Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LH(N)/A) of the type A ...neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.
Antigen-specific B and T cell responses against myelin basic protein, as well as responses against β-islet-cells or joint tissue, are commonly found both in patients with autoimmune disease and in ...normal control subjects with disease-associated HLA-DR/DQ alleles. Thus, autoreactive immune responses are not disease-specific; however, the presence of certain autoantibodies may have prognostic value and may aid in disease management.
ABSTRACT
The research presented in this poster is concerned with the ways in which people use information to build identities for themselves online with reference to the themes of personal reputation ...management. To date these two themes have been under‐explored together in the research literature, both in general and from an information science perspective. The poster content shares findings related to three areas of identity building: (1) the creation and use of online personas and identities; (2) the use of anonymity and pseudonyms through information sharing – or concealment – practices; and (3) the blurring or merging together of participants’ private and professional selves. This study used participant diaries and in‐depth, semi‐structured interviews with 45 UK‐based participants. The main finding presented here is that individuals present elements of their online persona or personality using online information, but that they do not do so with the intention of building identity. The findings explored in this presentation are contextualised with reference to identity building in the more formal setting of academic reputation management, i.e., through the use of citations.
Background: Tertiary Lymphoid Structures (TLSs) are common features of chronic inflammatory diseases including Sjogren’s syndrome (SS). We recently showed that these ectopic structures acquire ...secondary lymphoid organs properties and are capable of supporting B cell activation and autoantibody production including expression of activation-induced cytidine deaminase (AID) and Ig class switching. Dissecting TLSs dynamics in humans is technically and ethically challenging. Thus, we used the NOD mouse, a spontaneous model of autoimmune sialoadenitis, to characterize the cellular and molecular basis of autoreactive B cell activation and evolution of functional Ectopic Lymphoid Structures (ELS) in the chronically inflamed NOD salivary glands. Methods: Submandibular glands from 110 female NOD mice from 4 to 35 weeks of age were collected. Paired snap-frozen samples were analysed by immunohistochemistry (IHC) for T and B lymphocytes (CD3/CD20) to evaluate cell infiltration and the degree of B/T cell segregation. ELS were detected by staining for FDC-M1 (follicular dendritic cell networks), GL7 (germinal centre B cells) and AID (marker for ELS functionality). Characterization of B cell subsets within the infiltrates was carried out by immunostaining and by FACS analysis with CD19, CD21, CD23, B220, IgD, IgM, CD1d and CXCR5 antibodies. Quantitative TaqMan real-time PCR was performed to investigate the mRNA expression of ELS-related genes. Sex/age matched Balb/c and C57BL/6 mice were used as controls. Results: NOD infiltrates in glands displayed progressive features of ELS from week 8, with 75% of mice developing B/T cell segregation, FDC networks and GL7+ ectopic germinal centers from week 20. Evolution of TLSs was closely associated with mRNA upregulation of genes regulating ELS organization and function such as lymphoid chemokines CXCL13/CCL19 and their receptors CXCR5/CCR7, lymphotoxins and B cell survival factors BAFF and APRIL. In agreement with CXCL13/CXCR5 mRNA expression, B cells in infiltrates display strong CXCR5 expression and were mostly characterized by a follicular phenotype (B220+/IgD+/IgMlow/CD23+/CD21low) as demonstrated by both IHC and FACS analysis on isolated cells. Finally, functionality of ELS was demonstrated by expression of AID mRNA and protein within FDC networks, which paralleled the detection of circulating SS-related autoantibodies. Conclusions: This work provided the first in-depth characterization of cellular and molecular mechanisms underlying the evolution of functional TLSs within submandibular infiltrates of NOD mice. These data strongly support the hypothesis that B-cells can be activated within TLSs in the target organ and promote in situ autoantibody response. Overall, these data support the critical importance of ELS formation in chronic autoimmune inflammation and identified NOD mice as a suitable model to test therapeutic strategies aimed at modulating B cell functionality. Disclosure statement: All authors have declared no conflicts of interest.
Background: In an attempt to provide tools to assess patients with primary Sjögren’s Syndrome (pSS) in clinical trials, several outcome measures have been developed. There are significant differences ...between them. We aimed to compare recent proposals of activity and damage indices to assess outcome in patients with pSS. Methods: We analysed three activity indices—SCAI (Sjögren’s Systemic Clinical Activity Index), SSDAI (SS Disease Activity Index) and ESSDAI (EULAR SS Disease Activity Index)—and two damage indices—SSDDI (SS Disease Damage Index) and SSDI (SS Damage Index)—focusing on the number of patients included, basis for the development and type of index, their validity, reliability and sensitivity to change and comparative strengthens and weaknesses. Proposals for the assessment of perspectives of outcome (PROFAD, Profile of Fatigue and Discomfort; SF-36, Short-Form 36-item questionnaire and ESSPRI, EULAR Sjögren’s Syndrome Patients Reported Index) were considered. A revision of the literature complemented the analysis. Results: The size of cohorts was different between the studies and the patients included had mainly mild stable disease. Most indices were developed on the basis of outcome measures already validated for other autoimmune diseases. SSDAI and ESSDAI are global scores. SCAI is a composite score. ESSDAI measures systemic objective features being complemented by ESSPRI for the assessment of sicca complaints and subjective items. SSDAI and SCAI used single national cohort-based populations with resulting main limitations in validity. ESSDAI demonstrated limitations in reliability and sensitivity to change. Both damage indices demonstrated low or no correlation with the activity scores but both have limitations in content validity. Their methodology was different with respect to observation period and external validation. SSDI differentiates between the damage secondary to pSS and that derived from co-morbidities, but does not stratify each item according to the severity of damaged organ as SSDDI does. Time to complete the forms was not evaluated for any of the indices. Unresolved issues remain including the distinction between activity and damage for some items (e.g. fatigue and lymphoma), whether or not to include the identified prognostic factors for an adverse outcome and an agreed definition of flare in pSS. Conclusions: All indices analysed demonstrated a potentially useful benefit in addition to the panel of exocrine function and subjective outcome measures currently available for patients with pSS. Further studies are needed to assess reliability and sensitivity to change, particularly in response to therapy in order to validate their use in clinical trials for new therapeutic approaches. Improvements in knowledge with respect to the pathophysiology and clinical evolution of Sjögren’s syndrome are important to address some unresolved issues: the most troubling but probably also the most interesting, being the dichotomy of activity vs damage. Disclosure statement: All authors have declared no conflicts of interest.
This review examines the field of current HLA class II transgenic mouse models and the individual approaches applied in production of these mice. The majority of these mice have been created with the ...objective of obtaining a disease model with clinical features mimicking human autoimmune disease. The development process of a different type of HLA class II transgenic mice, which are designed to function as a substitute for a normal human immune system in studies of human autoantigens, is described. Several HLA‐DR4 transgenic lines with normally expressed HLA‐DR4 molecules have been produced. To obtain adequate positive selection of the HLA‐DR4‐restricted CD4+ T‐cell repertoire in these mice it is essential both to introduce a human CD4 transgene. and to delete the murine major histocompatibility complex (MHC) class II molecules. These HLA‐DR4 transgenic mice have been used to determine the immunogenic CD4+ T‐cell epitopes of several human autoantigenic proteins.
Despite substantial advances in our understanding of CD4 super(+)CD25 super(+) regulatory T cells, a possible equivalent regulatory subset within the CD8 super(+) T cell population has received less ...attention. We now describe novel human CD8 super(+)/TCR alpha beta super(+) T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS-activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I- restricted fashion, by proliferation and production of IL-4, IL-5, IL-13 and TGF beta 1, but not IFN- gamma . They constitutively expressed CD69 and CD25 as well as molecules associated with CD4 super(+)CD25 super(+) regulatory T cells, including cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and Foxp3. They suppressed IFN- gamma production and proliferation by CD4 super(+) T cells in vitro in a cell contact-dependent manner, which could be blocked using a CTLA-4-specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up-regulated in response to inflammation. We suggest that they are the CD8 super(+) counterparts of CD4 super(+)CD25 super(+) regulatory T cells. They resemble recently described CD8 super(+) regulatory cells in the rat that were able to abrogate graft-versus-host disease. Likewise, human HLA-restricted CD8 super(+) regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.
Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that
a catalytically active derivative (termed LH N /A) of the type A ...neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between
E. cristagalli lectin and either natively sourced LH N /A or recombinant LH N /A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of
neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH N /A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly
attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted
to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance
to the treatment of pain can be achieved.
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