Maria Allhorn

Platelet transcriptional profile and protein expression in patients with systemic lupus erythematosus: up-regulation of the type I interferon system is strongly associated with vascular disease

Patients with systemic lupus erythematosus (SLE) have a markedly increased risk to develop cardiovascular disease, and traditional cardiovascular risk factors fail to account for this increased risk. We used microarray to probe the platelet transcriptome in patients with SLE and healthy controls, and the gene and protein expression of a subset of differentially expressed genes was further investigated and correlated to platelet activation status. Real-time PCR was used to confirm a type I interferon (IFN) gene signature in patients with SLE, and the IFN-regulated proteins PRKRA, IFITM1 and CD69 (P < .0001) were found to be up-regulated in platelets from SLE patients compared with healthy volunteers. Notably, patients with a history of vascular disease had increased expression of type I IFN-regulated proteins as well as more activated platelets compared with patients without vascular disease. We suggest that interferogenic immune complexes stimulate production of IFNα that up-regulates the megakaryocytic type I IFN-regulated genes and proteins. This could affect platelet activation and contribute to development of vascular disease in SLE. In addition, platelets with type I IFN signature could be a novel marker for vascular disease in SLE.

Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress

Many nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila, Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up‐regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids.

Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

Background The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcγR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcγR and the Fc domain of IgG depend on the IgG glycosylation state. Methodology/Principal Findings Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcγR and to an erythroleukemic cell line, K562, expressing FcγRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcγRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. Conclusions/Significance We provide novel information about bacterial enzymatic modulation of the IgG/FcγR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes.

Pathological Conditions Involving Extracellular Hemoglobin: Molecular Mechanisms, Clinical Significance, and Novel Therapeutic Opportunities for alpha(1)-Microglobulin

Hemoglobin (Hb) is the major oxygen (O(2))-carrying system of the blood but has many potentially dangerous side effects due to oxidation and reduction reactions of the heme-bound iron and O(2). Extracellular Hb, resulting from hemolysis or exogenous infusion, is shown to be an important pathogenic factor in a growing number of diseases. This review briefly outlines the oxidative/reductive toxic reactions of Hb and its metabolites. It also describes physiological protection mechanisms that have evolved against extracellular Hb, with a focus on the most recently discovered: the heme- and radical-binding protein α(1)-microglobulin (A1M). This protein is found in all vertebrates, including man, and operates by rapidly clearing cytosols and extravascular fluids of heme groups and free radicals released from Hb. Five groups of pathological conditions with high concentrations of extracellular Hb are described: hemolytic anemias and transfusion reactions, the pregnancy complication pre-eclampsia, cerebral intraventricular hemorrhage of premature infants, chronic inflammatory leg ulcers, and infusion of Hb-based O(2) carriers as blood substitutes. Finally, possible treatments of these conditions are discussed, giving a special attention to the described protective effects of A1M.

The IgG specific endoglycosidase EndoS inhibits both cellular and complement mediated autoimmune hemolysis

EndoS from Streptococcus pyogenes is an immunomodulating enzyme that specifically hydrolyzes glycans from human immunoglobulin G and thereby affects antibody effector functions. Autoimmune hemolytic anemia is caused by antibody-mediated red blood cell (RBC) destruction and often resists treatment with corticosteroids that also cause frequent adverse effects. We show here that anti-RhD (anti-D) and rabbit anti-human-RBC antibodies (anti-RBC) mediated destruction of RBC, ie, phagocytosis, complement activation, and hemolysis in vitro and in vivo was inhibited by EndoS. Phagocytosis by monocytes in vitro was inhibited by pretreatment of anti-D with EndoS before sensitization of RBCs and abrogated by direct addition of EndoS to blood containing sensitized RBCs. The toxic effects of monocytes stimulated with anti-D-sensitized RBCs, as measured by interleukin-8 secretion and oxygen metabolite production, was restrained by EndoS. Agglutination of RBCs and complement-mediated hemolysis in vitro in whole human blood caused by rabbit anti-RBCs was inhibited by EndoS. Development of anemia in mice caused by a murine anti-RBC immunoglobulin G2a monoclonal autoantibody and complement activation and erythrophagocytosis by Kupffer cells in the liver were reduced by EndoS. Our data indicate that EndoS is a potential therapeutic agent that might be evaluated as an alternative to current treatment regimens against antibody-mediated destruction of RBCs.

IgG glycan hydrolysis by endoglycosidase S diminishes the proinflammatory properties of immune complexes from patients with systemic lupus erythematosus: A possible new treatment?

OBJECTIVE Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in several organ systems, related to the presence of circulating and tissue-deposited immune complexes (ICs) that stimulate leukocytes through Fcγ receptors (FcγR) with subsequent inflammation. Treatment with endoglycosidase S (EndoS), an IgG glycan-hydrolyzing bacterial enzyme from Streptococcus pyogenes, has shown beneficial effects in several experimental animal models of chronic inflammatory disease. This study was undertaken to investigate whether EndoS affects the proinflammatory properties of ICs and has the potential to be developed as a therapy for SLE. METHODS ICs purified from SLE patients or RNA-containing ICs formed in vitro were treated with EndoS and used in several assays reflecting different important features of SLE pathogenesis, such as phagocytosis by polymorphonuclear cells (PMNs) and plasmacytoid dendritic cells (PDCs), complement activation, and interferon-α (IFNα) production by PDCs. RESULTS EndoS treatment abolished all proinflammatory properties of the ICs investigated. This included FcγR-mediated phagocytosis by PDCs (P = 0.001) and subsequent production of IFNα (P = 0.002), IC-induced classical pathway of complement activation (P = 0.008), chemotaxis, and oxidative burst activity of PMNs (P = 0.002). EndoS treatment also had a direct effect on the molecular structure of ICs, causing decreased IC size and glycosylation. CONCLUSION Our findings indicate that EndoS treatment has prominent effects on several pathogenetically important IC-mediated events, and suggest that EndoS has the potential to be developed as a novel therapy for SLE.

EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

BackgroundThe endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS.ResultsWe could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity.ConclusionWe present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

ABSTRACT The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.

Sugar-free antibodies--the bacterial solution to autoimmunity?

The enzyme EndoS from Streptococcus pyogenes is an immunomodulatory molecule hydrolyzing the conserved glycans in the effector part of immunoglobulin G (IgG). EndoS is remarkably specific for IgG, and hydrolysis has profound effects on IgG effector functions. EndoS pretreatment of IgG, or direct administration to animals with experimental antibody‐mediated autoimmune diseases, inhibits development of disease or cures animals from established disease. The properties of EndoS make it a unique experimental tool and an attractive alternative to current therapies of conditions involving pathogenic antibodies. This review describes the discovery of EndoS, the effects of EndoS on IgG effector functions in vitro and in vivo, the biotechnological potential of EndoS, and the outcomes of EndoS treatment in animal models of autoimmunity.

EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein

Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.