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Células T γδ intraepiteliales intestinales y su influencia en la homeostasis intestinal: Estudio de la producción de CCL4 y flujos de calcio intracelular a través de la activación del complejo TCR γδ
Get PDFLas células T intraepiteliales intestinales (iIELs) están localizadas entre los enterocitos del epitelio intestinal y poseen un estado de activación intrínsecamente alto caracterizado por la expresión de CD69 y otros marcadores de activación. La activación del complejo TCR de las iIELs γδ induce la actividad citolítica, la producción de IFN-γ y una disminución de la expresión del complejo TCR de superficie; sin embargo, se desconoce el papel que juega el Ca2+ intracelular en su señalización y función. Por otra parte, la capacidad de homing de células T reguladoras (Tregs) a la lámina propia es importante en la mantención de la tolerancia inmune intestinal. Por lo tanto, el papel del TCRγδ fue estudiado en cuanto a la movilización de Ca2+ intracelular en iIELs y su relación con la expresión de la quimioquina CCL4. Esta quimioquina ha sido asociada a la inducción de homing de Tregs CCR5+ a diferentes tejidos.
De esta forma, nosotros describimos que las iIELs γδ y αβ producen CCL4 e IFN-γ de forma dependiente de CD3/TCR así como también dependiente de Ca2+/PKCs. CCL4 es producida fundamentalmente en las iIELs γδ de manera transitoria en comparación con las células T γδ de bazo. El aumento del Ca2+ intracelular per se como la estimulación del complejo TCR contribuyen a la activación celular dado por el aumento de la producción de CCL4 e IFN-γ en las subpoblaciones iIELs γδ y αβ ex vivo, respectivamente. Encontramos que el estado basal de activación intracelular de las iIELs, obtenidas de ratones reporteros γδ (H2B-EGFP) según las [Ca2+]i, es intrínsecamente elevado. Sin embargo, la amplitud de las [Ca2+]i fue menor respecto a la de células T sistémicas, en respuesta a la estimulación del TCR ex vivo con los mAbs anti-CD3 o anti-TCRγδ (clon GL3). La activación del complejo TCRγδ de ratones reportero γδ in vivo, que disminuye la expresión del complejo TCRγδ de superficie, redujo los niveles de [Ca2+]i basales en las iIELs γδ+CD8αα+ y γδ–CD8α+. Esta regulación negativa del complejo TCRγδ se asoció a una disminución en la funcionalidad de las iIELs γδ, debido a una refractariedad a la inducción de flujos de Ca2+ y producción de factores solubles en respuesta a la estimulación del complejo TCRγδ ex vivo.
La activación del TCRγδ in vivo tendió a aumentar el porcentaje de células T efectoras CCR5+ sin afectar el contenido de Tregs CCR5+ en la LP. Además, el contenido de células Treg CCR5+ en la LP en ratones TCRd-/- demostró ser reducido significativamente.
Estos resultados corresponden a los primeros estudios que han monitoreado directamente el estado de activación intracelular así como el flujo de Ca2+ en las iIELs γδ. También demuestran que la activación y subsecuente regulación negativa del complejo TCRγδ in vivo influye sobre los niveles de [Ca2+]i basal como en su movilización como también en la producción de CCL4 e IFN-γ. Por lo tanto, concluimos que la actividad y el nivel de expresión del TCR de las iIELs γδ son importantes para su funcionalidad. Estos resultados sugieren que las señales que toman contacto con el TCRγδ en las iIELs tienen una influencia sobre la composición de las células Treg y efectoras CCR5+ de la LP, posiblemente afectando la homeostasis de células T intestinales.The intestinal intraepithelial lymphocytes (iIELs) are located in-between the enterocytes of the gut epithelium. They have an intrinsic high activation state characterized by the expression of CD69 and other activation markers. The γδTCR complex activation in iIELs induces cytolyic activity, IFN-γ production and reduction of TCR complex surface expression, being unknown the intracellular Ca2+ role in its signaling and function. On the other hand, it has been described that regulatory (Tregs) T cells capability to home to lamina propria is important in the maintenance of the intestinal immune tolerance. Thus, studies that showing the γδTCR role in terms of intracellular Ca2+ mobilization in iIELs and their relation with CCL4 chemokine expression were carried out, in which CCL4 has been shown to be associated with CCR5+ Tregs homing to different tissues.
The γδ and αβ iIELs produce CCL4 and IFN-γ in a CD3/TCR- and Ca2+/PKCs dependent manner. CCL4 is transiently and principally produced in γδ iIELs but not in splenic γδ T cells. The increase in intracellular Ca2+ per se as well as TCR stimulation contributed to cellular activation and increased CCL4 and IFN-γ production ex vivo in γδ and αβ iIELs subpopulations, respectively. We found that the intracellular activation state based on basal [Ca2+]i in iIELs from γδ reporter mice (H2B-EGFP) is intrinsically high. However, [Ca2+]i amplitudes were lower than in systemic T cells, after ex vivo anti-CD3 or anti-γδ TCR (clone GL3) stimulation. The in vivo γδTCR complex activation in γδ reporter mice, that induces a γδTCR complex negative regulation, reduced the basal [Ca2+]i in γδ+CD8αα+ y γδ–CD8α iIELs. This γδTCR complex negative regulation is associated with a γδ iIELs functional reduction due to Ca2+ flux induction and soluble factor production refractariety through ex vivo γδ TCR complex activation.
The in vivo γδTCR activation tended to increase the percentage of LP CCR5+ effector T cells without CCR5+ Treg percentage impairment. Moreover, in contrast to in vivo γδTCR activation, studies in Tcrd-/- mice showed a significantly lower percentage in LP CCR5+ Tregs.
These results are the first studies that directly monitor the γδ iIEL’s intracellular activation state and Ca2+ flux in γδ iIELs. Furthermore, it was demonstrated that γδTCR complex negative regulation influenced the basal [Ca2+]i as well as CCL4 and IFN-γ production of γδ iIELs in vivo. Therefore, we conclude that the activity and expression levels of γδTCR in iIELs are important for their functionality. These results suggest that γδ TCR signals in iIELs have an influence on the composition of LP CCR5+ Tregs and effectors T cells and possibly affect the intestinal T cell homeostasis.Initiation and Intensification of Bilateral Cooperation. Deutsche Forschungsgemeinschaft, DFG (German Research Foundation). Julio-Octubre 2009. DFG: PR727/3 Deutsche Forschungsgemeinschaft, DFG (German Research Foundation) DFG: SFB621-A14. FONDECYT 1050451, 1070954Versión original del auto
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
Molekulare und zelluläre Analyse eines kreuzreaktiven hsp60 spezifischen T-Zellrezeptors in vitro und in vivo
No full textTitle
Table of Contents I
1. Table of Abbreviations 1
2\. 3. Introduction and Aims 3
4. Materials 24
5. Methods 31
6. Results 44
7. Discussion 74
8. Summary 87
9. Zusammenfassung 89
10. Reference List 91
11. Danksagung, CV, Publications, Erklärung 111
12. Appendix 116The goal of this work was the characterization of a TCR from a CD8+ T cell
clone that crossreacted with defined epitopes of both mycobacterial and murine
hsp60 and induced an autoimmune intestinal pathology. TCR analysis of this T
cell clone revealed the productive in-frame rearrangement of one TCR alpha and
two TCR beta genes. The question was addressed whether hsp60 crossrecognition
was directly linked to the surface expression of two TCRs by the same cell or
if a single TCR alpha/beta combination was sufficient for the crossrecognition
of the mycobacterial and murine hsp60. Therefore, the potentially dual TCR of
the hsp60 reactive T cell clone was dissected into single TCR by double
retroviral transduction of TCR deficient cell lines. Our data show that only
one of the two TCR alpha/beta combinations could constitute a functional cell
surface TCR and that posttranslational allelic exclusion of the second alpha
chain was achieved by its inability to pair with the TCR beta chain. In
conclusion, a single TCR is not only sufficient for crossrecognition with
peptides that share minimal sequence homology, moreover this promiscuous TCR
reactivity accounts also for the immunopathology of the original T cell clone.
Consequently, the rearranged alpha8 and the beta8 chain genes of the
functional hsp-crossreactive single TCR were employed for the generation of
TCR transgenic mice. In spite of their self-reactive potential, the alpha/beta
chain double transgenic T cells were not negatively selected during thymic
maturation. Like the parental T cell clone, the transgenic alpha/beta T cells
displayed the CD8 T cell coreceptor and were specific for mycobacterial hsp60
peptides. So far, the TCR transgenic mice do not show any signs of autoimmune
disease and the transgenic T cells were not spontaneously activated by self
hsp60 peptides. Thus, the hsp specific TCR transgenic mice represent a
valuable tool to study the potentially harmful activation of selfreactive T
cells in vivo, e.g. by mycobacterial infection and thereby may provide new
information about the link between infection and autoimmunity. In addition,
using MHC class I tetrameters, the recognized mycobacterial hsp60 peptide was
shown to be a relevant CD8 epitope during an immune response to mycobacterial
infection. Hence, these transgenic mice may serve to study CD8+ T cell
responses against mycobacterial infections. Transgenic expression of the
second in-frame rearranged but non-pairing TCR alpha7 chain in TCR alpha chain
deficient mice unveiled that this TCR alpha chain was generally unable to form
a normal alpha/beta TCR. Surprisingly, augmented frequencies of unconventional
CD4+ TCR alpha- beta+ T cells were found in these mice. This unusual CD4+ TCR
alpha- beta+ T cell population is associated with the development of IBD
resembling human UC in TCR alpha chain deficient mice. However, the
significance of CD4+ TCR alpha- beta+ T cells in immunocompetent individuals
remains unclear. We found that an in-frame rearranged but non-pairing TCR
alpha chain stabilizes newly synthesized TCR beta chains in TCR alpha chain
deficient mice. This lead to increased frequencies of CD4+ TCR alpha- beta+ T
cells and exaggerated the course of IBD. Furthermore, it was shown that CD4+
TCR alpha- beta+ T cells are chronically activated. In this model,
physiological TCR alpha chain rearrangement can promote the formation of
chronically activated CD4+ TCR alpha- beta+ T cells. Thus, we conclude that
these T cells are present under physiological conditions and play a role in
the etiology of UC.Ziel dieser Arbeit war die Charakterisierung des T-Zellrezeptors (TZR) eines
Hsp-spezifischen CD8+ T-Zellklons, der eine autoimmune Entzündungsreaktion im
Dünndarm verursacht. Diese T-Zellen, Klon UZ3/4, kreuzreagieren mit
definierten Peptiden des mykobakteriellen und murinen Hsp60. Zu Beginn wurden
die in dem T-Zellklon UZ3/4 rearrangierten TZR-Gene, zwei alpha und eine beta
Kette, kloniert und charakterisiert. Es wurde der Frage nachgegangen, ob beide
alpha Ketten mit der beta Kette einen funktionellen TZR bilden können und ein
sogenannter "dualer TZR" an der Kreuzreaktivität dieser T-Zellen beteiligt
ist. Die Analyse TZR defizienter Thymoma-Zelllinien, die mit den verschiedenen
alpha und beta TZR Kombinationen stabil transfiziert wurden, zeigte, dass ein
singulärer promisker TZR für die Peptiderkennung sowohl der mykobakteriellen
als auch der murinen Hsp60 Peptide ausreicht. Obwohl die zweite TZR alpha
Kette des T-Zellklons UZ3/4 im Leseraster rearrangiert war, konnte sie keinen
funktionellen TZR an der Zelloberfläche exprimieren. Basierend auf diesen in
vitro Befunden wurden im Rahmen dieser Arbeit verschiedene TZR-transgene Mäuse
hergestellt, die den funktionellen Hsp60 spezifischen TZR (TZR alpha8 und TZR
beta8 Kette) konstitutiv in ihren T-Lymphozyten exprimieren. Es zeigte sich,
dass diese TZR transgenen T-Zellen trotz ihres autoreaktiven Potenzials nicht
durch thymische Selektionsmechanismen deletiert werden. Wie der T-Zellklon
UZ3/4 weisen diese T-Zellen die Expression des Korezeptors CD8 auf und
erkennen spezifisch mykobakterielles Hsp60 Peptid. Nach dem jetzigen Stand der
Untersuchungen tritt bei diesen Tieren keine spontane intestinale
Autoimmunreaktion auf, d.h. die transgenen T-Zellen werden nicht allein durch
die Erkennung von Peptiden des murinen Hsp60 aktiviert. Somit stellen diese
Tiere ein Modell zur Untersuchung der Aktivierung potenziell autoreaktiver
CD8+ T-Zellen dar. Besonders informativ ist hierbei die Frage, ob eine durch
diese T-Zellen vermittelte Autoimmunreaktion durch eine mykobakterielle
Infektion induziert werden kann. Weiterhin konnte mit Hilfe von MHC-
Klasse-I-Tetrameren gezeigt werden, dass auch in normalen Mäusen das durch den
T-Zellklon UZ3/4 erkannte Epitop im mykobakteriellen Hsp60 relevant für eine
CD8+ T-Zellantwort gegen Mykobakterien ist. Deshalb eignen sich die TZR
transgenen Mäuse auch generell zum Studium der CD8+ T-Zellantwort gegen
Mykobakterien. Die Expression der zweiten TZR alpha Kette (TZR alpha7) des
T-Zellklons UZ3/4 in den T-Lymphozyten TZR defizienter Mäuse demonstrierte,
dass diese TZR Kette grundsätzlich nicht im Stande war einen normalen
alpha/beta TZR zu bilden. Überraschenderweise wurde in solchen Tieren jedoch
eine erhöhte Frequenz von unkonventionellen CD4+ TZR alpha- beta+ T-Zellen
gefunden. Letztere T-Zellpopulation ist in TZR defizienten Mäusen für die
Entstehung einer entzündlichen Dickdarmpathologie ähnlich der Ulcerativen
Colitis im Menschen verantwortlich. Entsprechend ist der Krankheitsverlauf
dieser Darmentzündung in Mäusen, die eine erhöhte Frequenz dieser
unkonventionellen T-Zellen zeigen, schneller und stärker ausgeprägt.
Interessanterweise waren die CD4+ TZR alpha- beta+ Zellen chronisch aktiviert.
Die Bedeutung solcher CD4+ TZR alpha- beta+ T-Zellen in immunkompetenten
Individuen ist jedoch unklar. Hier konnte gezeigt werden, dass eine nicht
paarungsfähige TZR alpha Kette neu gebildete TZR beta Ketten stabilisieren und
damit die Entwicklung von CD4+ TZR alpha- beta+ T-Zellen begünstigen kann. Wir
vermuten daher, dass solche T-Zellen auch unter physiologischen Bedingungen
vorkommen und eine Rolle in der Ätiologie der Ulcerativen Colitis spielen
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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