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Immune regulation by human colonic bacteria and short-chain fatty acids
No full textThe intestinal microbiota plays a crucial role in the homeostasis of the human gastrointestinal tract by maintaining an anti-inflammatory status. Microbial imbalance in the gut, which is often referred to as ‘dysbiosis’, is known to be one of the major contributors to many human diseases, including inflammatory bowel disease (IBD). IBD is characterized by a chronic inflammation, but the current available treatment with anti-inflammatory drugs is often not effective. Therefore, the overarching aim of this thesis was to try to find bacterial strains or bacterial metabolites that have an immunomodulatory (i.e. an anti-inflammatory) function and may therefore be used as a therapy or treatment of IBD. In collaboration with the Rowett Institute, University of Aberdeen in Scotland, UK and the Department of Medical Microbiology, at the University Medical Centre Groningen, The Netherlands, a large number of colonic anaerobic bacteria were isolated from healthy patients. In our lab, we cultured more than 100 of these different strains of bacteria under strictly anaerobic conditions. We observed their growth characteristics and investigated their immunomodulatory properties. A high ratio between secreted anti-and pro-inflammatory cytokines (IL-10/IL-12 ratio) has been reported to be an indicator of positive correlation between in vitro trials and the attenuation of clinical symptoms in in vivo mouse models of colitis. Our initial screening therefore started with stimulation of peripheral mononuclear blood lymphocytes (PBMCs) with standardized concentrations of the bacteria or their culture supernatant. We determined the cytokine secretion of these PBMC after bacterial stimulation and determined the IL-10/IL-12 ratio. Additionally we checked if the viability of the PBMC was not affected by the bacterial strains or their metabolites which were secreted in the supernatant. As IBD is characterized by periods of remission and occasional flare-ups (periods of higher inflammation), we investigated both healthy and disease situations. To achieve this, we added heat inactivated bacteria (HIB) to our PBMC culture. These HIB triggered a strong induction of both pro-and anti- inflammatory cytokines, which is reminiscent of actual inflammation. Co-stimulation with both HIB and our bacterial strains enabled us to investigate the effect of the bacterial strains ‘during inflammation’. Additionally, we checked whether the bacterial strains were able to trigger NF-κB signalling via Toll like receptor (TLR) activation, which is one of the most known mechanisms to modulate immune response of the host. Finally, to assess oxidative stress, which is known to occur during flare-ups and damages the epithelial cells, we investigated whether the different bacterial strains were able to modulate nitric oxide (NO) secretion by a mouse macrophage (RAW 264.7) cell line. Again we were able to mimic an inflammatory situation by addition of bacterial lipopolysaccharides (LPS) to these cells and could thereby also investigate the effect of bacteria in an already inflamed gut. In Chapter 2 we summarize the characterisation of 68 different colonic anaerobic bacteria tested. Most importantly, we found that there is a large variation among the tested strains in their immunomodulatory properties. The variation in induction of cytokine and NO secretion as well as in the ability to trigger NF-κB signalling between strains was significant. Interestingly, the bacterially induced immune profiles were highly strain dependent and not characteristic for a specific bacterial species. We must therefore conclude that generalizations cannot be made easily and any newly discovered strain needs to be individually investigated to determine its immunomodulatory properties. However, we could identify three different immune profiles, resulting from bacterial stimulation. The first ‘immunostimulatory’ profile was characterized by strongly inducing cytokine secretion in PBMCs. Most of these strains also elicited relatively high concentrations of NO secretion and strong NF-κB signalling after TLR activation. The second ‘immunomodulatory’ profile was characterized by induction of only moderate amounts of cytokine secretion. However, when an inflammatory status was mimicked (addition of HIB), these strains were able to attenuate the secretion of pro- inflammatory cytokines. The final ‘immuno suppressive’ or ‘silent’ profile was characterized by a low capacity to induce cytokine or NO secretion. More importantly, when HIB was added as an inflammatory stimulus, these strains attenuated the resulting pro-inflammatory cytokine response. Several studies showed that there is a negative correlation between the relative abundance of F. prausnitzii and the disease severity of IBD, therefore a causal connection has been suggested. Indeed, several in vivo studies have shown that addition of certain strains of F. prausnitzii could attenuate clincal symptoms of colitis in mice. We therefore focused on another 28 bacterial strains that all belonged to the species of F. prausnitzii in Chapter 3. After thorough in vitro investigation we have to conclude that the immunomodulatory properties are really strain specific, as the tested properties of the strains (cytokine, NO secretion and NF-κB signaling via TLR activation) do not correlate with genomic phylogenetic clusters. Among the different strains tested, we found all three different immune profiles that were observed in Chapter 2. Moreover, the general assumption that all F. prausnitzii strains induce strong IL-10 secretion was found to not be universally true, as there were some ‘silent’ strains that hardly induced any IL-10 secretion. Interestingly, one of the strains that was the strongest activator of NF-κB signaling, hardly induced any cytokine secretion, which suggest some specific mechanism to prevent downstream effects. Indeed this strain manifested a ‘silent’ profile and as such was considered to be of interest for further in vivo trials. During the in vitro screening procedures described above we observed that the culture supernatant of all strains tested was able to attenuate HIB induced cytokine secretion. Further investigations showed that the original growth medium (without any bacteria present), and especially the short chain fatty acids (SCFA) in the medium also triggered this attenuation effect. SCFA were reported to have immunomodulatory effects, but the studies were not all consistent. Most studies showed that butyrate induced IL-10 secretion by immune cells and thereby triggered regulatory T cell differentiation. In Chapter 4 we compared the effects of acetate and butyrate on different immune cell mechanisms. We found that butyrate decreased cell viability when administered at higher concentrations, whereas similar, or even higher concentrations of acetate did not affect cell viability. Interestingly, although both acetate and butyrate were able to attenuate HIB induced secretion of pro- inflammatory cytokines, only acetate was able to increase the anti-inflammatory cytokine IL-10. More importantly, butyrate actually decreased the secretion of the anti-inflammatory IL-10 in vitro in both PBMC as well as CD14+ monocytes. This decreased IL-10 secretion could result in an overall more inflammatory response (lower IL-10/IL-12 ratio) compared to the response triggered by acetate. To investigate how butyrate and acetate elicited their effects, we tried to determine the mechanism by which they affected cytokine secretion. As G protein-coupled receptors (GPCR) are known to mediate the effect of SCFA, we blocked the expression of GPCR in monocyte-derived dendritic cells (MDDC) with specific inhibitors. The attenuating effects of SCFA on HIB induced cytokine secretion were still observed, suggesting that modulation of cytokine secretion used an GPCR-independent mechanism. Butyrate (and to a lesser extent acetate) are also known to affect histone acetylation and could thereby modulate gene transcription. We found that butyrate indeed increased histone acetylation, which may point to a mechanism used in modulation of cytokines secretion. Interestingly, NF- κB activation was also found to be differentially modulated by acetate compared to butyrate, although the underlying mechanism has not yet been elucidated. As we found clear effects of both acetate and butyrate on cytokine secretion by PBMC and CD14+ monocyte, we wondered if other biological pathways would be affected as well. We therefore stimulated CD14+ monocytes with acetate and butyrate (with or without co- stimulation with HIB) and investigated the resulting transcription profile in Chapter 5. Our monocytes showed the same attenuation of pro-inflammatory cytokine secretion in HIB induced samples as was seen in the PBMC and CD14+ monocyte. Acetate did not cause major effects in the number of regulated genes, but butyrate significantly affected the regulation of many different (immune) pathways and genes therein. To compare the effects that SCFA might have on epithelial cells with the effects we found on immune cells, we investigated the effect of SCFA on an ex vivo 3D porcine ileum organoid model in Chapter 6. We exposed the organoid cells to acetate and butyrate and performed a transcriptomic analysis. Similar to the results of the transcriptomic analysis of CD14+ monocyte, butyrate proved to elicit greater changes in gene expression compared to acetate and substantially affected apoptosis and cell-cycle related pathways. In contrast, acetate mainly affected cellular metabolism process- related pathways, suggesting a less damaging effect on gut epithelial function compared to butyrate. To conclude our studies, we tested several bacterial strains that appeared promising in the in vitro trials, in an in vivo mouse DSS-induced colitis model in Chapter 7. We observed attenuation of the clinical symptoms after addition of these ‘silent’ strains, which confirm our hypothesis that the bacterial strains that induced an ‘silent’ profile would be able to reduce colonic inflammation. In the last chapter, Chapter 8, we summarize and discuss the combined results from this thesis in the context of other studies regarding host- microbe interactions, probiotics and the effects of SCFA. We explain how these findings contribute to a better understanding of the immunomodulatory properties of colonic anaerobic bacterial strains and their metabolites and provide suggestions for future research, and reflect on the overall aim of this thesis
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Communication Dynamics Between First-Year Doctoral Students and Supervisors
Get PDFBuilding and maintaining a relationship with the supervisor is crucial for a doctoral student to guarantee stable progress. One of the important aspects is owing to both written and spoken communication, for example, in email and direct conversations. This study examines sample communication activitie of three Indonesian doctoral students and their supervisors to unfold the construction of shared values between them. An appraisal system analyses the linguistic mechanism of engagement, attitude, and graduation systems through which power and relationship are built between first-year doctoral students and their supervisors
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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