1,720,996 research outputs found
Characterization of H+,K+-ATPase T cell epitopes in human autoimmune gastritis
No full textHuman autoimmune gastritis (AIG) is an organ-specific inflammatory disorder leading to gastric atrophy and pernicious anemia. Gastric H+,K(+)-ATPase was identified as the autoantigen in both human disease and experimental murine AIG (EAIG). Studies of EAIG significantly contributed to current knowledge of human AIG, but to what extent EAIG mimics AIG is still debated, and the autoantigenic epitopes in AIG are yet unknown. This study aimed to identify the H+,K(+)-ATPase epitopes recognized by gastric T cell clones from AIG patients, to define their TCR Vbeta usage and epitope-induced cytokine response. Sixteen H+,K(+)-ATPase-reactive CD4+ gastric T cell clones of four AIG patients were tested for proliferation to overlapping 15-mer peptides spanning the a and beta chains of H+,K(+)-ATPase. We identified 6 epitopes in the a chain and 5 in the beta chain; TCR Vbeta usage was not restricted. Four (36%) of the 11 H+,K(+)-ATPase epitopes recognized in AIG were found to overlap with epitopes that are relevant in EAIG, including a previously described gastritogenic epitope. Gastric T cell recognition of the peptide epitopes resulted in secretion of Th1 cytokines. Our data suggest a striking similarity between human AIG and EAIG, at the epitope level, with regard to cytokine secretion and likely also with regard to pathogenic mechanism
Endogenous stress proteins as targets for anti-inflammatory T cells
No full textStress proteins such as heat shock proteins (Hsp) are important controllers of both cellular and immune homeostasis. Enhanced Hsp expression can be observed in virtually every inflammatory condition and has been proposed by us and others to lead to local activation of Hsp-specific anti-inflammatory T cells. Amongst the various Hsp, particularly Hsp70 is highly stress inducible. But stress inducibility of Hsp70 is decreased at increasing age. Therefore, we hypothesized in this thesis that when expression of the self-antigen is reduced, Hsp mediated immunoregulation fails and that boosting either Hsp-specific T cell responses or HSP expression itself, can restore or enhance immune homeostasis. First we explored in in vitro systems if the immune system can actually respond to enhanced Hsp70 levels. Using flow cytometry, Hsp70-specific CD4+ T cell hybridomas and reporter cell lines we found that Hsp70 is highly stress inducible in immune cells and that Hsp70-specific T cells can recognize enhanced Hsp70 expression. Furthermore, this strategy yielded the identity of several new food-derived enhancers of stress-induced Hsp70 expression, amongst them carvacrol, found in thyme and oregano. Then we proceeded to investigate the effect of boosting Hsp70-specific T cell responses with exogenous Hsp70 in the proteoglycan-induced arthritis (PGIA model). We demonstrate that intraperitoneal immunization with mycobacterial (Mt) Hsp70 suppresses PGIA. Moreover, we provide evidence for the mechanism of Hsp-specific regulatory T cells by our finding that IL-10 is essential for suppression. Since mucosal antigen administration will be an appealing way of tolerance induction, we studied the cellular basis of mucosal tolerance in the PGIA model and confirmed that also nasal administration of Mt Hsp70 ameliorates PGIA. Subsequently, the immunodominant T cell epitopes of Mt Hsp70 were identified. One of the highly conserved epitopes (peptide C1) was found to induce IL-10 and to suppress PGIA upon intranasal administration. We uncovered in addition, that this peptide is a good binder (in silico) to the human HLA and that human CD4+ T cell can recognize the peptide, illustrating the relevance for clinical application in humans. To boost Hsp70 expression directly, Hsp70 levels were manipulated with the food constituent carvacrol. Carvacrol potently enhanced stress-induced Hsp70 expression both in vitro and upon intragastric administration in vivo. Up-regulation of Hsp70 was sensed by Hsp70-specific T cell hybridomas. Also in vivo we show that carvacrol enhances Hsp70-specific T cell responses. Subsequently, intragastric administration of carvacrol suppressed PGIA and this protection could be transferred to naïve recipients with T cells, thus providing the first evidence that boosting endogenous Hsp can be translated into immune regulation. Furthermore, this data demonstrate that enhancing immune fitness through food components such as carvacrol is feasible and would allow relatively easy and safe intervention means. In summary the findings presented in this thesis show that boosting Hsp70 expression or the Hsp70-specific T cell response can modulate the immune system and enhance immune homeostasis, thereby illustrating that endogenous Hsp can function as targets for anti-inflammatory T cells, which will contribute to development of new or refinement of existing intervention strategies to treat autoimmune disease
Endogenous stress proteins as targets for anti-inflammatory T cells
No full textStress proteins such as heat shock proteins (Hsp) are important controllers of both cellular and immune homeostasis. Enhanced Hsp expression can be observed in virtually every inflammatory condition and has been proposed by us and others to lead to local activation of Hsp-specific anti-inflammatory T cells. Amongst the various Hsp, particularly Hsp70 is highly stress inducible. But stress inducibility of Hsp70 is decreased at increasing age. Therefore, we hypothesized in this thesis that when expression of the self-antigen is reduced, Hsp mediated immunoregulation fails and that boosting either Hsp-specific T cell responses or HSP expression itself, can restore or enhance immune homeostasis. First we explored in in vitro systems if the immune system can actually respond to enhanced Hsp70 levels. Using flow cytometry, Hsp70-specific CD4+ T cell hybridomas and reporter cell lines we found that Hsp70 is highly stress inducible in immune cells and that Hsp70-specific T cells can recognize enhanced Hsp70 expression. Furthermore, this strategy yielded the identity of several new food-derived enhancers of stress-induced Hsp70 expression, amongst them carvacrol, found in thyme and oregano. Then we proceeded to investigate the effect of boosting Hsp70-specific T cell responses with exogenous Hsp70 in the proteoglycan-induced arthritis (PGIA model). We demonstrate that intraperitoneal immunization with mycobacterial (Mt) Hsp70 suppresses PGIA. Moreover, we provide evidence for the mechanism of Hsp-specific regulatory T cells by our finding that IL-10 is essential for suppression. Since mucosal antigen administration will be an appealing way of tolerance induction, we studied the cellular basis of mucosal tolerance in the PGIA model and confirmed that also nasal administration of Mt Hsp70 ameliorates PGIA. Subsequently, the immunodominant T cell epitopes of Mt Hsp70 were identified. One of the highly conserved epitopes (peptide C1) was found to induce IL-10 and to suppress PGIA upon intranasal administration. We uncovered in addition, that this peptide is a good binder (in silico) to the human HLA and that human CD4+ T cell can recognize the peptide, illustrating the relevance for clinical application in humans. To boost Hsp70 expression directly, Hsp70 levels were manipulated with the food constituent carvacrol. Carvacrol potently enhanced stress-induced Hsp70 expression both in vitro and upon intragastric administration in vivo. Up-regulation of Hsp70 was sensed by Hsp70-specific T cell hybridomas. Also in vivo we show that carvacrol enhances Hsp70-specific T cell responses. Subsequently, intragastric administration of carvacrol suppressed PGIA and this protection could be transferred to naïve recipients with T cells, thus providing the first evidence that boosting endogenous Hsp can be translated into immune regulation. Furthermore, this data demonstrate that enhancing immune fitness through food components such as carvacrol is feasible and would allow relatively easy and safe intervention means. In summary the findings presented in this thesis show that boosting Hsp70 expression or the Hsp70-specific T cell response can modulate the immune system and enhance immune homeostasis, thereby illustrating that endogenous Hsp can function as targets for anti-inflammatory T cells, which will contribute to development of new or refinement of existing intervention strategies to treat autoimmune disease
Adjuvanted vaccines: Aspects of immunosafety and modes of action
No full textNew developments in vaccine design shift towards safe, though sometimes less immunogenic, subunit and synthetic antigens. Therefore, the majority of current vaccines require adjuvants to increase immunogenicity. Most adjuvants available were developed empirically and their mode of action is only partly elucidated. Earlier work shows that adjuvants exert their effect mainly on the innate immune system. To improve vaccines, more knowledge on adjuvants’ modes of action and their effect on innate immune responses at the site of application are required. Furthermore, immune responses that take place after vaccination are sometimes linked with the development or exacerbation of autoimmune diseases (AID). It is highly debated íf vaccines indeed induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale, more research is warranted. The aim of this thesis was to study working mechanisms of vaccines and adjuvants locally (innate immune system) as well as systemically (adaptive immune system) to identify possible risks on severe side effects of vaccination and potentially improve vaccine effectiveness. A panel of human and experimental adjuvants was tested in mice for their local effects and ability to initiate adaptive responses. Rapid but transient immune-stimulatory environments were induced by most adjuvants. Antigen-uptake and -presentation by APC and maturation of APC were differentially affected by distinct adjuvants. The choice of adjuvant determined the outcome of the initiated adaptive immune response and we showed that the route of administration of vaccines (intranasal or intradermal) also influences the response induced. Two potential mechanisms on how AID could result from vaccination were explored; (1) bystander activation and (2) disturbances in regulatory T cells (Treg). During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. Our study demonstrated that for a model vaccine (comprising the unsafe adjuvant Complete Freund’s adjuvant) not the adjuvant, but the vaccine-antigen likely induced limited bystander responses. No evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation. Regulatory T cells (Treg) function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. Both in men and mice, safe vaccines were shown to have only minimal impact on Treg frequencies and characteristics and even an unsafe stimulus (CFA) did not result in significant (long term) effects on Treg of mice. More knowledge on adjuvants, both their modes of action and aspects of immunosafety, will aid in the development of new, improved and safe(r) vaccines that will provide good protection against infectious diseases without any unnecessary side effects
Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography
No full textReversed-phase high-performance liquid chromatography with microbore columns (50 × 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1–0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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