Publications by authors named "John P Atkinson"

166 Publications

Dengue and the Lectin Pathway of the Complement System.

Viruses 2021 Jun 24;13(7). Epub 2021 Jun 24.

Molecular Biology of Dengue and Flaviviruses Research Team, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 12120, Thailand.

Dengue is a mosquito-borne viral disease causing significant health and economic burdens globally. The dengue virus (DENV) comprises four serotypes (DENV1-4). Usually, the primary infection is asymptomatic or causes mild dengue fever (DF), while secondary infections with a different serotype increase the risk of severe dengue disease (dengue hemorrhagic fever, DHF). Complement system activation induces inflammation and tissue injury, contributing to disease pathogenesis. However, in asymptomatic or primary infections, protective immunity largely results from the complement system's lectin pathway (LP), which is activated through foreign glycan recognition. Differences in N-glycans displayed on the DENV envelope membrane influence the lectin pattern recognition receptor (PRR) binding efficiency. The important PRR, mannan binding lectin (MBL), mediates DENV neutralization through (1) a complement activation-independent mechanism via direct MBL glycan recognition, thereby inhibiting DENV attachment to host target cells, or (2) a complement activation-dependent mechanism following the attachment of complement opsonins C3b and C4b to virion surfaces. The serum concentrations of lectin PRRs and their polymorphisms influence these LP activities. Conversely, to escape the LP attack and enhance the infectivity, DENV utilizes the secreted form of nonstructural protein 1 (sNS1) to counteract the MBL effects, thereby increasing viral survival and dissemination.
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http://dx.doi.org/10.3390/v13071219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310334PMC
June 2021

Membrane cofactor protein (MCP; CD46): deficiency states and pathogen connections.

Curr Opin Immunol 2021 May 15;72:126-134. Epub 2021 May 15.

Division of Rheumatology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, 63110, USA. Electronic address:

Membrane cofactor protein (MCP; CD46), a ubiquitously expressed complement regulatory protein, serves as a cofactor for serine protease factor I to cleave and inactivate C3b and C4b deposited on host cells. However, CD46 also plays roles in human reproduction, autophagy, modulating T cell activation and effector functions and is a member of the newly identified intracellular complement system (complosome). CD46 also is a receptor for 11 pathogens ('pathogen magnet'). While CD46 deficiencies contribute to inflammatory disorders, its overexpression in cancers and role as a receptor for some adenoviruses has led to its targeting by oncolytic agents and adenoviral-based therapeutic vectors, including coronavirus disease of 2019 (COVID-19) vaccines. This review focuses on recent advances in identifying disease-causing CD46 variants and its pathogen connections.
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http://dx.doi.org/10.1016/j.coi.2021.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8123722PMC
May 2021

Increased complement activation is a distinctive feature of severe SARS-CoV-2 infection.

bioRxiv 2021 Feb 23. Epub 2021 Feb 23.

Division of Rheumatology, John T. Milliken Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.

Complement activation has been implicated in the pathogenesis of severe SARS-CoV-2 infection. However, it remains to be determined whether increased complement activation is a broad indicator of critical illness (and thus, no different in COVID-19). It is also unclear which pathways are contributing to complement activation in COVID-19, and, if complement activation is associated with certain features of severe SARS-CoV-2 infection, such as endothelial injury and hypercoagulability. To address these questions, we investigated complement activation in the plasma from patients with COVID-19 prospectively enrolled at two tertiary care centers. We compared our patients to two non-COVID cohorts: (a) patients hospitalized with influenza, and (b) patients admitted to the intensive care unit (ICU) with acute respiratory failure requiring invasive mechanical ventilation (IMV). We demonstrate that circulating markers of complement activation (i.e., sC5b-9) are elevated in patients with COVID-19 compared to those with influenza and to patients with non-COVID-19 respiratory failure. Further, the results facilitate distinguishing those who are at higher risk of worse outcomes such as requiring ICU admission, or IMV. Moreover, the results indicate enhanced activation of the alternative complement pathway is most prevalent in patients with severe COVID-19 and is associated with markers of endothelial injury (i.e., Ang2) as well as hypercoagulability (i.e., thrombomodulin and von Willebrand factor). Our findings identify complement activation to be a distinctive feature of COVID-19, and provide specific targets that may be utilized for risk prognostication, drug discovery and personalized clinical trials.

Summmary: Complement has been implicated in COVID-19. However, whether this is distinctive of COVID-19 remains unanswered. Ma et al report increased complement activation in COVID-19 compared to influenza and non-COVID respiratory failure, and demonstrate alternative pathway activation as a key marker of multiorgan failure and death.
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http://dx.doi.org/10.1101/2021.02.22.432177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924264PMC
February 2021

CD46 and Oncologic Interactions: Friendly Fire against Cancer.

Antibodies (Basel) 2020 Nov 2;9(4). Epub 2020 Nov 2.

Division of Rheumatology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110, USA.

One of the most challenging aspects of cancer therapeutics is target selection. Recently, CD46 (membrane cofactor protein; MCP) has emerged as a key player in both malignant transformation as well as in cancer treatments. Normally a regulator of complement activation, CD46 is co-expressed as four predominant isoforms on almost all cell types. CD46 is highly overexpressed on a variety of human tumor cells. Clinical and experimental data support an association between increased CD46 expression and malignant transformation and metastasizing potential. Further, CD46 is a newly discovered driver of metabolic processes and plays a role in the intracellular complement system (complosome). CD46 is also known as a pathogen magnet due to its role as a receptor for numerous microbes, including several species of measles virus and adenoviruses. Strains of these two viruses have been exploited as vectors for the therapeutic development of oncolytic agents targeting CD46. In addition, monoclonal antibody-drug conjugates against CD46 also are being clinically evaluated. As a result, there are multiple early-phase clinical trials targeting CD46 to treat a variety of cancers. Here, we review CD46 relative to these oncologic connections.
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http://dx.doi.org/10.3390/antib9040059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709105PMC
November 2020

Targeting complement activation in COVID-19.

Blood 2020 10;136(18):2000-2001

Washington University School of Medicine.

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http://dx.doi.org/10.1182/blood.2020008925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596848PMC
October 2020

The beneficial and pathogenic roles of complement in COVID-19.

Cleve Clin J Med 2020 Oct 28. Epub 2020 Oct 28.

Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO

We briefly summarize the complement system and its functions in immunity and disease. We present data supporting the requirement of complement to resolve COVID-19, and discuss how complement overactivation later in severe disease could drive multiorgan damage characteristic of fatal COVID-19.
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http://dx.doi.org/10.3949/ccjm.87a.ccc065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079550PMC
October 2020

Lesion evolution and neurodegeneration in RVCL-S: A monogenic microvasculopathy.

Neurology 2020 10 4;95(14):e1918-e1931. Epub 2020 Sep 4.

From the Department of Neurology (A.L.F., V.W.C., S.F., M.B.M., A.M.B., V.B., Y.T., P.K., Y.C., J.H., J.-M.L.), Mallinckrodt Institute of Radiology (A.L.F., J.-M.L., H.A.), Department of Ophthalmology (M.G.G.), and Department of Medicine (M.B., M.K.L., D.H., J.J.M., J.P.A.), Division of Rheumatology, Washington University School of Medicine, St. Louis, MO; Department of Radiology (D.L.), The Johns Hopkins University School of Medicine, Baltimore, MD; and Department of Neurology (J.C.J.), Icahn School of Medicine at Mount Sinai, New York, NY.

Objective: To characterize lesion evolution and neurodegeneration in retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) using multimodal MRI.

Methods: We prospectively performed MRI and cognitive testing in RVCL-S and healthy control cohorts. Gray and white matter volume and disruption of white matter microstructure were quantified. Asymmetric spin echo acquisition permitted voxel-wise oxygen extraction fraction (OEF) calculation as an in vivo marker of microvascular ischemia. The RVCL-S cohort was included in a longitudinal analysis of lesion subtypes in which hyperintense lesions on fluid-attenuated inversion recovery (FLAIR), T1-postgadolinium, and diffusion-weighted imaging were delineated and quantified volumetrically.

Results: Twenty individuals with RVCL-S and 26 controls were enrolled. White matter volume and microstructure declined faster in those with RVCL-S compared to controls. White matter atrophy in RVCL-S was highly linear (ρ = -0.908, < 0.0001). Normalized OEF was elevated in RVCL-S and increased with disease duration. Multiple cognitive domains, specifically those measuring working memory and processing speed, were impaired in RVCL-S. Lesion volumes, regardless of subtype, progressed/regressed with high variability as a function of age, while FLAIR lesion burden increased near time to death ( < 0.001).

Conclusion: RVCL-S is a monogenic microvasculopathy affecting predominantly the white matter with regard to atrophy and cognitive impairment. White matter volumes in RVCL-S declined linearly, providing a potential metric against which to test the efficacy of future therapies. Progressive elevation of white matter OEF suggests that microvascular ischemia may underlie neurodegeneration in RVCL-S.
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http://dx.doi.org/10.1212/WNL.0000000000010659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682842PMC
October 2020

Clearance of amyloid-beta with bispecific antibody constructs bound to erythrocytes.

Alzheimers Dement (N Y) 2020 27;6(1):e12067. Epub 2020 Aug 27.

Department of Medicine and Division of Rheumatology Washington University St. Louis Missouri USA.

We propose use of bispecific monoclonal antibody (mAb) complexes bound to erythrocytes to redress the lack of efficacy of anti-amyloid beta mAbs in Alzheimer's disease treatment. Our paradigm leverages erythrocyte complement receptor 1 to promote rapid and quantitative removal of amyloid beta from the circulation, and its subsequent removal from the brain as well.
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http://dx.doi.org/10.1002/trc2.12067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453147PMC
August 2020

Local complement activation is associated with primary graft dysfunction after lung transplantation.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

Department of Medicine, Perlman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

BACKGROUNDThe complement system plays a key role in host defense but is activated by ischemia/reperfusion injury (IRI). Primary graft dysfunction (PGD) is a form of acute lung injury occurring predominantly due to IRI, which worsens survival after lung transplantation (LTx). Local complement activation is associated with acute lung injury, but whether it is more reflective of allograft injury compared with systemic activation remains unclear. We proposed that local complement activation would help identify those who develop PGD after LTx. We also aimed to identify which complement activation pathways are associated with PGD.METHODSWe performed a multicenter cohort study at the University of Pennsylvania and Washington University School of Medicine. Bronchoalveolar lavage (BAL) and plasma specimens were obtained from recipients within 24 hours after LTx. PGD was scored based on the consensus definition. Complement activation products and components of each arm of the complement cascade were measured using ELISA.RESULTSIn both cohorts, sC4d and sC5b-9 levels were increased in BAL of subjects with PGD compared with those without PGD. Subjects with PGD also had higher C1q, C2, C4, and C4b, compared with subjects without PGD, suggesting classical and lectin pathway involvement. Ba levels were higher in subjects with PGD, suggesting alternative pathway activation. Among lectin pathway-specific components, MBL and FCN-3 had a moderate-to-strong correlation with the terminal complement complex in the BAL but not in the plasma.CONCLUSIONComplement activation fragments are detected in the BAL within 24 hours after LTx. Components of all 3 pathways are locally increased in subjects with PGD. Our findings create a precedent for investigating complement-targeted therapeutics to mitigate PGD.FUNDINGThis research was supported by the NIH, American Lung Association, Children's Discovery Institute, Robert Wood Johnson Foundation, Cystic Fibrosis Foundation, Barnes-Jewish Hospital Foundation, Danish Heart Foundation, Danish Research Foundation of Independent Research, Svend Andersen Research Foundation, and Novo Nordisk Research Foundation.
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http://dx.doi.org/10.1172/jci.insight.138358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526453PMC
September 2020

The complement system in COVID-19: friend and foe?

JCI Insight 2020 08 6;5(15). Epub 2020 Aug 6.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, USA.

Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic and a disruptive health crisis. COVID-19-related morbidity and mortality have been attributed to an exaggerated immune response. The role of complement activation and its contribution to illness severity is being increasingly recognized. Here, we summarize current knowledge about the interaction of coronaviruses with the complement system. We posit that (a) coronaviruses activate multiple complement pathways; (b) severe COVID-19 clinical features often resemble complementopathies; (c) the combined effects of complement activation, dysregulated neutrophilia, endothelial injury, and hypercoagulability appear to be intertwined to drive the severe features of COVID-19; (d) a subset of patients with COVID-19 may have a genetic predisposition associated with complement dysregulation; and (e) these observations create a basis for clinical trials of complement inhibitors in life-threatening illness.
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http://dx.doi.org/10.1172/jci.insight.140711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455060PMC
August 2020

President Kennedy's Adrenals and My Brother's Death.

Authors:
John P Atkinson

Am J Med 2020 07 7;133(7):876-877. Epub 2020 Apr 7.

Internal Medicine Department/Rheumatology Division, Washington University School of Medicine, St. Louis, Mo. Electronic address:

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http://dx.doi.org/10.1016/j.amjmed.2020.03.007DOI Listing
July 2020

Impaired tumor necrosis factor-α secretion by CD4 T cells during respiratory syncytial virus bronchiolitis associated with recurrent wheeze.

Immun Inflamm Dis 2020 03 4;8(1):30-39. Epub 2020 Jan 4.

The Division of Pulmonary, Critical Care and Sleep Medicine, University of Kansas School of Medicine, Kansas City, Kansas.

Background: Infants with severe respiratory syncytial virus (RSV) bronchiolitis have an increased risk of recurrent wheezing and asthma. We aimed to evaluate the relationships between regulatory T cell (Treg) percentage and cytokine production of in vitro-stimulated CD4+ T cells during acute bronchiolitis and the development of recurrent wheezing in the first 3 years of life.

Methods: We obtained peripheral blood from 166 infants hospitalized with their first episode of RSV-confirmed bronchiolitis. Granzyme B (GZB) expression, and interleukin-10, interferon-γ, tumor necrosis factor-α (TNF-α), IL-4, and IL-5 production by in vitro anti-CD3/CD28- and anti-CD3/CD46-activated CD4+ T cells, and percentage of peripheral Treg (CD4+CD25 Foxp3 ) cells were measured by flow cytometry. Wheezing was assessed every 6 months. Recurrent wheezing was defined as three or more episodes following the initial RSV bronchiolitis.

Results: Sixty-seven percent (n = 111) of children had wheezing after their initial RSV infection, with 30% having recurrent wheezing. The percentage of peripheral Treg (CD4+CD25 Foxp3 ) cells was not significantly different between the wheezing groups. Decreased TNF-α production from anti-CD3/CD28- and anti-CD3/CD46- activated CD4+ T cells was observed in the recurrent wheezers, compared with nonwheezers (p = .048 and .03, respectively). There were no significant differences in the GZB+ CD4+ T cells and production of other inflammatory cytokines between these groups.

Conclusions: We demonstrated lower TNF-α production by in vitro stimulated CD4+ T cells during severe RSV bronchiolitis in children that subsequently developed recurrent wheezing, compared with children with no subsequent wheeze. These findings support the role of CD4+ T cell immunity in the development of subsequent wheezing in these children.
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http://dx.doi.org/10.1002/iid3.281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016853PMC
March 2020

Rare mutations in the complement regulatory gene CSMD1 are associated with male and female infertility.

Nat Commun 2019 10 11;10(1):4626. Epub 2019 Oct 11.

Department of Genetics, Washington University School of Medicine, St. Louis, MO, 63110, USA.

Infertility in men and women is a complex genetic trait with shared biological bases between the sexes. Here, we perform a series of rare variant analyses across 73,185 women and men to identify genes that contribute to primary gonadal dysfunction. We report CSMD1, a complement regulatory protein on chromosome 8p23, as a strong candidate locus in both sexes. We show that CSMD1 is enriched at the germ-cell/somatic-cell interface in both male and female gonads. Csmd1-knockout males show increased rates of infertility with significantly increased complement C3 protein deposition in the testes, accompanied by severe histological degeneration. Knockout females show significant reduction in ovarian quality and breeding success, as well as mammary branching impairment. Double knockout of Csmd1 and C3 causes non-additive reduction in breeding success, suggesting that CSMD1 and the complement pathway play an important role in the normal postnatal development of the gonads in both sexes.
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http://dx.doi.org/10.1038/s41467-019-12522-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789153PMC
October 2019

A Multimodality Approach to Assessing Factor I Genetic Variants in Atypical Hemolytic Uremic Syndrome.

Kidney Int Rep 2019 Jul 9;4(7):1007-1017. Epub 2019 Apr 9.

Department of Medicine, Division of Rheumatology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.

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http://dx.doi.org/10.1016/j.ekir.2019.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609824PMC
July 2019

Fat-Produced Adipsin Regulates Inflammatory Arthritis.

Cell Rep 2019 06;27(10):2809-2816.e3

Division of Anatomic and Molecular Pathology, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Division of Bone and Mineral Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA; Shriners Hospitals for Children, St. Louis, MO 63110, USA. Electronic address:

We explored the relationship of obesity and inflammatory arthritis (IA) by selectively expressing diphtheria toxin in adipose tissue yielding "fat-free" (FF) mice completely lacking white and brown fat. FF mice exhibit systemic neutrophilia and elevated serum acute phase proteins suggesting a predisposition to severe IA. Surprisingly, FF mice are resistant to K/BxN serum-induced IA and attendant bone destruction. Despite robust systemic basal neutrophilia, neutrophil infiltration into joints of FF mice does not occur when challenged with K/BxN serum. Absence of adiponectin, leptin, or both has no effect on joint disease, but deletion of the adipokine adipsin (complement factor D) completely prevents serum-induced IA. Confirming that fat-expressed adipsin modulates the disorder, transplantation of wild-type (WT) adipose tissue into FF mice restores susceptibility to IA, whereas recipients of adipsin-deficient fat remain resistant. Thus, adipose tissue regulates development of IA through a pathway in which adipocytes modify neutrophil responses in distant tissues by producing adipsin.
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http://dx.doi.org/10.1016/j.celrep.2019.05.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643993PMC
June 2019

Reply.

Arthritis Rheumatol 2019 09 19;71(9):1590-1592. Epub 2019 Jul 19.

Washington University School of Medicine, Saint Louis, MO.

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http://dx.doi.org/10.1002/art.40923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716999PMC
September 2019

Development and Optimization of an ELISA to Quantitate C3(H O) as a Marker of Human Disease.

Front Immunol 2019 4;10:703. Epub 2019 Apr 4.

Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, United States.

Discovery of a C3(HO) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(HO), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(HO). We initially utilized this assay to determine baseline C3(HO) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(HO) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(HO) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(HO) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(HO). To determine the potential use of C3(HO) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(HO) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(HO) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(HO) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(HO) as a diagnostic and prognostic biomarker in human diseases.
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http://dx.doi.org/10.3389/fimmu.2019.00703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458276PMC
September 2020

Hyperfunctional complement C3 promotes C5-dependent atypical hemolytic uremic syndrome in mice.

J Clin Invest 2019 03 4;129(3):1061-1075. Epub 2019 Feb 4.

Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.
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http://dx.doi.org/10.1172/JCI99296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391106PMC
March 2019

Thiol isomerase ERp57 targets and modulates the lectin pathway of complement activation.

J Biol Chem 2019 03 22;294(13):4878-4888. Epub 2019 Jan 22.

From the Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115,

ER protein 57 (ERp57), a thiol isomerase secreted from vascular cells, is essential for complete thrombus formation , but other extracellular ERp57 functions remain unexplored. Here, we employed a kinetic substrate-trapping approach to identify extracellular protein substrates of ERp57 in platelet-rich plasma. MS-based identification with immunochemical confirmation combined with gene ontology enrichment analysis revealed that ERp57 targets, among other substrates, components of the lectin pathway of complement activation: mannose-binding lectin, ficolin-2, ficolin-3, collectin-10, collectin-11, mannose-binding lectin-associated serine protease-1, and mannose-binding lectin-associated serine protease-2. Ficolin-3, the most abundant lectin pathway initiator in humans, circulates as disulfide-linked multimers of a monomer. ERp57 attenuated ficolin-3 ligand recognition and complement activation by cleaving intermolecular disulfide bonds in large ficolin-3 multimers, thereby reducing multimer size and ligand-binding affinity. We used MS to identify the disulfide-bonding pattern in ficolin-3 multimers and the disulfide bonds targeted by ERp57 and found that Cys and Cys in the N-terminal region of ficolin-3 form the intermolecular disulfide bonds in ficolin-3 multimers that are reduced by ERp57. Our results not only demonstrate that ERp57 can negatively regulate complement activation, but also identify a control mechanism for lectin pathway initiation in the vasculature. We conclude that extensive multimerization in large ficolin-3 multimers leads to a high affinity for ligands and strong complement-activating potential and that ERp57 suppresses complement activation by cleaving disulfide bonds in ficolin-3 and reducing its multimer size.
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http://dx.doi.org/10.1074/jbc.RA118.006792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442054PMC
March 2019

Association of immune response with efficacy and safety outcomes in adults with phenylketonuria administered pegvaliase in phase 3 clinical trials.

EBioMedicine 2018 Nov 23;37:366-373. Epub 2018 Oct 23.

BioMarin Pharmaceutical Inc., 105 Digital Dr, Novato, CA 94949, United States.

Background: This study assessed the immunogenicity of pegvaliase (recombinant Anabaena variabilis phenylalanine [Phe] ammonia lyase [PAL] conjugated with polyethylene glycol [PEG]) treatment in adults with phenylketonuria (PKU) and its impact on safety and efficacy.

Methods: Immunogenicity was assessed during induction, upward titration, and maintenance dosing regimens in adults with PKU (n = 261). Total antidrug antibodies (ADA), neutralizing antibodies, immunoglobulin (Ig) M and IgG antibodies against PAL and PEG, IgG and IgM circulating immune complex (CIC) levels, complement components 3 and 4 (C3/C4), plasma Phe, and safety were assessed at baseline and throughout the study. Pegvaliase-specific IgE levels were measured in patients after hypersensitivity adverse events (HAE).

Findings: All patients developed ADA against PAL, peaking by 6 months and then stabilizing. Most developed transient antibody responses against PEG, peaking by 3 months, then returning to baseline by 9 months. Binding of ADA to pegvaliase led to CIC formation and complement activation, which were highest during early treatment. Blood Phe decreased over time as CIC levels and complement activation declined and pegvaliase dosage increased. HAEs were most frequent during early treatment and declined over time. No patient with acute systemic hypersensitivity events tested positive for pegvaliase-specific IgE near the time of the event. Laboratory evidence was consistent with immune complex-mediated type III hypersensitivity. No evidence of pegvaliase-associated IC-mediated end organ damage was noted.

Interpretation: Despite a universal ADA response post-pegvaliase administration, adult patients with PKU achieved substantial and sustained blood Phe reductions with a manageable safety profile. FUND: BioMarin Pharmaceutical Inc.
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http://dx.doi.org/10.1016/j.ebiom.2018.10.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286649PMC
November 2018

Association of Blood Concentrations of Complement Split Product iC3b and Serum C3 With Systemic Lupus Erythematosus Disease Activity.

Arthritis Rheumatol 2019 03 24;71(3):420-430. Epub 2019 Jan 24.

Washington University School of Medicine, Saint Louis, Missouri.

Objective: To examine correlations between blood levels of complement split product iC3b and serum component C3 with clinically meaningful changes in disease activity in patients with systemic lupus erythematosus (SLE).

Methods: A total of 159 consecutive patients with SLE, diagnosed according to the American College of Rheumatology or Systemic Lupus International Collaborating Clinics classification criteria, were enrolled in CASTLE (Complement Activation Signatures in Systemic Lupus Erythematosus), a prospective observational study. Patients with 1-7 study visits were included in this longitudinal analysis. In addition, 48 healthy volunteers were enrolled to establish a normal reference value for the ratio of blood iC3b to serum C3 concentrations. Serum C3 and C4 levels were measured by nephelometry, and blood iC3b levels were measured by a lateral flow assay. SLE disease activity was monitored with the Responder Index 50 instrument of the SLE Disease Activity Index 2000.

Results: Relative changes in the iC3b:C3 ratio, levels of anti-double-stranded DNA (anti-dsDNA) antibodies, and use of a supraphysiologic dose of prednisone (>7.5 mg/day) each independently correlated with SLE disease activity, as determined in multilevel multiple logistic regression analyses. Only the iC3b:C3 ratio was significantly associated with clinically meaningful improvements in disease activity among patients with SLE who were receiving a supraphysiologic dose of prednisone. The iC3b:C3 ratio outperformed C3 and C4 levels with regard to discriminating active SLE from inactive SLE, and major flares from no disease activity. The iC3:C3 ratio, anti-dsDNA antibody levels, erythrocyte sedimentation rate, and use of a supraphysiologic prednisone dose were each independently associated with the presence of lupus nephritis, whereas none of these measures was associated with SLE rash. The association of the iC3b:C3 ratio with lupus nephritis was independent of other observed clinical manifestations.

Conclusion: The ratio of blood iC3b to serum C3 concentrations correlates with the extent of SLE disease activity and with clinically meaningful changes in disease activity in patients with SLE. Furthermore, the iC3b:C3 ratio may discriminate between active and inactive SLE, and between major flares and no active disease.
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http://dx.doi.org/10.1002/art.40747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393208PMC
March 2019

Intracellular C3 Protects Human Airway Epithelial Cells from Stress-associated Cell Death.

Am J Respir Cell Mol Biol 2019 02;60(2):144-157

2 Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri.

The complement system provides host defense against pathogens and environmental stress. C3, the central component of complement, is present in the blood and increases in BAL fluid after injury. We recently discovered that C3 is taken up by certain cell types and cleaved intracellularly to C3a and C3b. C3a is required for CD4 T-cell survival. These observations made us question whether complement operates at environmental interfaces, particularly in the respiratory tract. We found that airway epithelial cells (AECs, represented by both primary human tracheobronchial cells and BEAS-2B [cell line]) cultured in C3-free media were unique from other cell types in that they contained large intracellular stores of de novo synthesized C3. A fraction of this protein reduced ("storage form") but the remainder did not, consistent with it being pro-C3 ("precursor form"). These two forms of intracellular C3 were absent in CRISPR knockout-induced C3-deficient AECs and decreased with the use of C3 siRNA, indicating endogenous generation. Proinflammatory cytokine exposure increased both stored and secreted forms of C3. Furthermore, AECs took up C3 from exogenous sources, which mitigated stress-associated cell death (e.g., from oxidative stress or starvation). C3 stores were notably increased within AECs in lung tissues from individuals with different end-stage lung diseases. Thus, at-risk cells furnish C3 through biosynthesis and/or uptake to increase locally available C3 during inflammation, while intracellularly, these stores protect against certain inducers of cell death. These results establish the relevance of intracellular C3 to airway epithelial biology and suggest novel pathways for complement-mediated host protection in the airway.
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http://dx.doi.org/10.1165/rcmb.2017-0405OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376412PMC
February 2019

TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia.

Brain Pathol 2018 11 10;28(6):806-821. Epub 2018 Oct 10.

Department of Medicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO.

Background: Mutations in the three-prime repair exonuclease 1 (TREX1) gene have been associated with neurological diseases, including Retinal Vasculopathy with Cerebral Leukoencephalopathy (RVCL). However, the endogenous expression of TREX1 in human brain has not been studied.

Methods: We produced a rabbit polyclonal antibody (pAb) to TREX1 to characterize TREX1 by Western blotting (WB) of cell lysates from normal controls and subjects carrying an RVCL frame-shift mutation. Dual staining was performed to determine cell types expressing TREX1 in human brain tissue. TREX1 distribution in human brain was further evaluated by immunohistochemical analyses of formalin-fixed, paraffin-embedded samples from normal controls and patients with RVCL and ischemic stroke.

Results: After validating the specificity of our anti-TREX1 rabbit pAb, WB analysis was utilized to detect the endogenous wild-type and frame-shift mutant of TREX1 in cell lysates. Dual staining in human brain tissues from patients with RVCL and normal controls localized TREX1 to a subset of microglia and macrophages. Quantification of immunohistochemical staining of the cerebral cortex revealed that TREX1 microglia were primarily in the gray matter of normal controls (22.7 ± 5.1% and 5.5 ± 1.9% of Iba1 microglia in gray and white matter, respectively) and commonly in association with the microvasculature. In contrast, in subjects with RVCL, the TREX1 microglia were predominantly located in the white matter of normal appearing cerebral cortex (11.8 ± 3.1% and 38.9 ± 5.8% of Iba1 microglia in gray and white matter, respectively). The number of TREX1 microglia was increased in ischemic brain lesions in central nervous system of RVCL and stroke patients.

Conclusions: TREX1 is expressed by a subset of microglia in normal human brain, often in close proximity to the microvasculature, and increases in the setting of ischemic lesions. These findings suggest a role for TREX1 microglia in vessel homeostasis and response to ischemic injury.
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http://dx.doi.org/10.1111/bpa.12626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404532PMC
November 2018

Preface to the Special issue for the 27th International complement workshop.

Mol Immunol 2018 10 27;102:1-2. Epub 2018 Jul 27.

University of Colorado School of Medicine, Auroro, CO, USA.

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http://dx.doi.org/10.1016/j.molimm.2018.07.010DOI Listing
October 2018

Timing and mechanism of conceptus demise in a complement regulatory membrane protein deficient mouse.

Am J Reprod Immunol 2018 10 20;80(4):e12997. Epub 2018 Jun 20.

Department of Medicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO, USA.

Problem: Crry is a widely expressed type 1 transmembrane complement regulatory protein in rodents which protects self-tissue by downregulating C3 activation. Crry concepti produced by Crry  × Crry matings are attacked by maternal complement system leading to loss before day 10. The membrane attack complex is not the mediator of this death. We hypothesized that the ability of C3b to engage the alternative pathway's feedback loop relatively unchecked on placental membranes induces the lesion yielding the demise of the Crry mouse.

Method Of Study: We investigated the basis of Crry conceptus demise by depleting maternal complement with cobra venom factor and blocking antibodies. We monitored their effects primarily by genotyping and histologic analyses.

Results: We narrowed the critical period of the complement effect from 6.5 to 8.5 days post-coitus (dpc), which is immediately after the conceptus is exposed to maternal blood. Deposition by 5.5 dpc of maternal C3b on the placental vasculature lacking Crry yielded loss of the conceptus by 8.5 dpc. Fusion of the allantois to the chorion during placental assembly did not occur, fetal vessels originating in the allantois did not infiltrate the chorioallantoic placenta, the chorionic plate failed to develop, and the labyrinthine component of the placenta did not mature.

Conclusion: Our data are most consistent with the deposition of C3b being responsible for the failure of the allantois to fuse to the chorion leading to subsequent conceptus demise.
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http://dx.doi.org/10.1111/aji.12997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160323PMC
October 2018

Contribution of Adipose-Derived Factor D/Adipsin to Complement Alternative Pathway Activation: Lessons from Lipodystrophy.

J Immunol 2018 04 12;200(8):2786-2797. Epub 2018 Mar 12.

Division of Endocrinology, Metabolism and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;

Factor D (FD) is an essential component of the complement alternative pathway (AP). It is an attractive pharmaceutical target because it is an AP-specific protease circulating in blood. Most components of the complement activation pathways are produced by the liver, but FD is highly expressed by adipose tissue. Two critical questions are: 1) to what degree does adipose tissue contribute to circulating FD levels and 2) what quantity of FD is sufficient to maintain a functional AP? To address these issues, we studied a novel mouse strain with complete lipodystrophy (LD), the fld mouse with partial LD, an FD-deficient mouse, and samples from lipodystrophic patients. FD was undetectable in the serum of LD mice, which also showed minimal AP function. Reconstitution with purified FD, serum mixing experiments, and studies of partial LD mice all demonstrated that a low level of serum FD is sufficient for normal AP activity in the mouse system. This conclusion was further supported by experiments in which wild-type adipose precursors were transplanted into LD mice. Our results indicate that almost all FD in mouse serum is derived from adipose tissue. In contrast, FD levels were reduced ∼50% in the sera of patients with congenital generalized LD. Our studies further demonstrate that a relatively small amount of serum FD is sufficient to facilitate significant time-dependent AP activity in humans and in mice. Furthermore, this observation highlights the potential importance of obtaining nearly complete inhibition of FD in treating alternative complement activation in various autoimmune and inflammatory human diseases.
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http://dx.doi.org/10.4049/jimmunol.1701668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893424PMC
April 2018

Transcriptional Profiling of Synovial Macrophages Using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis.

Arthritis Rheumatol 2018 06 3;70(6):841-854. Epub 2018 May 3.

Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Objective: Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA.

Methods: Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients.

Results: Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy.

Conclusion: Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.
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http://dx.doi.org/10.1002/art.40453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984677PMC
June 2018

The complement system in the airway epithelium: An overlooked host defense mechanism and therapeutic target?

J Allergy Clin Immunol 2018 05 12;141(5):1582-1586.e1. Epub 2018 Jan 12.

Division of Rheumatology, Department of Medicine, Washington University in St Louis, St Louis, Mo. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.11.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955701PMC
May 2018

Protective Low-Frequency Variants for Preeclampsia in the Fms Related Tyrosine Kinase 1 Gene in the Finnish Population.

Hypertension 2017 08 26;70(2):365-371. Epub 2017 Jun 26.

From the Immunobiology, Research Programs Unit (A.I.L., S.M.), Molecular Neurology, Research Programs Unit (J.K.), and Institute for Molecular Medicine Finland/HiLIFE Unit (P.H., K.A., M.P., H.L.), University of Helsinki, Finland; Medical and Clinical Genetics (A.I.L., H.L.), Bacteriology and Immunology (A.I.L., S.M.), Obstetrics and Gynaecology (K.A., S.H., H.L.), and Children's Hospital (E.K), University of Helsinki and Helsinki University Hospital, Finland; Folkhälsan Institute of Genetics (J.K.), University of Helsinki, Finland; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA (E.D., M.I.K., M.D.); Department of Medicine, Division of Rheumatology (M.T., E.D.O.R., J.P.A.) and Department of Genetics (E.D.O.R.), Washington University School of Medicine, St. Louis, MO; Neurosurgery of Neuro Center, Kuopio University Hospital, Finland (M.I.K.); Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital and Harvard Medical School, Boston (M.I.K.); Unit of Genetics and Biomarkers (K.A.), Department of Health (M.P., E.K.), Chronic Disease Prevention Unit, Department of Health (E.K.), and Department of Government Services (A.P.), National Institute for Health and Welfare, Helsinki, Finland; The Estonian Genome Center, University of Tartu, Estonia (M.P.); PEDEGO Research Unit, MRC Oulu, University of Oulu and Oulu University Hospital, Finland (E.K., A.P.); Department of Biosciences and Nutrition, Karolinska Institutet, Solna, Sweden (J.K.); Department of Medical and Molecular Genetics, King's College, London, United Kingdom (J.K.); Division of Cardiovascular Medicine, University of Cambridge, United Kingdom (K.K.); Department of Medicine, Hospital for Special Surgery-Weill Cornell Medicine, New York, NY (J.E.S.); and Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston (M.D.).

Preeclampsia is a common pregnancy-specific vascular disorder characterized by new-onset hypertension and proteinuria during the second half of pregnancy. Predisposition to preeclampsia is in part heritable. It is associated with an increased risk of cardiovascular disease later in life. We have sequenced 124 candidate genes implicated in preeclampsia to pinpoint genetic variants contributing to predisposition to or protection from preeclampsia. First, targeted exomic sequencing was performed in 500 preeclamptic women and 190 controls from the FINNPEC cohort (Finnish Genetics of Preeclampsia Consortium). Then 122 women with a history of preeclampsia and 1905 parous women with no such history from the National FINRISK Study (a large Finnish population survey on risk factors of chronic, noncommunicable diseases) were included in the analyses. We tested 146 rare and low-frequency variants and found an excess (observed 13 versus expected 7.3) nominally associated with preeclampsia (<0.05). The most significantly associated sequence variants were protective variants rs35832528 (E982A; =2.49E-4; odds ratio=0.387) and rs141440705 (R54S; =0.003; odds ratio=0.442) in Fms related tyrosine kinase 1. These variants are enriched in the Finnish population with minor allele frequencies 0.026 and 0.017, respectively. They may also be associated with a lower risk of heart failure in 11 257 FINRISK women. This study provides the first evidence of maternal protective genetic variants in preeclampsia.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.117.09406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535812PMC
August 2017
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