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1.
J Immunol ; 200(8): 2542-2553, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29531170

ABSTRACT

Activation of Fc receptors and complement by immune complexes is a common important pathogenic trigger in many autoimmune diseases and so blockade of these innate immune pathways may be an attractive target for treatment of immune complex-mediated pathomechanisms. High-dose IVIG is used to treat autoimmune and inflammatory diseases, and several studies demonstrate that the therapeutic effects of IVIG can be recapitulated with the Fc portion. Further, recent data indicate that recombinant multimerized Fc molecules exhibit potent anti-inflammatory properties. In this study, we investigated the biochemical and biological properties of an rFc hexamer (termed Fc-µTP-L309C) generated by fusion of the IgM µ-tailpiece to the C terminus of human IgG1 Fc. Fc-µTP-L309C bound FcγRs with high avidity and inhibited FcγR-mediated effector functions (Ab-dependent cell-mediated cytotoxicity, phagocytosis, respiratory burst) in vitro. In addition, Fc-µTP-L309C prevented full activation of the classical complement pathway by blocking C2 cleavage, avoiding generation of inflammatory downstream products (C5a or sC5b-9). In vivo, Fc-µTP-L309C suppressed inflammatory arthritis in mice when given therapeutically at approximately a 10-fold lower dose than IVIG, which was associated with reduced inflammatory cytokine production and complement activation. Likewise, administration of Fc-µTP-L309C restored platelet counts in a mouse model of immune thrombocytopenia. Our data demonstrate a potent anti-inflammatory effect of Fc-µTP-L309C in vitro and in vivo, likely mediated by blockade of FcγRs and its unique inhibition of complement activation.


Subject(s)
Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/immunology , Autoimmune Diseases/immunology , Complement System Proteins/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/immunology , Receptors, Fc/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Cell Line , Complement Activation/immunology , Humans , Inflammation/immunology , Male , Mice , Mice, Inbred BALB C , Phagocytosis/immunology , Receptors, IgG/immunology
2.
PLoS One ; 11(8): e0161826, 2016.
Article in English | MEDLINE | ID: mdl-27561008

ABSTRACT

In relation to the recent trials of Intravenous Immunoglobulin (IVIG) in Alzheimer's Disease (AD) it was demonstrated that different IgG preparations contain varying amounts of natural anti-amyloid ß (Aß) antibodies as measured by ELISA. We therefore investigated the relevance of ELISA data for measuring low-affinity antibodies, such as anti-Aß. We analysed the binding of different commercial Immunoglobulin G (IgG) preparations to Aß, actin and tetanus toxoid in different binding assays to further investigate the possible cause for observed differences in binding to Aß and actin between different IgG preparations. We show that the differences of commercial IgG preparations in binding to Aß and actin in ELISA assays are artefactual and only evident in in vitro binding assays. In functional assays and in vivo animal studies the different IVIG preparations exhibited very similar potency. ELISA data alone are not appropriate to analyse and rank the binding capacity of low-affinity antibodies to Aß or other endogenous self-antigens contained in IgG preparations. Additional analytical methods should be adopted to complement ELISA data.


Subject(s)
Antigens/immunology , Autoantigens/immunology , Immunoglobulin G/immunology , Immunoglobulins, Intravenous/immunology , Actins/immunology , Actins/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/metabolism , Animals , Antibody Affinity/drug effects , Antibody Affinity/immunology , Antigens/metabolism , Autoantigens/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Herpesvirus 3, Human/immunology , Herpesvirus 3, Human/metabolism , Humans , Immunoglobulin G/metabolism , Immunoglobulins, Intravenous/pharmacology , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Binding/drug effects , Protein Binding/immunology , Rats , Tetanus Toxoid/immunology , Tetanus Toxoid/metabolism
3.
J Biol Chem ; 289(39): 27080-27089, 2014 Sep 26.
Article in English | MEDLINE | ID: mdl-25118284

ABSTRACT

Passive immunization with anti-amyloid-ß peptide (Aß) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aß-specific DARPins. We further showed their ability to delay Aß aggregation and prevent Aß-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aß-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aß levels. These findings demonstrate the therapeutic potential of Aß-specific DARPins for the treatment of Alzheimer disease.


Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Protein Precursor/antagonists & inhibitors , Amyloid beta-Protein Precursor/metabolism , Ankyrin Repeat , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/genetics , Animals , Humans , Mice , Mice, Transgenic , Recombinant Proteins/pharmacology
4.
PLoS One ; 8(5): e63162, 2013.
Article in English | MEDLINE | ID: mdl-23696796

ABSTRACT

Intravenous Immunoglobulin (IVIG) has been proposed as a potential therapeutic for Alzheimer's disease (AD) and its efficacy is currently being tested in mild-to-moderate AD. Earlier studies reported the presence of anti-amyloid beta (Aß) antibodies in IVIG. These observations led to clinical studies investigating the potential role of IVIG as a therapeutic agent in AD. Also, IVIG is known to mediate beneficial effects in chronic inflammatory and autoimmune conditions by interfering with various pathological processes. Therefore, we investigated the effects of IVIG and purified polyclonal Aß-specific antibodies (pAbs-Aß) on aggregation, toxicity and phagocytosis of Aß in vitro, thus elucidating some of the potential mechanisms of action of IVIG in AD patients. We report that both IVIG and pAbs-Aß specifically bound to Aß and inhibited its aggregation in a dose-dependent manner as measured by Thioflavin T assay. Additionally, IVIG and the purified pAbs-Aß inhibited Aß-induced neurotoxicity in the SH-SY5Y human neuroblastoma cell line and prevented Aß binding to rat primary cortical neurons. Interestingly, IVIG and pAbs-Aß also increased the number of phagocytosing cells as well as the amount of phagocytosed fibrillar Aß by BV-2 microglia. Phagocytosis of Aß depended on receptor-mediated endocytosis and was accompanied by upregulation of CD11b expression. Importantly, we could also show that Privigen dose-dependently reversed Aß-mediated LTP inhibition in mouse hippocampal slices. Therefore, our in vitro results suggest that IVIG may have an impact on different processes involved in AD pathogenesis, thereby promoting further understanding of the effects of IVIG observed in clinical studies.


Subject(s)
Amyloid beta-Peptides/metabolism , Immunoglobulins/metabolism , Microglia/cytology , Microglia/metabolism , Phagocytosis/physiology , Amyloid beta-Peptides/genetics , Animals , CD11b Antigen/metabolism , Cell Line, Tumor , Chromatography, Affinity , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulins/genetics , Immunohistochemistry , Mice , Microscopy, Atomic Force
5.
PLoS One ; 6(4): e18296, 2011 Apr 05.
Article in English | MEDLINE | ID: mdl-21483675

ABSTRACT

Amyloid-beta peptide (Aß)-directed active and passive immunization therapeutic strategies reduce brain levels of Aß, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aß-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aß, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aß-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aß fibril formation and Aß-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aß from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aß-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aß-directed immunotherapy can reduce Aß deposition in brain vessels in transgenic mice.


Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/immunology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Single-Chain Antibodies/administration & dosage , Single-Chain Antibodies/pharmacology , Administration, Intranasal , Alzheimer Disease/metabolism , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Complementarity Determining Regions/genetics , Disease Models, Animal , Escherichia coli/genetics , Female , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Protein Multimerization/drug effects , Protein Structure, Secondary , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Solubility , Time Factors
6.
Neurobiol Aging ; 32(10): 1827-38, 2011 Oct.
Article in English | MEDLINE | ID: mdl-19942317

ABSTRACT

Mitochondrial dysfunction and oxidative stress play an important role in ageing and have been implicated in several age-related neurodegenerative conditions including Alzheimer's disease (AD) and other tauopathies characterized by the presence of intracellular accumulations of the hyperphosphorylated microtubule-associated protein tau. To study the interaction between mitochondrial dysfunction and tau pathology in vivo, we generated a novel mouse model by crossbreeding two existing lines: the Harlequin (Hq) mutant mice which suffer from mitochondrial dysfunction and oxidative stress due to a lack of the mitochondrial apoptosis-inducing factor (AIF), and the P301L tau transgenic mice, a mouse model of human tau pathology. Combined expression of the Hq mouse mutation and the tau transgene in the Tau/Hq double mutant mice led to an increase in tau pathology and apoptotic neurodegeneration when compared to single expression of the two mutations. Neurodegeneration was most prominent in the dentate gyrus and was significantly increased in the cerebellum leading to aggravated motor deficits. Functional activity measurements of the mitochondrial respiratory chain (MRC) in the Tau/Hq mice revealed early decreased activities of multiple MRC complexes and depleted ATP levels which preceded neurodegeneration and elevated oxidative stress markers. These results suggest an age-dependent mutual reinforcement of the tau pathology and mitochondrial dysfunction in vivo, which may contribute to neurodegeneration in patients suffering from AD and other age-related tauopathies.


Subject(s)
Electron Transport Complex I/genetics , Mitochondrial Diseases/genetics , Mutation/genetics , Nerve Degeneration/genetics , Tauopathies/genetics , tau Proteins/genetics , Adenosine Triphosphate/metabolism , Age Factors , Animals , Apoptosis/genetics , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Brain/metabolism , Brain/ultrastructure , Caspase 3/metabolism , Electron Transport Complex I/deficiency , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Genetic Predisposition to Disease , Humans , In Situ Nick-End Labeling , Leucine/genetics , Lipid Peroxidation/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Mitochondria/genetics , Mitochondria/ultrastructure , Movement Disorders/genetics , Movement Disorders/physiopathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Oxidative Stress/genetics , Proline/genetics , Proton Pumps/metabolism , Silver Staining/methods , Statistics, Nonparametric
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