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Kuerten S.,University of Cologne | Schickel A.,University of Cologne | Kerkloh C.,University of Cologne | Recks M.S.,University of Cologne | And 4 more authors.
Acta Neuropathologica | Year: 2012

While the role of T cells has been studied extensively in multiple sclerosis (MS), the pathogenic contribution of B cells has only recently attracted major attention, when it was shown that B cell aggregates can develop in the meninges of a subset of MS patients and were suggested to be correlates of late-stage and more aggressive disease in this patient population. However, whether these aggregates actually exist has subsequently been questioned and their functional significance has remained unclear. Here, we studied myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which is one of the few animal models for MS that is dependent on B cells. We provide evidence that B cell aggregation is reflective of lymphoid neogenesis in the central nervous system (CNS) in MBP-PLP-elicited EAE. B cell aggregation was present already few days after disease onset. With disease progression CNS B cell aggregates increasingly displayed the phenotype of tertiary lymphoid organs (TLOs). Our results further imply that these TLOs were not merely epiphenomena of the disease, but functionally active, supporting intrathecal determinant spreading of the myelin-specific T cell response. Our data suggest that the CNS is not a passive "immune-privileged" target organ, but rather a compartment, in which highly active immune responses can perpetuate and amplify the autoimmune pathology and thereby autonomously contribute to disease progression. © 2012 Springer-Verlag.


Kuerten S.,University of Cologne | Lehmann P.V.,Case Western Reserve University | Lehmann P.V.,Cellular Technology Ltd
Journal of Interferon and Cytokine Research | Year: 2011

Experimental autoimmune encephalomyelits (EAE) has been widely studied as a model for multiple sclerosis (MS). EAE also holds a special place in basic autoimmune research. It is induced by immunizing healthy, naïve mice with neuroantigen. Unlike in spontaneous autoimmune models, one can therefore clearly define the initiation time point, the inducing antigen, the circumstances of the immunization that elicit a pathogenic-or nonpathogenic-T cell response, and many other parameters that are required for the induction and perpetuation of autoimmune central nervous system pathology. In the following, we will provide an overview of our current understanding of the discrete steps that lead to the pathogenesis of EAE, and we will highlight several junctions at which the perpetuation or abortive course of the disease is defined. It has become abundantly clear that the induction of a pathogenic CD4 + T cell response is a necessary requirement for the induction of EAE. However, many downstream mechanisms need to be considered if we want to understand the pathomechanisms that define the variable outcomes of EAE, and by inference, of MS. © 2011, Mary Ann Liebert, Inc.


Dittrich M.,University of Würzburg | Lehmann P.V.,Cellular Technology Ltd
Methods in Molecular Biology | Year: 2012

Cytokine ELISPOT assays have emerged as a powerful tool for the detection of rare antigen-specific T cells in freshly isolated cell material, such as blood. While ELISPOT assays allow one to directly visualize and count extremely low frequencies of cytokine-secreting T cells among millions of nonsecreting bystander cells, the interpretation of ELISPOT data can become ambiguous when (a) spot numbers in antigen-containing wells are low, (b) spot counts in negative control wells are elevated, and particularly (c) when both of the above occur simultaneously. Thus, the primary task, even before statistics are employed, must be the optimization of the basic assay parameters and reagents such that the assay yields low background signal in the negative-control wells and the maximal number of antigen-induced spots in test wells, i.e., the signal-to-noise ratio is maximized. Furthermore, the use of proper spot-size gating parameters for data analysis is indispensable for screening out irrelevant background spots, and thus increasing the signal-to-noise ratio. The goal of most ELISPOT experiments is to identify positive T-cell responses as defined by a significantly elevated spot count in antigen-stimulated wells over the nonstimulated medium-control or negative-control antigen. In this chapter, we conclude that-with some limitations-the T-Test and related statistical methods which rely on the assumption of normal distribution are suitable for identifying positive ELISPOT results. © 2012 Springer Science+Business Media, LLC.


Hundgeburth L.C.,University of Cologne | Wunsch M.,University of Cologne | Rovituso D.,University of Cologne | Recks M.S.,University of Cologne | And 3 more authors.
Clinical Immunology | Year: 2013

So far, studies of the human autoimmune disease multiple sclerosis (MS) have largely been hampered by the absence of a pathogenic B cell component in its animal model, experimental autoimmune encephalomyelitis (EAE). To overcome this shortcoming, we have previously introduced the myelin basic protein (MBP)-proteolipid protein (PLP) MP4-induced EAE, which is B cell and autoantibody-dependent. Here we show that MP4-immunized wild-type C57BL/6 mice displayed a significantly lower disease incidence when their complement system was transiently depleted by a single injection of cobra venom factor (CVF) prior to immunization. Considering the underlying pathomechanism, our data suggest that the complement system is crucial for MP4-specific antibodies to trigger CNS pathology. Demyelinated lesions in the CNS were colocalized with complement depositions. In addition, B cell deficient JHT mice reconstituted with MP4-reactive serum showed significantly attenuated clinical and histological EAE after depletion of complement by CVF. The complement system was also critically involved in the generation of the MP4-specific T and B cell response: in MP4-immunized wild-type mice treated with CVF the MP4-specific cytokine and antibody response was significantly attenuated compared to untreated wild-type mice. Taken together, we propose two independent mechanisms by which the complement system can contribute to the pathology of autoimmune encephalomyelitis. Our data corroborate the role of complement in triggering antibody-dependent demyelination and antigen-specific T cell immunity and also provide first evidence that the complement system can modify the antigen-specific B cell response in EAE and possibly MS. © 2012 Elsevier Inc.


Lehmann P.V.,Cellular Technology Ltd | Zhang W.,Cellular Technology Ltd
Methods in Molecular Biology | Year: 2012

The T cell system plays an essential role in infections, allergic reactions, tumor and transplant rejection, as well as autoimmune diseases. It does so by the selective engagement of different antigen-specific effector cell lineages that differentially secrete cytokines and other effector molecules. These T cell subsets may or may not have cytolytic activity, can preferentially migrate to different tissues, and display variable capabilities to expand clonally. The quest of T cell immune diagnostics is to understand which specific effector function and T cell lineage is associated with a given clinical outcome, be it positive or adverse. No single assay can measure all of the relevant parameters. In this chapter, we review the unique contributions that ELISPOT assays can make toward understanding T cell-mediated immunity. ELISPOT assays have an unsurpassed sensitivity in detecting low frequency antigen-specific T cells that secrete effector molecules, including granzyme and perforin. They provide robust, highly reproducible data-even by first time users. Because ELISPOT assays require roughly tenfold less cell material than flow cytometry, ELISPOT is ideally suited for all measurements requiring parallel testing under multiple conditions. These include defining (a) T cell reactivity to individual peptides of extensive libraries, thereby establishing the fine-specificity of the response, and determinant mapping; (b) reactivity to different concentrations of the antigen in serial dilutions to measure the avidity of the T cell response; or (c) different secretory products released by T cells which define their respective effector lineage/functions. Further, because T cells survive ELISPOT assays unaffected, they can be retested for the acquisition of additional information in follow-up assays. These strengths of ELISPOT assays the weaknesses of flow cytometry-based measurements. Thus, the two assays systems compliment each other in the quest to understand T cell-mediated immunity in vivo. © 2012 Springer Science+Business Media, LLC.


Zhang W.,Cellular Technology Ltd | Lehmann P.V.,Cellular Technology Ltd
Methods in Molecular Biology | Year: 2012

ELISPOT results used to be evaluated visually which, however, is inevitably subjective, inaccurate, and cumbersome. Even when applying automated image analysis to this end, the results are highly variable if the counting parameters are set subjectively. Since objective, accurate, and reproducible measurements are fundamental to science, major efforts have been undertaken over the last decade at CTL to understand the scientific principles behind ELISPOT data and to develop "intelligent" image analysis algorithms based on these principles. Thus, a spot recognition and gating algorithm was developed to automatically recognize the signatures of defined cell populations, such as T cells, discerning them from irrelevant cell types and noise. In this way, the science of ELISPOT data analysis has been introduced, permitting exact frequency measurement against background. As ELISPOT assays become a gold standard for monitoring antigen-specific T-cell immunity in clinical trials, the need has surfaced to make ELISPOT data transparent, reproducible, and tamper-proof, complying with Good Laboratory Practice (GLP) and Code for Federal Regulations (CFR) Part 11 guidelines. Flow cytometry-based and other immune monitoring assay platforms face the same challenge. In this chapter, we provide an overview of how CTL's ImmunoSpot ® platform for ELISPOT data analysis, management, and documentation meets these challenges. © 2012 Springer Science+Business Media, LLC.


Karulin A.Y.,Cellular Technology Ltd | Lehmann P.V.,Cellular Technology Ltd
Methods in Molecular Biology | Year: 2012

Over the past decade, ELISPOT has become well-established as a mainstream technology for the study of immune responses in vivo mainly due to its unique ability to detect rare antigen-specific lymphocytes ex vivo. The primary readout for ELISPOT assays has traditionally been the measurement of the frequency of analyte-secreting cells within a test population. While it has been generally appreciated that ELISPOT is a high-information-content assay system in which spot morphologies provide additional valuable information on the amount of analyte secreted by individual cells as well as the kinetics of the secretory process, the precise relationships involved have not been fully characterized and the specific relevant information-conveyed by spot morphologies has remained largely unexplored. In an attempt to bridge this gap, we formulated an in silico kinetic model for spot formation and derived a solution for the model in both a general and a numerical form. Both solutions suggested a logarithmic relationship between spot size and cell productivity. This chapter involves an in-depth analysis of the relationship between observed spot morphologies and cells' secretory functions (as well as an examination of additional assay parameters), and predictions based on the mathematical model are verified under experimental assay conditions where possible. © 2012 Springer Science+Business Media, LLC.


Lehmann P.V.,Cellular Technology Ltd | Rottlaender A.,University of Würzburg | Kurten S.,University of Würzburg
Pharmazie | Year: 2015

One of the major goals of biomedical research is to reveal the pathomechanisms that lead to a disease on a level on which diagnostic criteria and causal therapies can be designed. The understanding and treatment of multiple sclerosis (MS) is still far from this goal, but exciting developments are on the way. MS is thought to be an autoimmune disease that is mediated by brain tissue-reactive lymphocytes, T cells and B cells, but so far these lymphocytes could not be reliably detected. This article highlights recent developments that permit the detection of autoreactive B cells in MS, the implications of this finding for early diagnosis of the disease, monitoring its activity, and eventually for gaining insight into the specific immune pathology that drives MS. © 2015, Govi-Verlag Pharmazeutischer Verlag GmbH. All rights reserved.


Embodiments of this invention include methods for detecting in vitro the presence in peripheral blood mononuclear cells (PBMCs), and in serum or plasma, of antibodies reactive to and of lymphocytes that are responsive to CNS antigens associated with Multiple Sclerosis (MS). These CNS antigens include, but are not limited to whole brain lysate and the myelin antigens myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), MOG peptides (MOGps), proteolipid protein (PLP), and PLP peptides (PLPps). Stimulating PBMCs from patients with MS by CNS antigens cause B-lymphocytes to produce antibodies specific for CNS antigen, and causes T-lymphocytes to produce T-lymphocyte-specific cytokines, including interferon gamma (IFN-y), interleukin-2 (IL-2), or interleukin-17 (IL-17). In contrast, stimulating PBMCs from subjects without MS do not produce such responses.


Trademark
Cellular Technology Ltd | Date: 2011-01-25

Laboratory equipment, namely, automated bioassay analyzers and parts therefor, and operating software for automated image acquisition and analysis of bioassays for scientific and medical research.

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