Patty Atkinson - Academia.edu (original) (raw)

Papers by Patty Atkinson

Research paper thumbnail of A quantitative lateral flow assay to detect complement activation in blood

Analytical Biochemistry, 2015

Complement is a major effector arm of the innate immune system that responds rapidly to pathogens... more Complement is a major effector arm of the innate immune system that responds rapidly to pathogens or altered self. The central protein of the system, C3, participates in an amplification loop that can lead to rapid complement deposition on a target and, if excessive, can result in host tissue damage. Currently, complement activation is routinely monitored by assessing total C3 levels, which is an indirect and relatively insensitive method. An alternative approach would be to measure downstream C3 activation products such as C3a or iC3b. However, in vitro activation can produce falsely elevated levels of these biomarkers. To circumvent this issue, a lateral flow immunoassay system was developed that measures iC3b in whole blood, plasma and serum and avoids in vitro activation by minimizing sample handling. This assay system returns results in 15 minutes and specifically measures iC3b while having minimal cross-reactivity to other C3 split products. While evaluating the potential of this assay, it was observed that circulating iC3b levels can distinguish healthy individuals from those with complement activation-associated diseases. This tool is engineered to provide an improved method to assess complement activation at pointof-care and could facilitate studies to monitor disease progression in a variety of inflammatory conditions.

Research paper thumbnail of Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation

Immunity, Jan 15, 2015

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mu... more TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune def...

Research paper thumbnail of Complement system on the attack in autoimmunity

Journal of Clinical Investigation, 2003

The author has declared that no conflict of interest exists. Nonstandard abbreviations used: anti... more The author has declared that no conflict of interest exists. Nonstandard abbreviations used: antiphospholipid syndrome (APS); alternative pathway (AP); complement component 5a (C5a); rheumatoid arthritis (RA).

Research paper thumbnail of Too much of a good thing at the site of tissue injury: the instructive example of the complement system predisposing to thrombotic microangiopathy

Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program, 2011

The interplay between the complement and coagulation systems is just beginning to be explored and... more The interplay between the complement and coagulation systems is just beginning to be explored and characterized. This interaction, however, is ancient. For example, if endotoxin is added to the hemolymph of the horseshoe crab, a protease is activated that triggers both the coagulation and complement systems. However, in extant mammals, these 2 cascades have diverged. These infamous "terrible C's" are the scourge of many a medical student (and possibly even a few hematologists). They also are intimately involved in the pathophysiology of thrombomicroangiopathies (TMAs). The complement system generates a procoagulant microenvironment and the coagulation system forms a clot in the renal microvasculature, and thus the 2 systems are partners in mediating multiple pathophysiological conditions.

Research paper thumbnail of Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

Molecular Immunology, 2015

Autoantibody formation against Factor H (FH) is found in 7-10% of patients who are diagnosed with... more Autoantibody formation against Factor H (FH) is found in 7-10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.

Research paper thumbnail of Autoimmunity: Homeostasis of Innate Immunity Gone Awry

Journal of Clinical Immunology, 2012

Research paper thumbnail of Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited

Clinical Immunology, 2012

C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a ... more C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient's serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient's serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient's C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, reviews the literature on C4-NeF and we comment on its detection in the clinical laboratory.

Research paper thumbnail of Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

Journal of Experimental Medicine, 2010

The complement system plays an essential protective role in the initial defense against many micr... more The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutra...

Research paper thumbnail of Defective Complement Inhibitory Function Predisposes to Renal Disease

Annual Review of Medicine, 2013

The role of the complement system in mediating human renal disease has long been recognized in im... more The role of the complement system in mediating human renal disease has long been recognized in immune-complex excess syndromes such as systemic lupus erythematosus and in dense deposit disease in which no immunoglobulin (Ig) is present. Over the past 15 years, mutations in complement regulatory genes have been demonstrated to predispose to thrombotic microangiopathies including atypical hemolytic uremic syndrome, C3 and C1q glomerulopathies, and preeclampsia. Excessive complement activation on an endothelial cell, due to either an autoantibody or a regulatory protein deficiency, sets up a procoagulant state in these diseases as well as in the antiphospholipid syndrome. Knowledge of the genes involved and the functional consequences of alterations in their structure has led to therapy that blocks complement activation.

Research paper thumbnail of Acquired C1 esterase inhibitor deficiency in two patients presenting with a lupus‐like syndrome and anticardiolipin antibodies

Arthritis Care & Research, 2002

Research paper thumbnail of Systemic humoral autoimmunity but joint-specific inflammation: The syndrome of rheumatoid arthritis

Arthritis & Rheumatism, 2007

Research paper thumbnail of High-resolution structures of module pairs 10–11, 11–12 and 12–13 combined with SAXS data for 10–15 and 10–18 constructs to define architecture of factor H

Molecular Immunology, 2010

Complement receptor type 1 (CR1, CD35) is the erythrocyte's immune adherence receptor, vital for ... more Complement receptor type 1 (CR1, CD35) is the erythrocyte's immune adherence receptor, vital for clearance of C3b-and C4bopsonised particles. It also regulates alternative and classical complement pathways. Within its 30 CCP-module ectodomain, modules 1-3 comprise functional site 1 whilst two near-identical copies of site 2 occupy modules 8-10 and 15-17. Sites 1 and 2 have similar sequences but interact differentially with complement cascade proteins. Site 1 has a preference for C4b; site 2 prefers C3b. Moreover, site 1 preferentially mediates convertase decay acceleration whilst site 2 is more effective at factor I cofactor activity. The 3D structure of site 2 was solved previously but the structure of site 1 remains undetermined despite much effort. We have persisted with long-term efforts to solve the 3D structure of site 1. Expression of a codon-optimised gene in Pichia pastoris circumvented previous difficulties with protein yield. We prepared multiple-mg quantities of double-isotopically labelled (13C and 15N) samples of CR1 modules 1-2 (CR1 1-2) and CR1 1-3. The CR1 1-2 sample was employed to collect an extensive set of triple-resonance NMR spectra that were of sufficient quality to allow near-complete assignment of backbone and sidechain nuclei. Based on these assignments, distance restraints were extracted from nuclear Overhauser effect-spectra, which allowed a high-resolution 3D structure of CR1 1-2 to be calculated. This NMRderived structure and a newly refined, previously solved, structure of CR1 2-3 were supplemented by small-angle X-ray scattering data that helped to establish intermodular angles within the doublemodule constructs and within CR1 1-3, thus helping to morph the structures of CR1 1-2 and CR1 2-3 into a structure of site 1. Intermodular flexibility was characterised using NMR-based relaxation experiments. The new structure allows interpretation of previously performed homologous reverse-substitution experiments between sites 1 and 2.

Research paper thumbnail of A quantitative lateral flow assay to detect complement activation in blood

Analytical Biochemistry, 2015

Complement is a major effector arm of the innate immune system that responds rapidly to pathogens... more Complement is a major effector arm of the innate immune system that responds rapidly to pathogens or altered self. The central protein of the system, C3, participates in an amplification loop that can lead to rapid complement deposition on a target and, if excessive, can result in host tissue damage. Currently, complement activation is routinely monitored by assessing total C3 levels, which is an indirect and relatively insensitive method. An alternative approach would be to measure downstream C3 activation products such as C3a or iC3b. However, in vitro activation can produce falsely elevated levels of these biomarkers. To circumvent this issue, a lateral flow immunoassay system was developed that measures iC3b in whole blood, plasma and serum and avoids in vitro activation by minimizing sample handling. This assay system returns results in 15 minutes and specifically measures iC3b while having minimal cross-reactivity to other C3 split products. While evaluating the potential of this assay, it was observed that circulating iC3b levels can distinguish healthy individuals from those with complement activation-associated diseases. This tool is engineered to provide an improved method to assess complement activation at pointof-care and could facilitate studies to monitor disease progression in a variety of inflammatory conditions.

Research paper thumbnail of Cytosolic Nuclease TREX1 Regulates Oligosaccharyltransferase Activity Independent of Nuclease Activity to Suppress Immune Activation

Immunity, Jan 15, 2015

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mu... more TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune def...

Research paper thumbnail of Complement system on the attack in autoimmunity

Journal of Clinical Investigation, 2003

The author has declared that no conflict of interest exists. Nonstandard abbreviations used: anti... more The author has declared that no conflict of interest exists. Nonstandard abbreviations used: antiphospholipid syndrome (APS); alternative pathway (AP); complement component 5a (C5a); rheumatoid arthritis (RA).

Research paper thumbnail of Too much of a good thing at the site of tissue injury: the instructive example of the complement system predisposing to thrombotic microangiopathy

Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program, 2011

The interplay between the complement and coagulation systems is just beginning to be explored and... more The interplay between the complement and coagulation systems is just beginning to be explored and characterized. This interaction, however, is ancient. For example, if endotoxin is added to the hemolymph of the horseshoe crab, a protease is activated that triggers both the coagulation and complement systems. However, in extant mammals, these 2 cascades have diverged. These infamous "terrible C's" are the scourge of many a medical student (and possibly even a few hematologists). They also are intimately involved in the pathophysiology of thrombomicroangiopathies (TMAs). The complement system generates a procoagulant microenvironment and the coagulation system forms a clot in the renal microvasculature, and thus the 2 systems are partners in mediating multiple pathophysiological conditions.

Research paper thumbnail of Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS)

Molecular Immunology, 2015

Autoantibody formation against Factor H (FH) is found in 7-10% of patients who are diagnosed with... more Autoantibody formation against Factor H (FH) is found in 7-10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.

Research paper thumbnail of Autoimmunity: Homeostasis of Innate Immunity Gone Awry

Journal of Clinical Immunology, 2012

Research paper thumbnail of Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited

Clinical Immunology, 2012

C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a ... more C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient's serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient's serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient's C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, reviews the literature on C4-NeF and we comment on its detection in the clinical laboratory.

Research paper thumbnail of Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

Journal of Experimental Medicine, 2010

The complement system plays an essential protective role in the initial defense against many micr... more The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutra...

Research paper thumbnail of Defective Complement Inhibitory Function Predisposes to Renal Disease

Annual Review of Medicine, 2013

The role of the complement system in mediating human renal disease has long been recognized in im... more The role of the complement system in mediating human renal disease has long been recognized in immune-complex excess syndromes such as systemic lupus erythematosus and in dense deposit disease in which no immunoglobulin (Ig) is present. Over the past 15 years, mutations in complement regulatory genes have been demonstrated to predispose to thrombotic microangiopathies including atypical hemolytic uremic syndrome, C3 and C1q glomerulopathies, and preeclampsia. Excessive complement activation on an endothelial cell, due to either an autoantibody or a regulatory protein deficiency, sets up a procoagulant state in these diseases as well as in the antiphospholipid syndrome. Knowledge of the genes involved and the functional consequences of alterations in their structure has led to therapy that blocks complement activation.

Research paper thumbnail of Acquired C1 esterase inhibitor deficiency in two patients presenting with a lupus‐like syndrome and anticardiolipin antibodies

Arthritis Care & Research, 2002

Research paper thumbnail of Systemic humoral autoimmunity but joint-specific inflammation: The syndrome of rheumatoid arthritis

Arthritis & Rheumatism, 2007

Research paper thumbnail of High-resolution structures of module pairs 10–11, 11–12 and 12–13 combined with SAXS data for 10–15 and 10–18 constructs to define architecture of factor H

Molecular Immunology, 2010

Complement receptor type 1 (CR1, CD35) is the erythrocyte's immune adherence receptor, vital for ... more Complement receptor type 1 (CR1, CD35) is the erythrocyte's immune adherence receptor, vital for clearance of C3b-and C4bopsonised particles. It also regulates alternative and classical complement pathways. Within its 30 CCP-module ectodomain, modules 1-3 comprise functional site 1 whilst two near-identical copies of site 2 occupy modules 8-10 and 15-17. Sites 1 and 2 have similar sequences but interact differentially with complement cascade proteins. Site 1 has a preference for C4b; site 2 prefers C3b. Moreover, site 1 preferentially mediates convertase decay acceleration whilst site 2 is more effective at factor I cofactor activity. The 3D structure of site 2 was solved previously but the structure of site 1 remains undetermined despite much effort. We have persisted with long-term efforts to solve the 3D structure of site 1. Expression of a codon-optimised gene in Pichia pastoris circumvented previous difficulties with protein yield. We prepared multiple-mg quantities of double-isotopically labelled (13C and 15N) samples of CR1 modules 1-2 (CR1 1-2) and CR1 1-3. The CR1 1-2 sample was employed to collect an extensive set of triple-resonance NMR spectra that were of sufficient quality to allow near-complete assignment of backbone and sidechain nuclei. Based on these assignments, distance restraints were extracted from nuclear Overhauser effect-spectra, which allowed a high-resolution 3D structure of CR1 1-2 to be calculated. This NMRderived structure and a newly refined, previously solved, structure of CR1 2-3 were supplemented by small-angle X-ray scattering data that helped to establish intermodular angles within the doublemodule constructs and within CR1 1-3, thus helping to morph the structures of CR1 1-2 and CR1 2-3 into a structure of site 1. Intermodular flexibility was characterised using NMR-based relaxation experiments. The new structure allows interpretation of previously performed homologous reverse-substitution experiments between sites 1 and 2.