estimated sensitivity value 75.7% based on the HSROC model. A hypothetical patient with a 56% prior probability of active. as opposed to inactive LN (the median prevalence across the 9 eligible studies) would have a post-test probability of 82.8% following a positive test result or 27.9% following a negative test result.
Conclusions: Although C1q antibodies are associated with lupus nephritis the post-test probabilities are not sufficiently convincing to provide reasonable certainty of the presence or absence of history of disease/active disease.
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Quirke A-M, Perry E, Cartwright A, Kelly C, De Sozya A, Eggleton P, Hutchinson D, Venables P (2014). Bronchiectasis: a Model for Chronic Bacterial Infection Inducing Autoimmunity in Rheumatoid Arthritis.
ARTHRITIS & RHEUMATOLOGY,
66, S185-S185.
Author URL.
Holley JE, Bremer E, Kendall AC, De Bruyn M, Helfrich W, Tarr JM, Newcombe J, Gutowski NJ, Eggleton P (2014). CD20+inflammatory T-cells are present in blood and brain of multiple sclerosis patients and can be selectively targeted for apoptotic elimination.
Multiple Sclerosis and Related Disorders,
3(5), 650-658.
Abstract:
CD20+inflammatory T-cells are present in blood and brain of multiple sclerosis patients and can be selectively targeted for apoptotic elimination
Background: a subset of T-cells expresses the B-cell marker CD20 and in rheumatoid arthritis
secretes Interleukin(IL)-17.IL-17 secreting T-cells(Th17)have also been implicated in the
inflammatory response in the central nervous system in multiple sclerosis(MS)and maybe a
potential target for elimination by biologic therapeutics.ScFvRit:sFasL comprises of a
rituximab-derived antibody fragment scFvRit genetically fused to human soluble FasL that
specifically eliminatedT-cells.
Objective: to determine the presence and phenotype of CD20+ T-cells in blood and brain of MS patients. Second, to determine whether scFvRit:sFasL can selectively eliminate CD20+
T-cells.After CD20-selective binding,scFvRit:sFasL is designed to trigger FasL-mediated activation-induced cell death of T-cells,but not B-cells.
Methods: Flow cytometry and immunohistochemistry were used to screen for CD20+ inflammatory T-cells in MS blood and brain tissue. ScFvRit:sFasL pro-apoptotic activity was evaluated by Annexin-V/PI staining followed by flow cytometry assessment.
Results: Peripheral blood(n=11) and chronic but not active lesions of MS patient brains (n=5) contained CD20+inflammatory T-cells. Activated CD20+ T-cells were predominantly CD4+ and secreted both IL-17 and INF-γ. ScFvRit:sFasL triggered CD20-restricted FasL-mediated activation-induced cell death in peripheral blood CD20+T-cells, but not CD20+ B-cells.
Conclusion: CD20+ inflammatory T-cells are present in blood and chronic brain lesions of MS patients. ScFvRit:sFasL selectively eliminated CD20+T-cells and may eliminate pathogenic T-cells without B-cell depletion.
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Aust J, Davison, J, Perry, E, Eggleton P, Hutchinson D, Chalmers J, Kelly C, De Soyza A (2014). Co-existent rheumatoid arthritis and non-cystic fibrosis bronchiectasis: Evidence for more severe pulmonary disease using the bronchiectasis severity index (BSI). ERS Annual Congress. 6th - 10th Sep 2014.
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Co-existent rheumatoid arthritis and non-cystic fibrosis bronchiectasis: Evidence for more severe pulmonary disease using the bronchiectasis severity index (BSI)
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Ryan BJ, Eggleton P (2014). Detection and characterization of autoantibodies against modified self-proteins in SLE sera after exposure to reactive oxygen and nitrogen species. In Eggleton P, Ward FJ (Eds.)
Systemic Lupus Erythematosus: Methods and Protocols, New York: Spinger, 163-171.
Abstract:
Detection and characterization of autoantibodies against modified self-proteins in SLE sera after exposure to reactive oxygen and nitrogen species
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Eggleton P, Bremer E (2014). Direct and indirect rituximab-induced T cell depletion: comment on the article by Mélet et Al.
Arthritis Rheumatol,
66(4).
Author URL.
Eggleton P, Bremer E (2014). Direct and indirect rituximab-induced T-cell depletion. Arthritis and Rheumatism
Eggleton P, Bremer E (2014). Direct and indirect rituximab-induced T-cell depletion: Comment on the article by Mélet et al.
Arthritis Rheum Author URL.
Perry L, Quirke A-M, Eggleton P, Kelly C, Hutchinson D, De-Soyza A, Venables P (2014). Diverse pulmonary insults lead to common anti-citrullinated peptide fine specificity profiles and may promote autoimmunity in RA. British Society for Rheumatology. 29th Apr - 1st May 2014.
Eggleton P, Ward FJ (2014). Foreword.
Holley JE, Bremer E, Kendall AC, de Bruyn M, Helfrich W, Tarr JM, Newcombe J, Gutowski NJ, Eggleton P (2014). Identification of CD20+ Th17 cells in the blood and brain of multiple sclerosis patients and possible elimination by novel anti-CD20 biological therapeutics.
Multiple Sclerosis and related disorders,
3(5).
Abstract:
Identification of CD20+ Th17 cells in the blood and brain of multiple sclerosis patients and possible elimination by novel anti-CD20 biological therapeutics
Background: a subset of T-cells expresses the B-cell marker CD20 and in rheumatoid arthritis secretes Interleukin (IL)-17. IL-17 secreting T-cells (Th17) have also been implicated in the inflammatory response in the central nervous system in multiple sclerosis (MS) and may be a potential target for elimination by biologic therapeutics. ScFvRit:sFasL comprises of a rituximab-derived antibody fragment scFvRit genetically fused to human soluble FasL that can eliminate T-cells.
Objective: to determine the presence and phenotype of CD20+ T-cells in blood and brain of MS patients. Second, to determine whether scFvRit:sFasL can selectively eliminate CD20+ T-cells. After CD20-selective binding, scFvRit:sFasL is designed to trigger FasL-mediated activation-induced cell death of T-cells, but not B-cells.
Methods: Flow cytometry and immunohistochemistry were used to screen for CD20+ inflammatory T-cells in MS blood and brain tissue. ScFvRit:sFasL pro-apoptotic activity was evaluated by Annexin-V/PI staining followed by flow cytometry assessment.
Results: Peripheral blood (n=11) and chronic but not active lesions of MS patient brains (n=5) contained CD20+ inflammatory T-cells. Activated CD20+ T-cells were predominantly CD4+ and secreted both IL-17 and INF-γ. ScFvRit:sFasL triggered CD20-restricted FasL-mediated activation-induced cell death in peripheral blood CD20+ T-cells, but not CD20+ B-cells.
Conclusion: CD20+ inflammatory T-cells are present in blood and chronic brain lesions of MS patients. ScFvRit:sFasL selectively eliminated CD20+ T-cells and may eliminate pathogenic T-cells without B-cell depletion.
Abstract.
Perry E, Eggleton P, De Soyza A, Htchinson D, Kelly C (2014). Increased disease activity, severity and autoantibody positivity in rheumatoid arthritis patients with co-existent bronchiectasis.
Arthritis Care and ResearchAbstract:
Increased disease activity, severity and autoantibody positivity in rheumatoid arthritis patients with co-existent bronchiectasis.
Background:
Patients with Rheumatoid Arthritis and co-existent Bronchiectasis (BRRA) have a 5 fold increased mortality compared to Rheumatoid Arthritis (RA) alone. Despite this, previous studies have found no difference in clinical and serological markers of RA disease severity between BRRA patients and RA alone. RA disease activity measures such as DAS28-CRP and modern autoantibodies, anti-cyclic citrullinated peptide antibodies (anti-CCP) however have not been studied.
Objectives:
To determine RA disease activity/severity measures and auto-antibody status comparing patients with BRRA and those with RA alone.
Methods:
Multi centre recruitment of 53 BRRA patients and 50 matched controls with RA alone. BRRA patients had HRCT proven bronchiectasis without any interstitial lung disease and ≥2 respiratory infections/year. RA alone patients had no clinical or radiological evidence of lung disease. All patients met the 2010 ACR/EULAR RA criteria. The affected joint counts, visual analogue score (global health) and CRP were used to calculate the DAS28-CRP. Serum rheumatoid factor (IgM) and anti-CCP were measured in all patients. Clinical records and radiology were reviewed and current disease modifying drugs, biologic drugs and hand and foot radiology findings recorded.
Results:
In BRRA, BR predated RA in 58% of patients. Higher DAS28 scores were observed in BRRA compared to RA alone(3.51 vs 2.59). Higher levels of RA autoantibody positivity were observed in the BRRA group than in RA alone for both anti-CCP (89 vs 46%) and for RF (79 vs 52%) (p=0.003). Where hand and foot radiology findings were recorded, 29/37 BRRA (78%) and 13/30 (43%) RA alone had evidence of erosive change (p=0.003). There were no significant differences between groups in smoking history or DMARD/biologic therapy.
Conclusions:
Increased levels of RA disease activity, severity and RA autoantibodies are demonstrated in patients with RA and co-existent bronchiectasis compared to patients with RA alone, despite lower tobacco exposure. Mechanistic research to explain this association is required.
Abstract.
Cottrell I, Khan A, Maqsood S, Thornes J, Eggleton P (2014). Meta-analysis as a diagnostic tool for predicting disease onset and/or activity in systemic lupus erythematosus. In Eggleton P, Ward FJ (Eds.)
Systemic lupus erythematosus: Methods and Protocols, New York: Springer, 249-259.
Abstract:
Meta-analysis as a diagnostic tool for predicting disease onset and/or activity in systemic lupus erythematosus
Abstract.
Quirke A-M, Perry E, Kelly C, de-Soyza A, Eggleton P, Hutchinson D, Venables P (2014). PATIENTS WITH BRONCHIECTASIS, WITH OR WITHOUT RHEUMATOID ARTHRITIS, HAVE AN ELEVATED ANTI-CITRULLINATED PEPTIDE ANTIBODIES (ACPA) RESPONSE.
Author URL.
Eggleton P, Ward FJ (2014). Preface.
Perry, E, Stenton, C, Kelly, C, Eggleton P, Hutchinson, D, De Soyza, A (2014). RA-autoantibodies as predictors of Rheumatoid Arthritis in non-CF Bronchiectasis patients.
European Respiratory Journal Full text.
Eggleton P, Ward FJ (eds)(2014).
Systemic Lupus Erythematosus., Springer.
Abstract:
Systemic Lupus Erythematosus
Abstract.
Perry E, Kelly C, Eggleton P, De Soyza A, Hutchinson D (2014). The Lung in ACPA-Positive Rheumatoid Arthritis: an Initiating Site of Injury?.
Rheumatology (Oxford),
53(7).
Abstract:
The Lung in ACPA-Positive Rheumatoid Arthritis: an Initiating Site of Injury?
Recent findings have highlighted the potential initiation of anti-citrullinated peptide antibodies (ACPA) in sites away from the joint. Periodontitis is an example of this concept. This process in the gums appears to be independent of smoking, the main environmental risk factor for ACPA-positive RA. There is an extensive literature regarding the potential role of smoking in the pathogenesis of ACPA-positive RA. As a consequence of this strong association, the lung has become the focus of research to determine if processes within the lung are linked to the generation of ACPA. Here we outline the current body of evidence and explore the hypothesis that the lung as an organ of immune defence has a role in the pathogenesis of the autoimmune disease ACPA-positive RA.
Abstract.
Perry E, Kelly C, Eggleton P, De Soyza A, Hutchinson D (2014). The lung in ACPA-positive rheumatoid arthritis: an initiating site of injury?.
Rheumatology (United Kingdom),
53(11), 1-11.
Abstract:
The lung in ACPA-positive rheumatoid arthritis: an initiating site of injury?
© the Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. Recent findings have highlighted the potential initiation of ACPA in sites away from the joint. Periodontitis is an example of this concept. This process in the gums appears to be independent of smoking, the main environmental risk factor for ACPA-positive RA. There is extensive literature regarding the potential role of smoking in the pathogenesis of ACPA-positive RA. As a consequence of this strong association, the lung has become the focus of research to determine whether processes within the lung are linked to the generation of ACPA. Here we outline the current body of evidence and explore the hypothesis that the lung as an organ of immune defence has a role in the pathogenesis of the autoimmune disease ACPA-positive RA.
Abstract.
2013
Wiersma V, He Y, Samplonius D, van Ginkel R, Gerssen J, Eggleton P, Bremer E, Helfrich W (2013). A CD47-blocking TRAIL fusion protein with dual pro-phagocytic and pro-apoptotic anticancer activity.
British Journal of HaematologyAbstract:
A CD47-blocking TRAIL fusion protein with dual pro-phagocytic and pro-apoptotic anticancer activity
Anti-CD47:TRAIL effectively blocks CD47-mediated "don't eat me" signaling, promotes rituximab-induced phagocytosis by granulocytes and triggers CD47-restricted apoptosis in malignant B-cells. This multifunctional therapeutic activity of antiCD47:sTRAIL may be of general use for optomising antibody-based cancer thherapy and serves as proof of concept for combining CD47-blockade with alternate effector principles that may synergise anticancer activity.
Abstract.
Full text.
Holley JE, Bremer E, Kendall AC, de Bruyn M, Helfrich W, Tarr JM, Newcombe J, Gutowski NJ, Eggleton P (2013). CD20+inflammatory T-cells are present in blood and brain of multiple sclerosis patients and can be selectively targeted for apoptotic elimination. Multiple Sclerosis and Related Disorders
Eggleton P, Michalak M (2013). Calreticulin for better or for worse, in sickness and in health, until death do us part.
Eggleton P, Michalak M (2013). Calreticulin for better, for worse, in sickness and in health, until death do us part. Cell Calcium, 54(2), 126-131.
Eggleton P, Nissim A, Ryan BJ, Whiteman M, Winyard PG (2013). Detection and isolation of human serum autoantibodies that recognize oxidatively modified autoantigens.
Free Radic Biol Med,
57, 79-91.
Abstract:
Detection and isolation of human serum autoantibodies that recognize oxidatively modified autoantigens.
The breakdown of human immune tolerance to self-proteins occurs by a number of mechanisms, including posttranslational modifications of host molecules by reactive oxygen, nitrogen, or chlorine species. This has led to great interest in detecting serum autoantibodies raised against small quantities of oxidatively modified host proteins in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis. Here, we provide protocols for the preparation and chemical characterization of oxidatively modified protein antigens and procedures for their use in immunoblotting and ELISAs that detect autoantibodies against these antigens in clinical samples. These gel electrophoresis- and plate reader-based immunochemical methods sometimes suffer from low analytical specificity and/or sensitivity when used for serum autoantibody detection. This is often because a single solid-phase protein (antigen) is exposed to a complex mixture of serum proteins that undergo nonspecific binding. Therefore more sensitive/specific techniques are required to detect autoantibodies specifically directed against oxidatively modified proteins. To address this, we describe novel affinity chromatography protocols by which purified autoantibodies are isolated from small volumes (
Abstract.
Author URL.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Eggleton P, Smerdon GR (2013). Different oxygen treatment pressures alter inflammatory gene expression in human endothelial cells.
Undersea and Hyperbaric Medicine,
40(2), 115-123.
Abstract:
Different oxygen treatment pressures alter inflammatory gene expression in human endothelial cells.
Hyperbaric oxygen has proven to be a useful treatment for chronic wounds. However, therapeutic conditions vary between treatment centers, and we wished to investigate the effects of different treatment pressures on cells under inflammatory conditions. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O2 at 1 atmosphere absolute (atm abs); PO2 ~ 2 kPa) in the presence of 0.5 μg/ml lipopolysaccharide and 1 ng/ml TNF-alpha for 24 hours, then treated with normobaric oxygen (NBO2; 95%O2/5%CO2 at 1.0 atm abs; PO2 ~ 96.3 kPa), hyperbaric oxygen (HBO2) at 1.5 atm abs (1.5HBO2; 96.7%O2/3.3%CO2 at 1.5 atm abs; PO2 ~ 147 kPa) and HBO2 at 2.4 atm abs (2.4 HBO2; 97.9% O2/2.1% CO2 at 2.4 atms; PO2 ~238 kPa). The mRNA expression of 92 genes was then analyzed, and we identified changes in genes involved in adhesion molecule expression, angiogenesis and tissue remodeling, intracellular signaling, and cellular oxygen responses and redox signaling. We noted differences in expression between different treatment pressures, highlighting the need for further research into the use of different therapeutic protocols in the treatment of inflammatory conditions such as chronic wounds.
Abstract.
Perry E, Hutchinson D, de-Soyza A, Eggleton P, Kelly C (2013). Disease activity and autoantibody positivity are increased in rheumatoid arthritis patients with co-existent bronchiectasis compared to those without.
Gooden MJM, Wiersma VR, Eggleton P, Samplonius DF, Gerssen J, Nijman HW, Niki T, Helfrich W, Bremer E (2013). Galectin-9 activates and expands human T-helper cells.
PLoS One,
8(5), 1-11.
Abstract:
Galectin-9 activates and expands human T-helper cells.
Galectin-9 (Gal-9) is known for induction of apoptosis in IFN-c and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-c positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5–30 nM) induced apoptosis in ,60% of T-cells after 1 day, but activated the surviving T- cells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from na ̈ıve towards central memory and IFN-c producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2) Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.
Abstract.
Full text.
Kendall AC, Whatmore JL, Winyard PG, Smerdon GR, Eggleton P (2013). Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation.
Wound Repair and Regeneration,
21(6), 860-868.
Abstract:
Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation
Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells (HUVEC) and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and TNF-α, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced Ë™NO-related protein modifications. The transnitrosating agent SNCEE (600µM) also reduced neutrophil adhesion to HUVEC monolayers, and the iNOS inhibitor 1400W (10µM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation.
Abstract.
Kendall AC, Whatmore JL, Winyard PG, Smerdon GR, Eggleton P (2013). Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation.
Wound Repair and Regeneration,
21(6), 860-868.
Abstract:
Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation
Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation. © 2013 by the Wound Healing Society.
Abstract.
Eggleton P (2013). Hypersensitivity: Immune Complex Mediated
(Type III). In (Ed)
Encyclopaedia of Life Sciences, Chichester: John Wiley & Sons.
Abstract:
Hypersensitivity: Immune Complex Mediated
(Type III)
Abstract.
Perry E, Kelly C, de-Soyza A, Moullaali T, Eggleton P, Hutchinson D (2013). NATURAL HISTORY, DISEASE CHARACTERISTICS AND AUTOANTIBODY POSITIVITY IN PATIENTS WITH BRONCHIECTASIS AND RA:IS THE LUNG AN INITIATING SITE OF AUTOIMMUNITY IN RHEUMATOID ARTHRITIS?.
Abstract:
NATURAL HISTORY, DISEASE CHARACTERISTICS AND AUTOANTIBODY POSITIVITY IN PATIENTS WITH BRONCHIECTASIS AND RA:IS THE LUNG AN INITIATING SITE OF AUTOIMMUNITY IN RHEUMATOID ARTHRITIS?
Abstract.
Szabó-Taylor KE, Nagy G, Eggleton P, Winyard PG (2013). Oxidative Stress in Rheumatoid Arthritis. In Alcaraz MJ (Ed)
Studies on Arthritis and Joint Disorders, Oxidative Stress in Applied Basic Research and Clinical Practice, Springer Science+Business Media, 145-167.
Abstract:
Oxidative Stress in Rheumatoid Arthritis
Abstract.
Eggleton P, Ward FJ (eds)(2013).
Systemic lupus erythematosus: Methods and Protocols. Exeter, Springer Science+Business Media, LLC.
Abstract:
Systemic lupus erythematosus: Methods and Protocols
Abstract.
Marut W, Jamier V, Kavian N, Servettaz A, Eggleton P, Winyard PG, Anwar A, Nicco C, Jacob C, Chéreau C, et al (2013). The natural organosulfur compound dipropyltetrasulfide prevents HOCL-induced systemic sclerosis in the mouse.
Arthritis Research and TherapyAbstract:
The natural organosulfur compound dipropyltetrasulfide prevents HOCL-induced systemic sclerosis in the mouse
Introduction: the aim of this study was to test the naturally occurring organosulfur compound dipropyltetrasulfide (DPTTS) found in plants, which has antibiotic and anti-cancer properties, as a treatment of HOCl-induced systemic sclerosis in the mouse.
Methods: the pro-oxidative, anti-proliferative and cytotoxic effects of DPTTS were evaluated ex vivo on fibroblasts from normal and HOCl-mice. In vivo, the anti-fibrotic and immunomodulating properties of DPTTS were evaluated in the skin and lungs of HOCl-mice.
Results: H2O2 production was higher in fibroblasts derived from HOCl-mice than in normal fibroblasts (P
Abstract.
Full text.
2012
Ferguson D, Smerdon GR, Eggleton P, Curnow A, Winyard PG (2012). Altering oxygen concentrations to enhance the efficacy of PpIX-based photodynamic cell killing.
Ferguson D, Smerdon GR, Eggleton P, Curnow A, Winyard PG (2012). Altering oxygen concentrations to enhance the efficacy of PpIX-based photodynamic cell killing.
FREE RADICAL BIOLOGY AND MEDICINE,
53, S127-S127.
Author URL.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Ferguson D, Eggleton P (2012). Different oxygen treatment pressures alter oxygen responses and redox signalling gene expression in human endothelial cells.
FREE RADICAL BIOLOGY AND MEDICINE,
53, S166-S166.
Author URL.
Szabó-Taylor K, Eggleton P, Turner CAL, Lo Faro ML, Tarr JM, Tóth S, Whiteman M, Haigh RC, Littlechild JA, Winyard PG, et al (2012). Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular
peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2,
compared with healthy human lymphocytes.
The International Journal of Biochemistry and Cell Biology,
44(8), 1223-1231.
Abstract:
Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular
peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2,
compared with healthy human lymphocytes
Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood
lymphocytes and extracellular fluid in healthy subjects and rheumatoid arthritis patients is poorly
described. In the present study, the median intracellular peroxiredoxin 2 protein content of
lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with
healthy subjects’ lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca.
8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. In the total
lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed
surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17+ve
lymphocytes were peroxiredoxin 2+ve (median, 39%). We suggest that crucial inflammatory cell
subsets, i.e. interleukin-17+ve T cells, exhibit increased exofacial redox-regulating enzymes and
that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic
inflammation.
Abstract.
Winyard PG, Nissim A, Whiteman M, Eggleton P (2012). Measurement of serum autoantibodies against oxidatively modified autoantigens in human autoimmune diseases.
Smallwood MJ, Jewell SA, Petrov PG, Winlove CP, Eggleton P, Winyard PG (2012). The role of phosphatidylserine externalisation and oxidation in C1q-dependent apoptotic cell clearance.
2011
Connolly M, Donnelly S, Eggleton P, Kavanagh E, Last J, Moran E, Whelan B (2011). Basic Science for Rheumatology. In Callan M (Ed) The Rheumatology Handbook, Imperial College Press, 1-38.
Kendall AC, Whatmore JL, Harries LW, Winyard PG, Smerdon GR, Eggleton P (2011). Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions.
Experimental Cell ResearchAbstract:
Changes in inflammatory gene expression induced by hyperbaric oxygen treatment in human endothelial cells under chronic wound conditions
Hyperbaric oxygen (HBO) therapy involves the inhalation of 100% oxygen, whilst inside a chamber at greater than atmospheric pressure. It is an effective treatment for chronic diabetic wounds, although the molecular mechanisms involved remain unclear. We hypothesised that HBO could alter inflammatory gene expression in human endothelial cells via a reactive oxygen/nitrogen species-mediated pathway. Endothelial cells were exposed to a chronic wound model comprising hypoxia (2% O(2) at 1 atmosphere absolute (ATA); PO(2) ~2kPa) in the presence of lipopolysaccharide and TNF-α for 24h, then treated with HBO for 90min (97.5% O(2) at 2.4 ATA; PO(2) ~237kPa). 5h post-HBO, 19 genes involved in adhesion, angiogenesis, inflammation and oxidative stress were downregulated. Notably, only angiogenin gene expression, which promotes both angiogenesis and nitric oxide production (reflected by increased eNOS protein expression in this study), was upregulated. This led to a decrease in endothelial IL-8 mRNA and protein, which could help alleviate inflammatory processes during chronic wound healing. This was no longer evident 22.5h post-HBO, demonstrating the importance of daily exposures in HBO treatment protocols. These studies indicate that elevated oxygen transiently regulates inflammatory gene expression in endothelial cells, which may enhance chronic wound healing.
Abstract.
Eggleton P, Tarr JM, Winyard PG, Haigh R, Viner N (2011). FREQUENCY OF TH17 CD20+CELLS IN THE BLOOD OF RHEUMATOID ARTHRITIS PATIENTS IS HIGHER COMPARED TO HEALTHY CONTROL SUBJECTS.
Author URL.
Eggleton P, Bremer E, Tarr J, De Bruyn M, Helfrich W, Kendall K, Haigh R, Viner N, Winyard PG (2011). Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects. Arthritis research and therapy
Eggleton P, Bremer E, Tarr JM, de Bruyn M, Helfrich W, Kendall A, Haigh RC, Viner NJ, Winyard PG (2011). Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects.
Arthritis Res Ther,
13(6).
Abstract:
Frequency of Th17 CD20+ cells in the peripheral blood of rheumatoid arthritis patients is higher compared to healthy subjects.
Rheumatoid arthritis (RA) is considered a T cell driven autoimmune disease, therefore, the ability of B cell depleting biologics, e.g. anti-CD20 antibodies, to alleviate RA is unclear. This study examined the proportions of IL-17-secreting lymphocytes in the blood of healthy subjects and RA patients and determined if Th17 cells belong to a CD20+ subset of T cells.
Abstract.
Author URL.
Full text.
Kendall A, Smerdon G, Harries LW, Winyard PG, Eggleton P, Whatmore J (2011). Hyperbaric Oxygen Therapy and chronic wound healing. In Middleton JE (Ed) Wound Healing: Process, Phases and Promoting, Nova Publishers, 145-177.
Winyard PG, Ryan B, Eggleton P, Nissim A, Taylor E, Lo Faro ML, Burkholz T, Szabó-Taylor KE, Fox B, Viner N, et al (2011). Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease.
Biochem Soc Trans,
39(5), 1226-1232.
Abstract:
Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease.
Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO•, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.
Abstract.
Author URL.
Eggleton P, Ryan B, Brown S, Johnson S, Viner N, Nissim A, Isenberg D, Haigh R, Winyard P (2011). POST-TRANSLATIONAL MODIFICATIONS OF C1Q LEAD TO ANTIGENICITY AND BREAKDOWN OF IMMUNE TOLERANCE IN SYSTEMIC LUPUS ERYTHEMATOSUS.
Author URL.
2010
Tarr1 JM, Young PJ, Morse R, Shaw DJ, Haigh R, Petrov PG, Johnson SJ, Winyard PG, Eggleton P (2010). A mechanism of release of calreticulin from cells during apoptosis.
J Molecular BiologyAbstract:
A mechanism of release of calreticulin from cells during apoptosis
Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca2+ homeostasis. CRT also exerts extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release remains unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here we show that under apoptotic stress conditions, the cytosolic CRT concentration increases and associates with phosphatidylserine (PS) in a Ca2+ dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT) which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH-groups by nitrogen species. During apoptosis, both CRT expression and the nitric oxide (NO) concentration increases. By using S-nitroso-L-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol (RSNO) dependent and caspase-independent manner. Furthermore, the CRT and PS relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS may explain how CRT acts as a bridging molecule during apoptotic cell clearance.
Abstract.
Full text.
Tarr JM, Young PJ, Morse R, Shaw DJ, Haigh R, Petrov PG, Johnson SJ, Winyard PG, Eggleton P (2010). A mechanism of release of calreticulin from cells during apoptosis.
J Mol Biol,
401(5), 799-812.
Abstract:
A mechanism of release of calreticulin from cells during apoptosis.
Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.
Abstract.
Full text.
Raffiq S, Frayling T, Vyse T, Cunninghame-Graham D, Eggleton P (2010). Assessing association of common variation in the C1q gene cluster with systemic lupus erythematosus. Clinical Experimental Immunology, 161(2), 284-289.
Gold LI, Eggleton P, Sweetwyne MT, Van Duyn LB, Greives MR, Naylor S-M, Michalak M, Murphy-Ullrich JE (2010). Calreticulin: non-endoplasmic reticulum functions in physiology and disease.
FASEB J,
24(3), 665-683.
Abstract:
Calreticulin: non-endoplasmic reticulum functions in physiology and disease.
Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I. Eggleton, P. Sweetwyne, M. T. Van Duyn, L. B. Greives, M. R. Naylor, S.-M. Michalak, M. Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.
Abstract.
Author URL.
Eggleton P, Harries LW, Alberigo G, Wordsworth P, Viner N, Haigh R, Donnelly S, Jones HW, O Conner TWE, Thomson AER, et al (2010). Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes.
Journal of Clinical Immunology,
30(5), 649-658.
Abstract:
Changes in apoptotic gene expression in lymphocytes from rheumatoid arthritis and systemic lupus erythematosus patients compared with healthy lymphocytes
Introduction Systemic lupus erythematosus (SLE) and rheumatoid arthritis have complex genetic traits, but in both autoimmune diseases, dysfunctional apoptosis appears to play a part in disease pathology. This study examined the levels of in vitro apoptosis in lymphocytes from healthy, rheumatoid arthritis (RA) and SLE individuals and related observed differences to their lymphocyte apoptosis gene profiles. Methods Materials and Methods Lymphocytes were assessed for cell death by nuclear pyknosis and DNA fragmentation. Control, SLE and RA apoptosis gene profiles were obtained by quantitative real time polymerase chain reaction (QRT-PCR) analysis.
Results and Discussion the mean levels of pyknosis in RA and SLE freshly isolated lymphocytes were significantly higher than in control lymphocytes. Ninety three apoptosis genes were analysed by QRT-PCR of mRNA from RA, SLE and healthy lymphocytes. We identified significant differences (p
Abstract.
Tarr JM, Winyard PG, Haigh R, Viner N, Eggleton P (2010). Extracellular calreticulin accumulates in the joints of rheumatoid arthritis patients and inhibits FasL (CD95L) mediated apoptosis of T cells.
Arthritis and Rheumatism,
62(10), 2919-2929.
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Shaw DJ, Morse R, Todd AG, Eggleton P, Lorson CL, Young PJ (2010). Identification of a self-association domain in the Ewing's sarcoma protein: a novel function for arginine-glycine-glycine rich motifs?.
J Biochem,
147(6), 885-893.
Abstract:
Identification of a self-association domain in the Ewing's sarcoma protein: a novel function for arginine-glycine-glycine rich motifs?
The Ewing's sarcoma (EWS) protein is a ubiquitously expressed RNA chaperone. The EWS protein localizes predominantly to the nucleus. Previous reports have suggested that the EWS protein is capable of dimerizing. However, to date this has not been confirmed. Here, using a novel panel of recombinant proteins, we have performed an in vitro biomolecular interaction analysis of the EWS protein. We have demonstrated that all three arginine-glycine-glycine (RGG) motifs are capable of binding directly to the survival motor neuron protein, a Tudor domain containing EWS binding partner. We have also confirmed EWS is capable of self-associating, and we have mapped this binding domain to the RGG motifs. We have also found that self-association may be required for EWS nuclear import. This is the first direct evidence of RGG domains being involved in self-association and has implications on all RGG-containing proteins.
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Author URL.
Kepp O, Gdoura A, Martins I, Panaretakis T, Schlemmer F, Tesniere A, Fimia GM, Ciccosanti F, Piacentini M, Eggleton P, et al (2010). Lysyl tRNA synthetase is required for the translocation of calreticulin to the cell surface in immunogenic death.
Cell Cycle,
9(15), 3072-3077.
Abstract:
Lysyl tRNA synthetase is required for the translocation of calreticulin to the cell surface in immunogenic death
In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.(1) Here, we report that yet another ER protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRT in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. These results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRT exposure.
Abstract.
Ryan B, Eggleton P, Brown S, Viner N, Nissim A, Isenberg D, Haigh R, Winyard PG (2010). Oxidative and Nitrative Modification of C1q Affects its Structure, Function and Antigenicity in Systemic Lupus Erythematosus.
Author URL.
Szabo-Taylor KE, Lo Faro ML, Eggleton P, Turner CAL, Tarr JM, Haigh RC, Littlechild JA, Whiteman M, Winyard PG (2010). Peroxiredoxin 2 in Human Inflammatory Joint Disease.
Author URL.
2009
Eggleton P, Haigh R, Viner N, Donnelly S, Harries LW, Alberigo G, Winyard PG (2009). DIFFERENCES IN APOPTOSIS GENE EXPRESSION IN LYMPHOCYTES FROM SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AND RHEUMATOID ARTHRITIS (RA) PATIENTS.
Author URL.
Tarr JM, Szabo K, Haigh RC, Viner NJ, Eggleton P, Winyard PG (2009). DOES TREATMENT WITH RITUXIMAB INDUCE APOPTOSIS IN T LYMPHOCYTES?.
Author URL.
Turner CA, Szabo KE, Haigh R, Tarr J, Littlechild JA, Eggleton P, Winyard PG (2009). EXPRESSION OF THE PEROXIREDOXIN-BASED ANTIOXIDANT SYSTEM IN PERIPHERAL BLOOD LYMPHOCYTES IN RHEUMATOID ARTHRITIS.
Author URL.
Gutowski NJ, Nejadhamzeeigilani Z, Eggleton P, Whatmore JL (2009). Factors released from lung tumour cells alter adhesion molecule expression and increase activation of human cerebral endothelial cells.
Author URL.
Shaw FL, Winyard PG, Smerdon GR, Bryson PJ, Moody AJ, Eggleton P (2009). Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules.
Clin Biochem,
42(6), 467-476.
Abstract:
Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules.
OBJECTIVES: to investigate the effect of hyperbaric oxygen (HBO) on platelet physiology. DESIGN AND METHODS: Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation. RESULTS: HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa). CONCLUSIONS: Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.
Abstract.
Author URL.
Shaw DJ, Morse R, Todd AG, Eggleton P, Lorson CL, Young PJ (2009). Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS).
Biochem Biophys Res Commun,
390(4), 1197-1201.
Abstract:
Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS).
The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.
Abstract.
Author URL.
Ryan B, Winyard PG, Viner N, Haigh R, Haas M, Nissim A, Isenberg D, Eggleton P (2009). OXIDATIVE MODIFICATIONS TO C1Q INCREASE THE SENSITIVITY OF AN ANTI-C1Q ELISA IN THE DIAGNOSIS OF SYSTEMIC LUPUS ERYTHEMATOSUS.
Author URL.
Szabo KE, Line K, Eggleton P, Littlechild JA, Winyard PG (2009). Structure and function of the human peroxiredoxin-based antioxidant system: the interplay between peroxierdoxins, thioredoxins, thioredoxin reductases, sulfiredoxins and sestrins. In Jacob C, Winyard PG (Eds.) Redox Signaling and Regulation in Biology and Medicine, Weinheim, Germany: Wiley-VCH.
Szabo KE, Line K, Eggleton P, Littlechild JA, Winyard PG (2009). Structure and function of the human peroxiredoxin-based antioxidant system: the interplay between peroxiredoxins, thioredoxins, thioredoxin reductases, sulfiredoxins and sestrins. In Jacob C, Winyard PG (Eds.) Redox Signaling and Regulation in Biology and Medicine, Weinheim: Wiley-VCH-Verlag, 151-192.
2008
Gutowski NJ, Holley JE, Eggleton P, Whatmore JL (2008). Changes in endothelial cell activation occur in multiple sclerosis lesion formation.
Author URL.
Winyard PG, Knight IA, Shaw FL, Rocks SA, Davies CA, Eggleton P, Haigh R, Whiteman M, Benjamin N (2008). Chapter 8 Determination of S-Nitrosothiols in Biological and Clinical Samples Using Electron Paramagnetic Resonance Spectrometry with Spin Trapping. Methods in Enzymology, 441, 151-160.
Eggleton P, Haigh R, Winyard PG (2008). Consequence of neo-antigenicity of the 'altered self'.
Rheumatology (Oxford),
47(5), 567-571.
Abstract:
Consequence of neo-antigenicity of the 'altered self'.
Post-translational modifications play a central role in determining the function of proteins. Such protein modifications come in a great variety of guises, and include phosphorylation, proteolysis, glycosylation, citrullination and oxidative modifications. In relation to inflammatory autoimmune diseases, some post-translational modifications appear to result in the generation of new antigens, and hence autoantibodies. Examples include: the induction of peptide immunogenicity by the spontaneous conversion of aspartic acid residues to isoaspartic acid; granzyme B-mediated cleavage of SLE autoantigens; the oxidative modification--on the surface of apoptotic cells--of lipids and proteins, rendering them immunogenic; and the presence of antibodies to oxidatively modified type II collagen and C1q in RA and SLE patients, respectively. The measurement of autoantibodies to citrullinated proteins has been verified as a very useful diagnostic tool in RA. Proteomics techniques, in principle, allow the detection of all types of in vivo protein modifications, and the increasing application of such technologies to the study of rheumatological diseases will further our understanding of autoantigenicity.
Abstract.
Author URL.
Winyard PG, Knight IA, Shaw FL, Rocks SA, Davies CA, Eggleton P, Haigh R, Whiteman M, Benjamin N (2008). Determination of S-nitrosothiols in biological and clinical samples using electron paramagnetic resonance spectrometry with spin trapping.
Methods Enzymol,
441, 151-160.
Abstract:
Determination of S-nitrosothiols in biological and clinical samples using electron paramagnetic resonance spectrometry with spin trapping.
S-Nitroso moieties, such as the S-nitroso group within S-nitrosated albumin, constitute a potential endogenous reservoir of nitric oxide (NO.) in human tissues and other biological systems. Moreover, S-nitroso compounds are under investigation as therapeutic agents in humans. Therefore, it is important to be able to detect S-nitrosothiols (RSNOs) in human extracellular fluids, such as plasma and synovial fluid, as well as other biological samples. This chapter describes a method for the determination of S-nitrosothiols in biofluids. The method is based on electron paramagnetic resonance (EPR) spectrometry, in combination with spin trapping using a ferrous ion complex of the iron chelator N-methyl-d-glucamine dithiocarbamate under alkaline conditions. This iron complex mediates the decomposition of RSNO to NO. as well as spin trapping the generated NO. The resulting spin adduct has a unique EPR signal that can be quantified.
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Szabo K, Tarr J, Eggleton P, Line K, Ryan B, Aksu K, Akcay Y, Haigh R, Littlechild J, Winyard P, et al (2008). Extracellular peroxiredoxin II in rheumatoid arthritis and other autoimmune conditions.
Author URL.
Shaw DJ, Eggleton P, Young PJ (2008). Joining the dots: production, processing and targeting of U snRNP to nuclear bodies.
Biochim Biophys Acta,
1783(11), 2137-2144.
Abstract:
Joining the dots: production, processing and targeting of U snRNP to nuclear bodies.
The spliceosome is the RNP complex than catalyses the removal of introns from the Uridine-rich small nuclear ribonucleoproteins (U snRNPs) that make up the main components of this complex. The production of these RNPs is an intricate process, involving several key stages. These include: 1) the transcription of the U snRNAs; 2) their nuclear export; 3) the cytoplasmic assembly of the U snRNP; 4) their nuclear import; 5) their processing within Cajal bodies and the nucleolus; and 6) their storage in interchromatin granule clusters (IGCs). This review focuses on each of these stages, discussing the key complexes involved as well as the trafficking and targeting mechanisms involved.
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Tarr JM, Haas M, Haigh R, Eggleton P, Winyard PG (2008). Measurement of calreticulin in plasma and synovial fluid from rheumatoid arthritis patients.
Author URL.
2007
Gutowski NJ, Holley JE, Eggleton P, Whatmore J (2007). Alteration in blood vessel density in multiple sclerosis lesions implies a role in brain scarring.
Author URL.
Ryan B, Szestakowska D, Viner N, Haigh R, Haas M, Nissim A, Isenberg D, Winyard P, Eggleton P (2007). Autoantibodies target oxidatively modified proteins of the C1q/calreticulin/CD91 apoptotic pathway in systemic lupus erythematosus.
Author URL.
Holley J, Eggleton P, Whatmore J, Gutowski N (2007). Temporal changes in endothelial cell activation occur in MS lesion formation.
NEURON GLIA BIOLOGY,
2, S35-S35.
Author URL.
Honoré C, Hummelshoj T, Hansen BE, Madsen HO, Eggleton P, Garred P (2007). The innate immune component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells.
Arthritis Rheum,
56(5), 1598-1607.
Abstract:
The innate immune component ficolin 3 (Hakata antigen) mediates the clearance of late apoptotic cells.
OBJECTIVE: Ficolin 3 (Hakata antigen), a collagen-like defense molecule, is a known autoantigen in patients with systemic lupus erythematosus (SLE). Recent studies have shown that other collagen-like defense molecules, such as C1q, mannose-binding lectin (MBL) and ficolin 2, bind to apoptotic cells and mediate their clearance by phagocytic cells. Dysfunction in this mechanism is regarded as an important contributor to the pathophysiology of SLE. Thus, we sought to determine whether ficolin 3 participates in the clearance of apoptotic cells. METHODS: a Jurkat T cell line was used as the source of dying host cells. The cells were rendered apoptotic or necrotic by incubation with etoposide or by heat shocking, respectively. Binding of ficolin 3 to the cells was analyzed by flow cytometry. The apoptotic cells were incubated with human monocyte-derived macrophages, and the effect of ficolin 3 on the adhesion/uptake was examined by flow cytometry. RESULTS: Ficolin 3 bound to a population of late apoptotic cells, while a strong and uniform binding to necrotic cells was observed. The binding properties differed from those of MBL and ficolin 2. Ficolin 3 binding to late apoptotic cells resulted in a significant increase in adhesion/uptake by macrophages. CONCLUSION: Ficolin 3 mediates the clearance of late apoptotic cells, which suggests that this protein is involved in the maintenance of tissue homeostasis and might play a protective role against the development of autoimmunity.
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2006
Eggleton P (2006). Antigen-Antibody Complexes. In (Ed) Encyclopaedia of Life Sciences.
Winyard PG, Eggleton P, Jones HW, Thomson AER, O'Connor TWE, Gruska AM, Chikanza IC, Williams MA, Harries L (2006). Apoptotic activity in patients with rheumatoid arthritis and systemic lupus erythematosus.
Author URL.
Eggleton P, Szestakowska D, Winyard PG, Viner N, Nissim A (2006). Generation of neo-antigenic epitopes recognised by autoimmune sera after the post-translational modification of human C1q by free radicals.
Author URL.
Donnelly S, Roake W, Brown S, Young P, Naik H, Wordsworth P, Isenberg DA, Reid KBM, Eggleton P (2006). Impaired recognition of apoptotic neutrophils by the C1q/calreticulin and CD91 pathway in systemic lupus erythematosus.
Arthritis Rheum,
54(5), 1543-1556.
Abstract:
Impaired recognition of apoptotic neutrophils by the C1q/calreticulin and CD91 pathway in systemic lupus erythematosus.
OBJECTIVE: a deficiency in a subcomponent of C1q can result in increased susceptibility to autoimmune diseases such as systemic lupus erythematosus (SLE). The monocyte endocytic receptor CD91 is implicated in the endocytosis of apoptotic neutrophils via interactions with C1q and calreticulin. In this clinical study, we studied the binding of C1q to leukocytes and determined whether C1q bound specifically to calreticulin and CD91 on cells undergoing apoptosis in SLE. METHODS: Proximal antibody phage display, calreticulin-transfected cells, and immunocytochemical and confocal techniques were used in a comprehensive analysis of direct binding of C1q to apoptotic neutrophils that were obtained from healthy individuals and from patients with SLE. In addition, apoptotic cellular systems were assessed in vitro. RESULTS: C1q appeared to colocalize to apoptotic blebs on the surface of leukocytes in association with both calreticulin and CD91, as determined by phage display and transfected cell studies. However, C1q did not bind to apoptotic cells isolated from SLE patients, despite the positivity of the cells for both calreticulin and CD91. Surface expression of calreticulin decreased on neutrophils as they aged, but increased on monocytes. In an apoptotic phagocytic assay, the addition of C1q and calreticulin significantly enhanced the phagocytosis of apoptotic cell debris by monocyte-derived cells. CONCLUSION: These observations indicate that neutrophils from SLE patients have a reduced ability to be recognized and removed by the C1q/calreticulin/CD91-mediated apoptotic pathway, despite the presence of main apoptotic recognition partners. This suggests that an additional component, as yet unidentified, acts as a C1q binding partner on apoptotic cells, and this component may be lacking in cells isolated from SLE patients.
Abstract.
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Tarr JM, Eggleton P, Winyard PG (2006). Nitric oxide and the regulation of apoptosis in tumour cells.
Curr Pharm Des,
12(34), 4445-4468.
Abstract:
Nitric oxide and the regulation of apoptosis in tumour cells.
Nitric oxide (NO) is a small, highly reactive, diffusible free radical which has been implicated in many physiological and pathophysiological processes. It has either pro-apoptotic or anti-apoptotic effects on cells, depending upon a host of factors. This review outlines some of the regulatory molecules and organelles involved in the apoptotic pathways that can be influenced by the presence of NO, including p53, Bcl-2, caspases, mitochondria, and heat shock proteins. The effects of NO on the apoptosis of tumour cells are also examined.
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Szestakowska D, Young P, Morse R, Viner N, Isenberg D, Nissim A, Eggleton P, Winyard P (2006). Role of oxidative protein modifications in the generation of neoantigens recognised by lupus patient sera.
Author URL.
Szestakowska D, Szabok E, Eggleton P, Opas M, Young P (2006). The complexities of calreticlulin from protein folding to disease prevention and therapeutic application. Calcium Binding Proteins, 1(2), 135-139.
Eggleton P (2006). The diversity of EF-hand calcium binding proteins. Calcium Binding Proteins, 1(1), 1-2.
Shaw FL, Eggleton P, Moody AJ, Smerdon GR, Bryson PJ, Winyard P (2006). The effect of hyperbaric oxygen on platelet S-nitrosothiol release and aggregation.
Author URL.
Tarr J, Eggleton P, Haigh R, Winyard P (2006). The effect of nitric oxide on the apoptosis of Jurkat T cells.
Author URL.
2005
Gutowski NJ, Holley JE, Eggleton P, Whatmore J (2005). Blood vessel density is increased in chronic MS lesions: Relationship to scarring.
EUROPEAN JOURNAL OF NEUROLOGY,
12, 129-129.
Author URL.
Gutowski NJ, Maskell L, Eggleton P, Whatmore JL (2005). Human lung tumour cells induce changes in human astrocyte phenotype in vitro: Relevance to metastasis evolution.
EUROPEAN JOURNAL OF NEUROLOGY,
12, 143-143.
Author URL.
Tarr J, Eggleton P (2005). Immune function of C1q and its modulators CD91 and CD93.
Crit Rev Immunol,
25(4), 305-330.
Abstract:
Immune function of C1q and its modulators CD91 and CD93.
C1q is a subcomponent of the first component of complement C1, which is a multimolecular complex comprising one molecule of C1q and two molecules each of the autoreactive proteases, C1r and C1s. This multimolecular complex triggers the classical pathway of complement. Advances in the past several years have provided a partial crystal structure of the C1q subunit. This, together with gene deletion of C1q, has allowed further insight into the multifunctional immune aspects of this molecule. Two C1q-mediated functions that have received intense scrutiny recently are C1q-mediated apoptotic clearance of cell debris and phagocytosis. This has led to a heightened search for specific receptors for the collagen-like region (CLR) as well as the globular heads. Two transmembrane proteins, CD91 and CD93, have been proposed to interact indirectly with the CLR of C1q, promoting apoptotic clearance and phagocytosis, respectively. The aim of this article is to provide an overview of the structural and functional information that implicates CD91 and CD93 in C1q-mediated functional effects.
Abstract.
Author URL.
Gutowski NJ, Holley JE, Eggleton P, Whatmore JL (2005). Increased blood vessel density in chronic multiple sclerosis lesions.
Author URL.
Gutowski NJ, Maskell L, Eggleton P, Whatmore JL (2005). Lung tumour cells induce changes in astrocyte phenotype in vitro: relevance to brain metastasis evolution.
Author URL.
Jounblat R, Clark H, Eggleton P, Hawgood S, Andrew PW, Kadioglu A (2005). The role of surfactant protein D in the colonisation of the respiratory tract and onset of bacteraemia during pneumococcal pneumonia.
Respir Res,
6Abstract:
The role of surfactant protein D in the colonisation of the respiratory tract and onset of bacteraemia during pneumococcal pneumonia.
We have shown previously that surfactant protein D (SP-D) binds and agglutinates Streptococcus pneumoniae in vitro. In this study, the role of SP-D in innate immunity against S. pneumoniae was investigated in vivo, by comparing the outcome of intranasal infection in surfactant protein D deficient (SP-D-/-) to wildtype mice (SP-D+/+). Deficiency of SP-D was associated with enhanced colonisation and infection of the upper and lower respiratory tract and earlier onset and longer persistence of bacteraemia. Recruitment of neutrophils to inflammatory sites in the lung was similar in both strains mice in the first 24 hrs post-infection, but different by 48 hrs. T cell influx was greatly enhanced in SP-D-/- mice as compared to SP-D+/+ mice. Our data provides evidence that SP-D has a significant role to play in the clearance of pneumococci during the early stages of infection in both pulmonary sites and blood.
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Full text.
Khamri W, Moran AP, Worku ML, Karim QN, Walker MM, Annuk H, Ferris JA, Appelmelk BJ, Eggleton P, Reid KBM, et al (2005). Variations in Helicobacter pylori lipopolysaccharide to evade the innate immune component surfactant protein D.
Infect Immun,
73(11), 7677-7686.
Abstract:
Variations in Helicobacter pylori lipopolysaccharide to evade the innate immune component surfactant protein D.
Helicobacter pylori is a common and persistent human pathogen of the gastric mucosa. Surfactant protein D (SP-D), a component of innate immunity, is expressed in the human gastric mucosa and is capable of aggregating H. pylori. Wide variation in the SP-D binding affinity to H. pylori has been observed in clinical isolates and laboratory-adapted strains. The aim of this study was to reveal potential mechanisms responsible for evading SP-D binding and establishing persistent infection. An escape variant, J178V, was generated in vitro, and the lipopolysaccharide (LPS) structure of the variant was compared to that of the parental strain, J178. The genetic basis for structural variation was explored by sequencing LPS biosynthesis genes. SP-D binding to clinical isolates was demonstrated by fluorescence-activated cell sorter analyses. Here, we show that H. pylori evades SP-D binding through phase variation in lipopolysaccharide. This phenomenon is linked to changes in the fucosylation of the O chain, which was concomitant with slipped-strand mispairing in a poly(C) tract of the fucosyltransferase a (fucT1) gene. SP-D binding organisms are predominant in mucus in vivo (P = 0.02), suggesting that SP-D facilitates physical elimination. Phase variation to evade SP-D contributes to the persistence of this common gastric pathogen.
Abstract.
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2004
Vucenik I, Passaniti A, Vitolo MI, Tantivejkul K, Eggleton P, Shamsuddin AM (2004). ANTI-ANGIOGENIC ACTIVITY OF INOSITOL HEXAPHOSPHATE (IP6).
ANTICANCER RESEARCH,
24(5D), 3477-3477.
Author URL.
Vucenik I, Passaniti A, Vitolo MI, Tantivejkul K, Eggleton P, Shamsuddin AM (2004). Anti-angiogenic activity of inositol hexaphosphate (IP6).
Carcinogenesis,
25(11), 2115-2123.
Abstract:
Anti-angiogenic activity of inositol hexaphosphate (IP6).
A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.
Abstract.
Author URL.
Vucenik I, Passaniti A, Eggleton P, Shamsuddin AKM (2004). Antiangiogenic activity of inositol hexaphosphate.
Author URL.
Jounblat R, Kadioglu A, Iannelli F, Pozzi G, Eggleton P, Andrew PW (2004). Binding and agglutination of Streptococcus pneumoniae by human surfactant protein D (SP-D) vary between strains, but SP-D fails to enhance killing by neutrophils.
Infect Immun,
72(2), 709-716.
Abstract:
Binding and agglutination of Streptococcus pneumoniae by human surfactant protein D (SP-D) vary between strains, but SP-D fails to enhance killing by neutrophils.
Recombinant human surfactant protein D (SP-D) expressed in Escherichia coli, consisting of the head and neck regions of the native molecule, bound to all strains of Streptococcus pneumoniae that were tested, but the extent of binding varied between strains of differing capsular serotypes. The recombinant protein expressed in the yeast Pichia pastoris did not bind. Full-length native SP-D aggregated pneumococci in a calcium-dependent manner that was inhibited by maltose acting as a competitive sugar. The ability of SP-D to modulate the uptake and killing of pneumococci by human neutrophils was also addressed. Neither recombinant truncated SP-D nor native full-length SP-D enhanced the killing of pneumococci by human neutrophils. Aggregation of pneumococci varied not only between strains of the same multilocus sequence type and different serotypes but also between strains of the same serotype. However, use of recombinant strains in which the serotype had been changed showed that the degree of aggregation was influenced by the capsular type. Indeed, a 19F serotype strain which was not aggregated by SP-D did exhibit aggregation when the original isogenic strain was capsule switched to capsular serotype 3. However, although our results show that SP-D is capable of aggregating most pneumococci, no correlation between the degree of aggregation and the capsule or multilocus sequence type of the pneumococcus was clearly apparent. Therefore, although the capsule serotype is not the only determinant of aggregation by SP-D, the data presented here indicate that it does have a role to play.
Abstract.
Author URL.
Castell L, Vance C, Abbott R, Marquez J, Eggleton P (2004). Granule localization of glutaminase in human neutrophils and the consequence of glutamine utilization for neutrophil activity. J Biol Chem, 279(14), 13305-13310.
2003
Eggleton P, Michalak M (2003).
Calreticulin., Kluwer Academic Pub.
Abstract:
Calreticulin
Abstract.
Eggleton P (2003). Stress protein-polypeptide complexes acting as autoimmune triggers.
Clin Exp Immunol,
134(1), 6-8.
Author URL.
2002
Murray E, Khamri W, Walker MM, Eggleton P, Moran AP, Ferris JA, Knapp S, Karim QN, Worku M, Strong P, et al (2002). Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection.
Infect Immun,
70(3), 1481-1487.
Abstract:
Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection.
Helicobacter pylori establishes persistent infection of gastric mucosa with diverse clinical outcomes. The innate immune molecule surfactant protein D (SP-D) binds selectively to microorganisms, inducing aggregation and phagocytosis. In this study, we demonstrated the expression of SP-D in gastric mucosa by reverse transcription-PCR and immunohistochemical analysis. SP-D is present at the luminal surface and within the gastric pits, with maximal expression at the surface. Levels of expression are significantly increased in H. pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used to demonstrate binding and agglutination of H. pylori by SP-D in a lectin-specific manner. These activities resulted in a 50% reduction in the motility of H. pylori, as judged on the basis of curvilinear velocity measured by using a Hobson BacTracker. Lipopolysaccharides extracted from three H. pylori strains were shown to bind SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding among the strains. SP-D may therefore play a significant role in the innate immune response to H. pylori infection.
Abstract.
Author URL.
Naik H, Donnelly S, Roake W, Wordsworth P, Reid KBM, Isenberg DA, Eggleton P (2002). Identification of apoptotic recognition molecules on leukocytes by phage display and microarray.
Author URL.
Elton CM, Smethurst PA, Eggleton P, Farndale RW (2002). Physical and functional interaction between cell-surface calreticulin and the collagen receptors integrin alpha2beta1 and glycoprotein VI in human platelets.
Thromb Haemost,
88(4), 648-654.
Abstract:
Physical and functional interaction between cell-surface calreticulin and the collagen receptors integrin alpha2beta1 and glycoprotein VI in human platelets.
Calreticulin is an abundant protein in the endoplasmic reticulum of most cells. In this study, flow cytometry and immunoprecipitation from surface-biotinylated platelets each provided direct evidence that calreticulin is also expressed on the surface of human platelets. Anti-calreticulin antibodies caused platelet activation, inducing FcgammaRIIa-independent platelet aggregation. In addition, these antibodies inhibited platelet adhesion to the integrin alpha2beta1-specific ligands, GFOGER-GPP and monomeric collagen I, and to the glycoprotein VI-specific ligand, CRP. Inhibition of platelet adhesion to these ligands was independent of integrin alphaIIbbeta3. In resting platelets, calreticulin was shown to interact with integrin alpha2beta1 and glycoprotein VI. Together, these data demonstrate that surface calreticulin is associated with collagen receptors on the platelet surface, where it may play a role in the modulation of the platelet-collagen interaction.
Abstract.
Author URL.
Goicoechea S, Pallero MA, Eggleton P, Michalak M, Murphy-Ullrich JE (2002). The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin.
J Biol Chem,
277(40), 37219-37228.
Abstract:
The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin.
Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.
Abstract.
Author URL.
Donnelly S, Naik H, Brown S, Savill J, Wordsworth P, Isenberg D, Reid KBM, Eggleton P (2002). The role of C1q and apoptotic bridging molecules in the recognition and clearance of apoptotic neutrophils in SLE.
Author URL.
Kang W, Nielsen O, Fenger C, Madsen J, Hansen S, Tornoe I, Eggleton P, Reid KBM, Holmskov U (2002). The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester.
Clin Exp Immunol,
130(3), 449-458.
Abstract:
The scavenger receptor, cysteine-rich domain-containing molecule gp-340 is differentially regulated in epithelial cell lines by phorbol ester.
Gp-340 is a glycoprotein belonging to the scavenger receptor cysteine rich (SRCR) group B family. It binds to host immune components such as lung surfactant protein D (SP-D). Recent studies found that gp-340 interacts directly with pathogenic microorganisms and induces their aggregation, suggesting its involvement in innate immunity. In order to investigate further its potential immune functions in the appropriate cell lines, the expression of gp-340 in four conventional immune cell lines (U937, HL60, Jurkat, Raji), and two innate immune-related epithelial cell lines (A549 derived from lung and AGS from stomach), was examined by RT-PCR and immunohistochemistry. The resting immune cell lines showed weak or no gp-340 mRNA expression; while the two epithelial cell lines expressed gp-340 at much higher level, which was differentially regulated by phorbol myristate acetate (PMA) treatment. In the A549 cells, gp-340 was up-regulated along with the PMA-induced proinflammatory expression of both IL-6 and IL-8. In AGS cells, PMA down-regulation of gp-340 was seen in parallel with an up-regulation of the two mature gastric epithelial specific proteins TFF1 (trefoil factor 1) and TFF2, which are implicated as markers of terminal differentiation. Analysis of the distribution of gp-340, together with the TFFs and SP-D in normal lung and gastric mucosa, supported further our in vitro data. We conclude that the differential regulation of gp-340 in the two epithelial cell lines by PMA indicates that gp-340 s involvement in mucosal defence and growth of epithelial cells may vary at different body locations and during different stages of epithelial differentiation.
Abstract.
Author URL.
2001
Kasper G, Brown A, Eberl M, Vallar L, Kieffer N, Berry C, Girdwood K, Eggleton P, Quinnell R, Pritchard DI, et al (2001). A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins.
Parasite Immunol,
23(3), 141-152.
Abstract:
A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins.
Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.
Abstract.
Author URL.
Goicoechea SM, Pallero AM, Eggleton P, Murphy-Ullrich JE (2001). Anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of calreticulin.
MOLECULAR BIOLOGY OF THE CELL,
12, 58A-59A.
Author URL.
Johnson S, Michalak M, Opas M, Eggleton P (2001). The ins and outs of calreticulin: from the ER lumen to the extracellular space.
Trends Cell Biol,
11(3), 122-129.
Abstract:
The ins and outs of calreticulin: from the ER lumen to the extracellular space.
Calreticulin was first isolated 26 years ago. Since its discovery as a minor Ca(2+)-binding protein of the sarcoplasmic reticulum, the appreciation of its importance has grown, and it is now recognized to be a multifunctional protein, most abundant in the endoplasmic reticulum (ER). The protein has well-recognized physiological roles in the ER as a molecular chaperone and Ca(2+)-signalling molecule. However, it has also been found in other membrane-bound organelles, at the cell surface and in the extracellular environment, where it has recently been shown to exert a number of physiological and pathological effects. Here, we will focus on these less-well-characterized functions of calreticulin.
Abstract.
Author URL.
2000
Eggleton P, Tenner AJ, Reid KB (2000). C1q receptors.
Clin Exp Immunol,
120(3), 406-412.
Author URL.
Llewellyn DH, Johnson S, Eggleton P (2000). Calreticulin comes of age.
Trends Cell Biol,
10(9), 399-402.
Author URL.
Andrin C, Corbett EF, Johnson S, Dabrowska M, Campbell ID, Eggleton P, Opas M, Michalak M (2000). Expression and purification of mammalian calreticulin in Pichia pastoris.
Protein Expr Purif,
20(2), 207-215.
Abstract:
Expression and purification of mammalian calreticulin in Pichia pastoris.
Calreticulin is a 46-kDa Ca(2+)-binding chaperone of the endoplasmic reticulum membranes. The protein binds Ca(2+) with high capacity, affects intracellular Ca(2+) homeostasis, and functions as a lectin-like chaperone. In this study, we describe expression and purification procedures for the isolation of recombinant rabbit calreticulin. The calreticulin was expressed in Pichia pastoris and purified to homogeneity by DEAE-Sepharose and Resource Q FPLC chromatography. The protein was not retained in the endoplasmic reticulum of Pichia pastoris but instead it was secreted into the external media. The purification procedures reported here for recombinant calreticulin yield homogeneous preparations of the protein by SDS-PAGE and mass spectroscopy analysis. Purified calreticulin was identified by its NH(2)-terminal amino acid sequences, by its Ca(2+) binding, and by its reactivity with anti-calreticulin antibodies. The protein contained one disulfide bond between (88)Cys and (120)Cys. CD spectral analysis and Ca(2+)-binding properties of the recombinant protein indicated that it was correctly folded.
Abstract.
Author URL.
Eggleton P, Ward FJ, Johnson S, Khamashta MA, Hughes GR, Hajela VA, Michalak M, Corbett EF, Staines NA, Reid KB, et al (2000). Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization.
Clin Exp Immunol,
120(2), 384-391.
Abstract:
Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization.
Extracellular calreticulin (CRT) as well as anti-CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in 'epitope spreading' to other autoantigens such as the Ro/SS-A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti-CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjögren's syndrome. Approximately 40% of all SLE patients were positive for anti-CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1-289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N-terminal half of the protein in 69% of the SLE sera from active disease patients, while the C-domain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P-domains. Sera from both healthy and disease controls and primary Sjögren's syndrome patients were non-reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N-terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.
Abstract.
Author URL.
Corbett EF, Michalak KM, Oikawa K, Johnson S, Campbell ID, Eggleton P, Kay C, Michalak M (2000). The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment.
J Biol Chem,
275(35), 27177-27185.
Abstract:
The conformation of calreticulin is influenced by the endoplasmic reticulum luminal environment.
In order to understand the dynamics of the endoplasmic reticulum (ER) luminal environment, we investigated the role of Ca(2+), Zn(2+), and ATP on conformational changes of calreticulin. Purified calreticulin was digested with trypsin in the presence or absence of Ca(2+), Zn(2+), and ATP. At low Ca(2+) concentration (
Abstract.
Author URL.
Goicoechea S, Orr AW, Pallero MA, Eggleton P, Murphy-Ullrich JE (2000). Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.
MOLECULAR BIOLOGY OF THE CELL,
11, 271A-271A.
Author URL.
Goicoechea S, Orr AW, Pallero MA, Eggleton P, Murphy-Ullrich JE (2000). Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.
J Biol Chem,
275(46), 36358-36368.
Abstract:
Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.
Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17-35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, binds a hep I affinity column, and neutralizes thrombospondin/hep I-mediated focal adhesion disassembly. Calreticulin on the cell surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-activated cell sorting analyses. Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calreticulin and preincubation of thrombospondin/hep I with glutathione S-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.
Abstract.
Author URL.
1999
Walker MM, Karim QN, Worku M, Murray E, Eggleton P, Reid KBM, Thursz MR (1999). Direct observation of kinetics and agglutination of Helicobacter pylori with the collectin, surfactant protein D.
GUT,
45, A41-A42.
Author URL.
Eggleton P (1999). Effect of IP6 on human neutrophil cytokine production and cell morphology.
Abstract:
Effect of IP6 on human neutrophil cytokine production and cell morphology
Abstract.
Eggleton P (1999). Effect of IP6 on human neutrophil cytokine production and cell morphology.
Abstract:
Effect of IP6 on human neutrophil cytokine production and cell morphology.
Abstract.
Author URL.
Walker M, Karim Q, Thursz M, Murray E, Eggleton P, Reid K, Worku M (1999). Kinetic impairment of Helicobacter pylori by the collectin surfactant protein D.
GUT,
44, A67-A67.
Author URL.
Karim QN, Thursz MR, Murray E, Eggleton P, Reid KB, Worku M, Walker MM (1999). Kinetic impairment of Helicobacter pylori by the collectin surfactant protein D.
GASTROENTEROLOGY,
116(4), A745-A745.
Author URL.
Eggleton P, Reid KB (1999). Lung surfactant proteins involved in innate immunity.
Curr Opin Immunol,
11(1), 28-33.
Abstract:
Lung surfactant proteins involved in innate immunity.
The two lung surfactant collectins, surfactant protein (SP)-A and SP-D, are involved in host defence against infectious and allergenic agents via enhancement of killing and clearance by macrophages and neutrophils. Recent gene-knockout, protein engineering and physiological studies have emphasised the roles that SP-A and SP-D play in acute inflammatory responses.
Abstract.
Author URL.
Eggleton P, Llewellyn DH (1999). Pathophysiological roles of calreticulin in autoimmune disease.
Scand J Immunol,
49(5), 466-473.
Abstract:
Pathophysiological roles of calreticulin in autoimmune disease.
Autoantibodies against the endoplasmic reticulum (ER) luminal protein, calreticulin are often present in sera from patients with systemic lupus erythematosus, rheumatic disease and various parasitic diseases including onchocerciasis. New information has revealed that calreticulin is implicated in a number of autoimmune processes, including molecular mimicry, epitope spreading, complement inactivation and stimulation of inflammatory mediators, such as nitric oxide production. Calreticulin also binds to the Ro/SS-A antigen complex, which is composed of at least three immunologically distinct proteins bound to a group of small cytoplasmic RNAs that together form a common target for autoimmune responses. Up-regulation of calreticulin at the protein and RNA levels can be triggered by cell stresses, including heat shock, exposure to heavy metals and perturbation of normal ER function, which may in some cases lead to its secretion from cells. Calreticulin is targeted by autoantibodies following its release into the extracellular environment, possibly as a result of cell death, or its presence at the cell surface in response to insults such as viral infection or ultraviolet irradiation. These findings suggest that calreticulin is not just an autoantigen, but plays an active role in the pathology of various autoimmune disease through determinant spreading.
Abstract.
Author URL.
Eggleton P, Murray E, Dodds A, Worku M, Karim Q, Walker M, Ferris J, Moran A, Appelmelk B, Reid K, et al (1999). Surfactant protein D binding to Helicobacter pylori lipopolysaccharide.
GUT,
45, A35-A35.
Author URL.
Griffiths A, Sumiya M, Summerfield J, Cheng S, Murray E, Eggleton P, Sim R, Walker M, Karim Q, Thursz M, et al (1999). The codon 52 mutation in the mannose binding lectin gene predispoes to Helicobacter pylori infection.
GUT,
44, A67-A67.
Author URL.
Griffiths AE, Sumiya M, Summerfield J, Cheng S, Murray E, Eggleton P, Sim R, Walker M, Karim N, Thursz M, et al (1999). The colon 52 mutation in the mannose binding lectin gene predisposes to Helicobacter pylori infection.
GASTROENTEROLOGY,
116(4), A727-A727.
Author URL.
1998
Eggleton P, Reid KB, Tenner AJ (1998). C1q--how many functions? How many receptors?.
Trends Cell Biol,
8(11), 428-431.
Abstract:
C1q--how many functions? How many receptors?
C1, the first component of the classical pathway of complement activation is a complex of three proteins called C1q, C1r and C1s. Normally, C1q binding to aggregated IgG molecules results in activation of the classical pathway of complement. However, C1q has a number of other observed functions, not directly related to complement, that could be mediated by recently identified binding proteins acting as cell-surface receptors or soluble modulators of C1q-mediated functions. This article discusses the various activities of C1q and the evidence that these functions might be influenced by both membrane-bound and soluble C1q-binding proteins.
Abstract.
Author URL.
Leigh LE, Ghebrehiwet B, Perera TP, Bird IN, Strong P, Kishore U, Reid KB, Eggleton P (1998). C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms.
Biochem J,
330 ( Pt 1), 247-254.
Abstract:
C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms.
C1q, the first component of the classical pathway of the complement system, interacts with various cell types and triggers a variety of cell-specific cellular responses, such as oxidative burst, chemotaxis, phagocytosis, etc. Different biological responses are attributed to the interaction of C1q with more than one putative cell-surface C1q receptor/C1q-binding protein. Previously, it has been shown that C1q-mediated oxidative burst by neutrophils is not linked to G-protein-coupled fMet-Leu-Phe-mediated response. In the present study, we have investigated neutrophil migration brought about by C1q and tried to identify the signal-transduction pathways involved in the chemotactic response. We found that C1q stimulated neutrophil migration in a dose-dependent manner, primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). This C1q-induced chemotaxis could be abolished by an inhibitor of G-proteins (pertussis toxin) and PtdIns(3,4,5)P3 kinase (wortmannin and LY294002). The collagen tail of C1q appeared to mediate chemotaxis. gC1qR, a C1q-binding protein, has recently been reported to participate in C1q-mediated chemotaxis of murine mast cells and human eosinophils. We observed that gC1qR enhanced binding of free C1q to adherent neutrophils and promoted C1q-mediated chemotaxis of neutrophils by nearly seven-fold. Our results suggests C1q-mediated chemotaxis involves gC1qR as well as G-protein-coupled signal-transduction mechanisms operating downstream to neutrophil chemotaxis.
Abstract.
Author URL.
Kovacs H, Campbell ID, Strong P, Johnson S, Ward FJ, Reid KB, Eggleton P (1998). Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation.
Biochemistry,
37(51), 17865-17874.
Abstract:
Evidence that C1q binds specifically to CH2-like immunoglobulin gamma motifs present in the autoantigen calreticulin and interferes with complement activation.
Calreticulin (CRT) is located predominantly in the endoplasmic reticulum (ER) of cells, where it functions as a quality control controller of protein folding. However, CRT is also a prevalent autoantigen in patients with systemic lupus erythematosus (SLE), where its release from the cell may arise as a results of dysfunctional apoptosis and inefficient removal of ER vesicles, which are an abundant source of CRT and other autoantigens. Indicative of this is the presence of autoantibodies against CRT in the sera of 40-60% of all SLE patients. Once released into the circulation, CRT might bind directly to C1q and we have suggested that this association may result in a defect in C1q-mediated clearance of antigen-antibody complexes. It has been previously shown that CRT under physiological salt conditions binds to the globular head of C1q. It is known that the globular head region of C1q binds to the CH2 domain in the Fc portion of immunoglobulin gamma (IgG). The N-terminal half of CRT contains a number of short regions of 7-10 amino acids that show sequence similarity to the putative C1q binding region in the CH2 domain of IgG. By use of a series of 92 overlapping CRT synthetic peptides, a number of C1q binding sites on the CRT molecule have been identified, including several containing a CH2-like motif similar to the ExKxKx C1q binding motif found in the CH2 domain of IgG. A number of these peptides were shown to inhibit binding of C1q to IgG and reduce binding of native CRT to C1q. Moreover, several of the peptides were capable of inhibiting the classical pathway of complement activation. These studies have identified specific binding sites on the CRT molecule for C1q and lend support to the hypothesis that interaction of CRT with C1q may interfere with the ability of C1q to associate with immune complexes in autoimmune-related disorders.
Abstract.
Author URL.
Prasad A, Eggleton P, Dodds A, Reid KBM (1998). Expression and immune function analysis of recombinant bovine conglutinin (BK), comprising of carbohydrate recognition domain and neck regions.
MOLECULAR IMMUNOLOGY,
35(6-7), 375-375.
Author URL.
Khamashta MA, Ward FJ, Johnson S, Hajela V, Staines N, Hughes GRV, Reid KBM, Eggleton P (1998). Fine specificity of autoantibodies to calreticulin: Epitope mapping and characterization.
ARTHRITIS AND RHEUMATISM,
41(9), S73-S73.
Author URL.
Kishore U, Leigh LE, Eggleton P, Strong P, Perdikoulis MV, Willis AC, Reid KB (1998). Functional characterization of a recombinant form of the C-terminal, globular head region of the B-chain of human serum complement protein, C1q.
Biochem J,
333 ( Pt 1), 27-32.
Abstract:
Functional characterization of a recombinant form of the C-terminal, globular head region of the B-chain of human serum complement protein, C1q.
The first step in the activation of the classical pathway of the complement system by immune complexes involves the binding of the six globular heads of C1q to the Fc regions of IgG or IgM. The globular heads of C1q are located C-terminal to the six triple-helical stalks present in the molecule; each head is considered to be composed of the C-terminal halves (3x136 residues) of one A-, one B- and one C-chain. It is not known if the C-terminal globular regions, present in each of the three types of chain, are independently folded modules (with each chain having distinct binding properties towards immunoglobulins) or whether the different binding functions of C1q are dependent upon a globular structure which relies on contributions from all three chains. As a first step towards addressing this question, we have expressed the globular head region (residues 87-226) of the C1q B-chain (ghB) as a soluble fusion protein with maltose-binding protein (MBP) in Escherichia coli. The affinity purified fusion protein, designated MBP-ghB, behaved as a dimer on gel filtration and bound preferentially to aggregated IgG rather than to IgM. It could also inhibit C1q-dependent haemolysis of both IgG- and IgM-sensitized erythrocytes. After its release from MBP, by use of Factor Xa, the free ghB exhibited a tendency to aggregate and come out of solution. Since MBP is known to be a monomeric molecule, the dimerization of the MBP-ghB fusion polypeptide is probably brought about by the ghB region, perhaps through hydrophobic interactions within the ghB region. The functional behaviour of MBP-ghB indicates that the globular regions of C1q may adopt a modular organization, i.e. each globular head of C1q may be composed of three structurally and functionally independent domains, thus retaining multivalency in the form of a heterotrimer.
Abstract.
Author URL.
Walker MM, Macdonald S, Thursz MR, Eggleton P, Reid KBM (1998). Surfactant protein D expression in normal and H-pylori infected human gastric antral mucosa.
GUT,
42, A77-A77.
Author URL.
MacDonald S, Thursz MR, Eggleton P, Reid KBM, Walker MM (1998). Surfactant protein D expression in normal and H-pylori-infected human gastric antral mucosa.
GASTROENTEROLOGY,
114(4), A1028-A1029.
Author URL.
Walker MM, Thursz MR, Karim QN, MacDonald S, Murray E, Eggleton P, Reid KBM (1998). The role of the collectin surfactant protein D in Helicobacter pylori infection.
GUT,
43, A16-A16.
Author URL.
Perkins VC, Marshall S, Snowden N, Eggleton P, Bird GA, Haeney M, Reid KBM (1998). Type I properdin deficiency: Novel cases, genetic heterogeneity and immune effector status.
MOLECULAR IMMUNOLOGY,
35(6-7), 376-376.
Author URL.
1997
Thiel S, Vorup-Jensen T, Stover CM, Schwaeble W, Laursen SB, Poulsen K, Willis AC, Eggleton P, Hansen S, Holmskov U, et al (1997). A second serine protease associated with mannan-binding lectin that activates complement.
Nature,
386(6624), 506-510.
Abstract:
A second serine protease associated with mannan-binding lectin that activates complement.
The complement system comprises a complex array of enzymes and non-enzymatic proteins that is essential for the operation of the innate as well as the adaptive immune defence. The complement system can be activated in three ways: by the classical pathway which is initiated by antibody-antigen complexes, by the alternative pathway initiated by certain structures on microbial surfaces, and by an antibody-independent pathway that is initiated by the binding of mannan-binding lectin (MBL; first described as mannan-binding protein) to carbohydrates. MBL is structurally related to the complement C1 subcomponent, C1q, and seems to activate the complement system through an associated serine protease known as MASP (ref. 4) or p100 (ref. 5), which is similar to C1r and C1s of the classical pathway. MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms, including bacteria, yeasts, parasitic protozoa and viruses, and exhibits antibacterial activity through killing mediated by the terminal, lytic complement components or by promoting phagocytosis. The level of MBL in plasma is genetically determined, and deficiency is associated with frequent infections in childhood, and possibly also in adults (for review, see ref. 6). We have now identified a new MBL-associated serine protease (MASP-2) which shows a striking homology with the previously reported MASP (MASP-1) and the two C1q-associated serine proteases C1r and C1s. Thus complement activation through MBL, like the classical pathway, involves two serine proteases and may antedate the development of the specific immune system of vertebrates.
Abstract.
Author URL.
Madan T, Eggleton P, Kishore U, Strong P, Aggrawal SS, Sarma PU, Reid KB (1997). Binding of pulmonary surfactant proteins a and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages.
Infect Immun,
65(8), 3171-3179.
Abstract:
Binding of pulmonary surfactant proteins a and D to Aspergillus fumigatus conidia enhances phagocytosis and killing by human neutrophils and alveolar macrophages.
To determine whether the lung surfactant proteins a (SP-A) and D (SP-D) are involved in the initial protective immunity against opportunistic pulmonary fungal infections caused by Aspergillus fumigatus, we performed a series of in vitro functional studies to see if SP-A and SP-D enhanced binding, phagocytosis, activation, and killing of A. fumigatus conidia by human alveolar macrophages and circulating neutrophils. Both SP-A and SP-D bound to carbohydrate structures on A. fumigatus conidia in a calcium-dependent manner. SP-A and SP-D were also chemoattractant and significantly enhanced agglutination and binding of conidia to alveolar macrophages and neutrophils. Furthermore, in the presence of SP-A and SP-D, the phagocytosis, oxidative burst, and killing of A. fumigatus conidia by neutrophils were significantly increased. These findings indicate that SP-A and SP-D may have an important immunological role in the early antifungal defense responses in the lung, through inhibiting infectivity of conidia by agglutination and by enhancing uptake and killing of A. fumigatus by phagocytic cells.
Abstract.
Author URL.
Eggleton P, Reid KB, Kishore U, Sontheimer RD (1997). Clinical relevance of calreticulin in systemic lupus erythematosus.
Lupus,
6(7), 564-571.
Abstract:
Clinical relevance of calreticulin in systemic lupus erythematosus.
Calreticulin is an abundant intracellular protein which is proposed to have numerous biological functions. However, there is increasing evidence to suggest that calreticulin plays a multifunctional role as an autoantigen present in patients with systemic lupus erythematosus. In this review we detail some of the recent evidence which indicate that calreticulin may play a supportive role in the formation of the autoantigen complex-Ro/SS-A. In addition, several proposed mechanisms of release and surface expression of calreticulin are described in relation to SLE mediated responses to the autoantigen. In particular, the generation of autoantibodies to specific regions of the protein and the ability of calreticulin to interfere with complement mediated inflammatory processes.
Abstract.
Author URL.
Ghebrehiwet B, Lu PD, Zhang W, Keilbaugh SA, Leigh LE, Eggleton P, Reid KB, Peerschke EI (1997). Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex.
J Immunol,
159(3), 1429-1436.
Abstract:
Evidence that the two C1q binding membrane proteins, gC1q-R and cC1q-R, associate to form a complex.
Two types of widely coexpressed, highly acidic, cell membrane binding proteins that display preferential domain specificity for C1q have been described: a 60-kDa calreticulin homologue, designated cC1q-R, that binds to the collagen-like "stalk" and a 33-kDa glycoprotein with affinity for the globular "heads" (gC1q-R). Although the two molecules are known to be coexpressed on all cell types examined to date and often coelute during purification, there is no direct evidence showing that they associate with each other either on the membrane or when examined in a purified system. In this report we present the first evidence that 1) biotinylated cC1q-R binds to recombinant as well as native gC1q-R, as assessed by solid phase ELISA; 2) binding sites for cC1q-R are located within N-terminal residues 76 through 93 of the mature form of gC1q-R and within residues 204 through 218; 3) this interaction is inhibited by two mAbs, 60.11 and 46.23, that recognize primarily epitopes within the N terminus of gC1q-R corresponding to residues 74 through 96 and by mAb 74.5.2 that recognizes epitopes within residues 204 through 218; and 4) biotinylated cC1q-R binds to microtiter-fixed Raji and K562 cells, and this interaction is inhibited by mAb 60.11. Furthermore, coimmunoprecipitation analysis of Raji cell membranes with anti-gC1q-R mAbs showed the presence of cC1q-R in addition to gC1q-R. Taken together, the evidence suggests that cC1q-R is able to form a complex with gC1q-R and may associate with gC1q-R on the cell surface.
Abstract.
Author URL.
Madan T, Kishore U, Shah A, Eggleton P, Strong P, Wang JY, Aggrawal SS, Sarma PU, Reid KB (1997). Lung surfactant proteins a and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils.
Clin Exp Immunol,
110(2), 241-249.
Abstract:
Lung surfactant proteins a and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils.
Aspergillus fumigatus is an opportunistic fungal pathogen which, in the immunocompetent host, causes allergic disorders such as allergic rhinitis, allergic sinusitis, hypersensitivity pneumonitis, and allergic bronchopulmonary Aspergillosis (ABPA). In the present study, the interaction of 3-week culture filtrate (3wcf) allergens and various purified glycosylated and non-glycosylated allergens of A. fumigatus with lung surfactant proteins, SP-A and SP-D, was investigated. Purified SP-A and SP-D, isolated from human bronchoalveolar lavage fluid, bound to the 3wcf allergens and purified allergens, gp55 and gp45, in a carbohydrate-specific and calcium-dependent manner. Both SP-A and SP-D did not bind to deglycosylated allergens, suggesting that the ability of SP-A and SP-D to bind certain allergens is mediated through their carbohydrate recognition domains, interacting with the carbohydrate residues on the allergen. Both SP-A and SP-D could inhibit the ability of allergen-specific IgE from Aspergillosis patients to bind these allergens, suggesting that SP-A and SP-D may be involved in the modulation of allergic sensitization and/or development of allergic reactions. The view that SP-A and SP-D play a protective role against airborne allergens is further supported by the demonstration of their ability to inhibit A. fumigatus allergen-induced histamine release from allergic patients' basophils.
Abstract.
Author URL.
Kishore U, Eggleton P, Reid KB (1997). Modular organization of carbohydrate recognition domains in animal lectins.
Matrix Biol,
15(8-9), 583-592.
Abstract:
Modular organization of carbohydrate recognition domains in animal lectins.
In spite of the great diversity of animal lectins, a common characteristic is their ability to bind sugars by means of discrete, modular carbohydrate recognition domains, CRDs. Three different groups of animal lectins-galectins, P-type and C-type lectins- have different types of CRDs which they arrange in a number of combinations, in three dimensions, in order to increase the affinity for oligosaccharides associated with glycoconjugates. The necessity of combining multiple CRDs in a native lectin molecule in order to increase the affinity for multiple ligands is of great importance physiologically, since many of the carbohydrate structures associated with proteins exist in a variety of different conformations. Recent work has clarified the structural basis for carbohydrate recognition by some of these lectins.
Abstract.
Author URL.
Wirthmueller U, Dewald B, Thelen M, Schäfer MK, Stover C, Whaley K, North J, Eggleton P, Reid KB, Schwaeble WJ, et al (1997). Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.
J Immunol,
158(9), 4444-4451.
Abstract:
Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.
Properdin is an important regulatory constituent of the complement system. In contrast to most other components of complement, biosynthesis of properdin is restricted to a few cell types only, i.e. monocytes/macrophages and peripheral blood T cells. This report demonstrates the presence of properdin mRNA in peripheral blood granulocytes and shows that properdin is stored in the granules of human neutrophils and secreted upon stimulation with TNF-alpha, C5a, IL-8, or FMLP. Subcellular fractionation using Percoll density gradients and Western blot analyses revealed that the bulk of properdin is contained in the secondary granules. Moreover, flow cytometric analyses indicated that properdin is present on the surface of neutrophils. In contrast to alternative pathway components, components of the classical pathway of complement activation, such as C2 and C4, were not detected. Our findings suggest that neutrophils can actively stabilize and amplify the alternative activation pathway of complement by secretion of properdin as part of the innate defense against microorganisms.
Abstract.
Author URL.
Kishore U, Sontheimer RD, Sastry KN, Zaner KS, Zappi EG, Hughes GR, Khamashta MA, Strong P, Reid KB, Eggleton P, et al (1997). Release of calreticulin from neutrophils may alter C1q-mediated immune functions.
Biochem J,
322 ( Pt 2), 543-550.
Abstract:
Release of calreticulin from neutrophils may alter C1q-mediated immune functions.
Calreticulin is an abundant intracellular protein which is involved in a number of cellular functions. During cytomegalovirus infection, as well as inflammatory episodes in autoimmune disease, calreticulin can be released from cells and detected in the circulation, where it may act as an immunodominant autoantigen in diseases such as systemic lupus erythematosus. Calreticulin is known to bind to the molecules of innate immunity, such as C1q, the first subcomponent of complement. However, the functional implications of C1q-calreticulin interactions are unknown. In the present study we sought to investigate, in greater detail, the interaction between these two proteins following the release of calreticulin from neutrophils upon stimulation. In order to pinpoint the regions of interaction, recombinant calreticulin and its discrete domains (N-, P- and C-domains) were produced in Escherichia coli. Both the N- and P-domains of calreticulin were shown to bind to the globular head regions of C1q. Calreticulin also appeared to alter C1q-mediated immune functions. Binding of calreticulin to C1q inhibited haemolysis of IgM-sensitized erythrocytes. Both the N- and P-domains of calreticulin were found to contain sites involved in the inhibition of C1q-induced haemolysis. Full-length calreticulin, and its N- and P-domains, were also able to reduce the C1q-dependent binding of immune complexes to neutrophils. We conclude that calreticulin, once released from neutrophils during inflammation, may not only induce an antigenic reaction, but, under defined conditions, may also interfere with C1q-mediated inflammatory processes.
Abstract.
Author URL.
Ghebrehiwet B, Lu PD, Zhang W, Keilbaugh SA, Leigh LEA, Eggleton P, Reid KBM, Peerschke EIB (1997). The C1q-binding membrane proteins, gC1q-R and cC1q-R, associate to form a metal ion-independent complex.
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY,
99(1), 1523-1523.
Author URL.
Kishore U, Sontheimer RD, Sastry KN, Zappi EG, Hughes GR, Khamashta MA, Reid KB, Eggleton P (1997). The systemic lupus erythematosus (SLE) disease autoantigen-calreticulin can inhibit C1q association with immune complexes.
Clin Exp Immunol,
108(2), 181-190.
Abstract:
The systemic lupus erythematosus (SLE) disease autoantigen-calreticulin can inhibit C1q association with immune complexes.
Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE. Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear. In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera. Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting. CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium. In order to further investigate the C1q-CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli. CRT binds to globular head region of C1q primarily via its N- and P-domains. The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region. CRT also altered C1q-mediated immune functions. The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q. Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q. We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.
Abstract.
Author URL.
1996
Ghebrehiwet B, Lu PD, Zhang W, Eggleton P, Leigh LAE, Reid KBM, Peerschke EJB (1996). Epitope analysis of gC1q-R using monoclonal antibodies.
FASEB JOURNAL,
10(6), 1901-1901.
Author URL.
Ghebrehiwet B, Lu PD, Zhang W, Lim BL, Eggleton P, Leigh LE, Reid KB, Peerschke EI (1996). Identification of functional domains on gC1Q-R, a cell surface protein that binds to the globular "heads" of C1Q, using monoclonal antibodies and synthetic peptides.
Hybridoma,
15(5), 333-342.
Abstract:
Identification of functional domains on gC1Q-R, a cell surface protein that binds to the globular "heads" of C1Q, using monoclonal antibodies and synthetic peptides.
A membrane protein (33 kDa) that binds to the globular "heads" of C1q (gC1q-R) has been recently described. The full length cDNA encoding gC1q-R has been cloned, expressed in E. coli and using the purified recombinant protein (rgC1q-R) as an immunogen, a panel of IgG monoclonal antibodies (MAb) has been produced by fusion of spleen cells from hyperimmunized BALB/c mice with NSO mouse myeloma partners. From this fusion, 60 anti-gC1q-R hybridomas were selected and evaluated for their ability to (1) discriminate between the mature form (MF) of gC1q-R (residues 74-282) and a truncated form (TF) lacking residues 74-95, which contains a major C1q binding site, (2) recognize two functionally defined synthetic peptides derived from the NH2-(XN18) and COOH-(XC15) terminus of gC1q-R, and (3) bind to microtiter well fixed intact Raji cells. Several clones were identified: MAbs 46.23 and 60.11 (IgG1 kappa), reacted strongly with ELISA plate-fixed intact Raji and K562 cells, MF, and the XN18 peptide, but had poor or no reactivity with TF; MAbs 74.5.2 > 25.15 (IgG1 kappa) recognized both MF and TF and are directed against epitopes in the XC15 peptide that contains a binding site for high-molecular-weight kininogen and Factor XII.
Abstract.
Author URL.
1995
Eggleton P, Wang L, Penhallow J, Crawford N, Brown KA (1995). Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.
Ann Rheum Dis,
54(11), 916-923.
Abstract:
Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.
OBJECTIVES: to determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. METHODS: Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. RESULTS: the production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. CONCLUSION: it is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.
Abstract.
Author URL.
Eggleton P, Ghebrehiwet B, Sastry KN, Coburn JP, Zaner KS, Reid KB, Tauber AI (1995). Identification of a gC1q-binding protein (gC1q-R) on the surface of human neutrophils. Subcellular localization and binding properties in comparison with the cC1q-R.
J Clin Invest,
95(4), 1569-1578.
Abstract:
Identification of a gC1q-binding protein (gC1q-R) on the surface of human neutrophils. Subcellular localization and binding properties in comparison with the cC1q-R.
Human neutrophils have multiple C1q-binding proteins. Direct ligand-binding studies with the globular domain of C1q and two-dimensional Western blot analysis revealed two gC1q-binding proteins (gC1q-R): a 33,000 M(r) protein (pI 4.5) mainly in the neutrophil plasma membrane and an 80,000-90,000 M(r) protein (pI 4.1-4.2) located mainly in the granules. Direct binding studies showed that C1q bound to this higher molecular weight protein under physiological conditions. In contrast, anti-cC1q-R antibody, which recognizes a protein binding to collagenous tails of C1q, detected only a 68,000 M(r) protein in the plasma membrane. Both the 33,000 and 68,000 M(r) receptors appear early on the surface of differentiating HL-60 cells. On mature neutrophils, surface expression of both C1q receptors was evident, but no upregulation was observed upon stimulation. Phorbol myristate acetate treatment of neutrophils downregulated both the receptors from cell surface, and significant amounts of soluble gC1q-R were in cell media supernatants, suggesting receptor shedding or secretion. gC1q-R, unlike cC1q-R, did not bind to other C1q-like ligands, namely mannose binding protein, surfactant protein-A, surfactant protein-D, or conglutinin under normal ionic conditions, suggesting a greater specificity for C1q than the "collectin" type receptor (cC1q-R). Rather, gC1q-R only bound purified C1q, and the binding was enhanced under low ionic conditions and in the absence of calcium. The role of C1q receptor shedding and its biologic consequence remain to be defined, but may contribute to the diversity of C1q-mediated responses observed in many cell types.
Abstract.
Author URL.
1994
EGGLETON P, SASTRY K, TAUBER AI, REID KBM, GHEBREHIWET B (1994). BINDING-SPECIFICITY OF 2 DIFFERENT C1Q RECEPTORS ON HUMAN NEUTROPHILS FOR C1Q AND C-TYPE LECTINS, AND ALTERATION IN RECEPTOR EXPRESSION DURING CELL ACTIVATION.
FASEB JOURNAL,
8(5), A766-A766.
Author URL.
Eggleton P, Lieu TS, Zappi EG, Sastry K, Coburn J, Zaner KS, Sontheimer RD, Capra JD, Ghebrehiwet B, Tauber AI, et al (1994). Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein.
Clin Immunol Immunopathol,
72(3), 405-409.
Abstract:
Calreticulin is released from activated neutrophils and binds to C1q and mannan-binding protein.
The Ca2+ storage protein calreticulin is associated with the endoplasmic reticulum and shares a high degree of amino acid homology with the surface receptor C1q-R. In this study, flow cytometric analysis detected calreticulin on the neutrophil surface, which decreased during stimulation probably as a consequence of shedding, as calreticulin was found by ELISA in the cell supernatants of stimulated cells. Antibodies raised against C1q-R and calreticulin demonstrated a high degree of immunological cross-reactivity for purified calreticulin as determined by dot blot analysis. Western blots of neutrophil subcellular fractions located calreticulin in both the cytosol and cell membrane fractions; C1q-R was largely confined to the cell membrane. Calreticulin and C1q-R both bind to C1q and mannan-binding protein. Therefore, calreticulin may be shed on cell activation and may be associated with the cell membrane, where it can potentially interact with C1q and serum lectins. The implications of this are discussed.
Abstract.
Author URL.
Eggleton P, Ghebrehiwet B, Coburn JP, Sastry KN, Zaner KS, Tauber AI (1994). Characterization of the human neutrophil C1q receptor and functional effects of free ligand on activated neutrophils.
Blood,
84(5), 1640-1649.
Abstract:
Characterization of the human neutrophil C1q receptor and functional effects of free ligand on activated neutrophils.
The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.
Abstract.
Author URL.
Hartshorn KL, Crouch EC, White MR, Eggleton P, Tauber AI, Chang D, Sastry K (1994). Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza a viruses.
J Clin Invest,
94(1), 311-319.
Abstract:
Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza a viruses.
We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins a and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza a viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.
Abstract.
Author URL.
LIEU TS, ZAPPI E, CAPRA JD, SONTHEIMER RD, GHEBREHIWET B, EGGLETON P, SASTRY K (1994). THE HUMAN CLQ RECEPTOR AND CALRETICULIN SHARE CROSS-REACTIVE EPITOPES.
JOURNAL OF INVESTIGATIVE DERMATOLOGY,
102(4), 590-590.
Author URL.
1993
EGGLETON P, SASTRYL K, GHEBREHIWET B, LIEU TS, ZAPPI EG, CAPRA JD, SONTHIEMER RD, TAUBER AI (1993). DETECTION OF CALRETICULIN ON THE SURFACE OF HUMAN NEUTROPHILS - IMMUNOLOGICAL CROSS-REACTIVITY WITH THE C1Q RECEPTOR.
BLOOD,
82(10), A509-A509.
Author URL.
LIEU TS, ZAPPI E, CAPRA JD, SONTHEIMER RD, GHEBREHIWET B, EGGLETON P, SASTRY K, TAUBER A (1993). THE HUMAN C1Q RECEPTOR AND CALRETICULIN SHARE CROSS-REACTIVE EPITOPES.
ARTHRITIS AND RHEUMATISM,
36(9), S241-S241.
Author URL.
1992
Eggleton P, Crawford N, Fisher D (1992). Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.
Eur J Cell Biol,
57(2), 265-272.
Abstract:
Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity.
Isolated blood neutrophils from normal healthy subjects were separated into fractions by sequential countercurrent distribution (CCD) in a charge-sensitive dextran/polyethylene glycol aqueous phase system. The neutrophils separated as a broad profile, and in a charged phase procedure the separation was based upon differences in cell surface electrokinetic properties, as confirmed by electrophoretic mobility measurements of fractions across the profile using analytical cytopherometry. The CCD cell fractions were generally pooled as three or four major subfractions for analysis of functional and metabolic differences. These included measurements of chemotaxis, phagocytosis, and respiratory burst. An inverse relationship was found between the electrophoretic mobility (EPM) of the subfraction pools and their functional competence, with the less electronegative cell fraction pools often as much as 2 to 3-fold more active than the more electronegative pools. This demonstration of electrokinetic and functional heterogeneity in 'resting' neutrophil subpopulations separated by CCD may reflect changes during their sojourn in the circulation that determine selective margination and recruitment of cells to inflammatory foci and sites of infection.
Abstract.
Author URL.
Eggleton P, Fisher D, Crawford N (1992). Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis.
J Leukoc Biol,
51(6), 617-625.
Abstract:
Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis.
Isolated blood neutrophils from healthy individuals have been separated by continuous flow electrophoresis (CFE) as a Gaussian-shaped profile extending over 12-15 fractions, on the basis of differences in cell surface electrical charge. The fractions were pooled into three or four subpopulations; the mean electrophoretic mobilities of the least and most electronegative cells were 0.96 and 1.22 microns/sec/V/cm, respectively. Each pool of neutrophils was analyzed for functional and biochemical differences. Expression of respiratory burst in terms of the rate of superoxide production by the least and most electronegative cells to fixed concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-7) M) or phorbol myristate acetate (PMA, 1 microgram/ml) revealed that the least electronegative cells generated superoxide anion (O2-) at approximately twice the rate of the most electronegative cells. However, when lower concentrations of fMLP were used (1-5 x 10(-9) M), the most electronegative cells were most active. The least electronegative cells were also the most active in terms of phagocytosis and chemotaxis. In accordance with these differences in motile function, the basal F-actin content of the least electronegative cell pool was greater than the F-actin levels found in the most electronegative cells and remained so upon stimulation with fMLP. Such neutrophil heterogeneity as detected by CFE may be important in selective margination and recruitment of cells to inflammatory foci and sites of infection and may in part represent subsets of cells within the circulation that are primed in vivo to respond to inflammatory stimuli.
Abstract.
Author URL.
Eggleton P (1992). Hunterian Institute.
Nature,
360(6401).
Author URL.
EGGLETON P (1992). PROSPECTS FOR GRADUATES.
NATURE,
355(6358), 292-292.
Author URL.
Eggleton P (1992). Prospects for graduates. Nature, 355
1991
Eggleton P, Penhallow J, Crawford N (1991). Differences in basal and formyl-Met-Leu-Phe-stimulated F-actin content of human blood neutrophil subpopulations separated by continuous-flow electrophoresis.
Biochem Soc Trans,
19(4), 1129-1130.
Author URL.
CRAWFORD N, EGGLETON P, FISHER D (1991). POPULATION HETEROGENEITY IN BLOOD NEUTROPHILS FRACTIONATED - BY CONTINUOUS-FLOW ELECTROPHORESIS (CFE) AND BY PARTITIONING IN AQUEOUS POLYMER 2-PHASE SYSTEMS (PAPS).
ACS SYMPOSIUM SERIES,
464, 190-205.
Author URL.
CRAWFORD N, EGGLETON P, FISHER D (1991). POPULATION HETEROGENEITY IN BLOOD NEUTROPHILS FRACTIONATED - BY CONTINUOUS-FLOW ELECTROPHORESIS (CFE) AND BY PARTITIONING IN AQUEOUS POLYMER 2-PHASE SYSTEMS (PAPS). In (Ed)
, 190-205.
Author URL.
Eggleton P, Penhallow J, Crawford N (1991). Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.
Biochim Biophys Acta,
1094(3), 309-316.
Abstract:
Priming action of inositol hexakisphosphate (InsP6) on the stimulated respiratory burst in human neutrophils.
After priming by a number of different host, bacterial and chemical agents, human neutrophils may be stimulated to produce a greater respiratory burst than would be elicited by the stimulus alone. Other neutrophil functions may be similarly enhanced by pre-exposure to a priming agent. We describe here a new extracellular role for inositol hexakisphosphate (InsP6) as a priming agent for a variety of human neutrophil functional responses. Preincubation of the cells with InsP6 alone (up to 250 microM) has no stimulatory effect upon the basal production of reactive oxygen intermediates but the response to a subsequent stimulus (FMLP, PMA or phagocytic particles) is substantially enhanced. Levels 100-200% higher than 'stimulus only' controls have been recorded. Peak enhancement of the FMLP-induced oxidative response occurs after 1-2 min preincubation with InsP6 and the effect is dose-dependent (maximum at approx. 100 microM InsP6). As others have shown FMLP stimulation of superoxide anion production has no external Ca2+ dependence but the presence of low levels of Ca2+ and Mg2+ (0.1 mM) during priming appears to be an essential requirement for full expression. Reports of intracellular concentrations of InsP6 in mammalian cells in the 30-100 microM range suggest that the local release of this inositol polyphosphate from damaged or effect cells could have a physiologically important modulatory role on neutrophil functions.
Abstract.
Author URL.
1990
CRAWFORD N, EGGLETON P, FISHER D (1990). POPULATION HETEROGENEITY IN BLOOD NEUTROPHILS FRACTIONATED BY CONTINUOUS-FLOW ELECTROPHORESIS (CFE) AND 2 PHASE AQUEOUS POLYMER PARTITIONING (TAPP).
ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY,
199, 64-IAEC.
Author URL.
1989
EGGLETON P, CRAWFORD N, FISHER D (1989). FUNCTIONAL-HETEROGENEITY IN HUMAN-NEUTROPHILS DETECTED BY CONTINUOUS-FLOW ELECTROPHORESIS AND COUNTER CURRENT DISTRIBUTION IN AQUEOUS POLYMER 2-PHASE SYSTEMS.
BIOCHEMICAL SOCIETY TRANSACTIONS,
17(4), 759-760.
Author URL.
CRAWFORD N, EGGLETON P, FISHER D (1989). METABOLIC, MOTILE FUNCTIONS AND RECEPTOR STATUS HETEROGENEITY IN BLOOD NEUTROPHIL SUBPOPULATIONS FRACTIONATED BY CONTINUOUS-FLOW ELECTROPHORESIS.
JOURNAL OF LEUKOCYTE BIOLOGY,
46(4), 331-331.
Author URL.
Eggleton P, Gargan R, Fisher D (1989). Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers.
J Immunol Methods,
121(1), 105-113.
Abstract:
Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers.
A simple, rapid and economical method for the isolation of polymorphonuclear leucocytes (PMNs) from whole blood is compared with dextran and dextran/Lymphoprep gradient techniques. The method eliminates the use of dextran and density gradient polymers such as Ficoll which have been shown to affect PMNs adversely. The technique is based on the lysis of red cells with isotonic ammonium chloride solution followed by differential centrifugation to separate the PMNs. This method gave a PMN yield of 73% (SD +/- 3.5) and a purity of 78% (SD +/- 2.5). Both morphology and functional activity were preserved, as assessed by bacterial phagocytosis and killing, chemotaxis, polarising response, superoxide production and adherence. In contrast, the dextran and dextran/Lymphoprep techniques gave yields of 50% and 15% with purities of 78% and 91% respectively. In a series of 14 PMN isolations, the differential centrifugation method gave an average yield of 63% with an average purity of 83%.
Abstract.
Author URL.
1986
SMITH GW, EGGLETON P (1986). SIMPLE C-REACTIVE PROTEIN ASSAYS FOR ROUTINE MICROBIOLOGY LABORATORY.
JOURNAL OF MEDICAL MICROBIOLOGY,
21(4), R11-R11.
Author URL.
1985
Smith GW, Eggleton P, Kibbler CC, Neville LO (1985). C-reactive protein and liver viability after transplantation. Lancet, 325(8430), 703-704.
Smith GW, Eggleton P, Kibbler CC, Neville LO (1985). C-reactive protein and liver viability after transplantation.
Lancet,
1(8430), 703-704.
Author URL.