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Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)
(2019)
Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human β-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.
Introduction: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
Methods: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.
Results: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.
Conclusions: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.
Background: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.
Methodology/Principal Findings: Here, we present evidence that raft-associated endocytic proteins (flotillins) are preassembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.
Conclusions: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.
Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.
Autoantibodies in sera from patients with autoimmune diseases have long been known and have become diagnostic tools. Analysis of their functional role again became popular with the availability of mice mutant for several genes of the complement and Fcγ receptor (FcγR) systems. Evidence from different inflammatory models suggests that both systems are interconnected in a hierarchical way. The complement system mediators such as complement component 5a (C5a) might be crucial in the communication between the complement system and FcγR-expressing cells. The split complement protein C5a is known to inactivate cells by its G-protein-coupled receptor and to be involved in the transcriptional regulation of FcγRs, thereby contributing to the complex regulation of autoimmune disease.
Impaired up-regulation of CD86 in B cells of "type A" common variable immunodeficiency patients
(2000)
Autoantikörper und das Komplementsystem - Bedeutung für die Pathogenese der Rheumatoiden Arthritis?
(2002)
Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function.
TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-kappaB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.
Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.
A soluble form of the complement receptor CD21 (sCD21) is shed from the lymphocyte surface. The sCD21 is able to bind all known ligands such as CD23, sCD23, Epstein-Barr virus and C3d in immune complexes. Here, we show the serum levels of sCD21 in sera the of antiphospholipid syndrome (APS) patients. Antiphospholipid syndrome is an autoimmune disorder in which autoantibodies cause heart attack, stroke and miscarriage. Antiphospholipid syndrome may appear as primary or in association with systemic lupus erythromatosus (SLE) and other autoimmune diseases. Here, we ask whether APS patients have different sCD21 titers compared to healthy persons and whether sCD21 levels correlate with the presence of anti-β2-GPI autoantibodies. We show that autoimmune APS patients have significantly reduced amounts of sCD21 in their sera, irrespective of the presence of anti-β2-GPI autoantibodies. In our APS patients cohort additional SLE, vasculities, DVT (deep vein thrombosis), fetal loss or thrombosis did not correlate to the reduced level of sCD21.