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Synthese und Charakterisierung von Limbusepithel-Amnion-Transplantaten aus langzeitorgankonservierten Hornhäuten und kryokonservierten AmnionmembranenHenkel, Tassilo 07 December 2010 (has links)
In dieser Arbeit wurden Methoden entwickelt und verglichen, um aus Corneoskleralringen langzeitorgankonservierter Hornhäute und intakten, kryokonservierten Amnionmembranen Limbusepithel-Amnion-Transplantate herzustellen. Als erfolgreichste Kultivierungsmethode stellte sich hierbei signifikant die Explantat-Technik mit nach unten gerichtetem Limbusepithel heraus. Hier konnte eine Auswachsrate von 42 % erzielt werden. Es wurde weiterhin gezeigt, dass das ausgewachsene, mehrschichtige Limbusepithel proliferationsfähige TACs (Transient Amplifying Cells) enthält.
Weiterhin konnten mittels Regressionsanalyse signifikante Zusammenhänge zwischen Spenderalter, Post-mortem-Zeit, Organkultur-Dauer und der Auswachsrate beschrieben werden. Kurzgefasst wurde die Vermutung bestätigt, dass jede Verlängerung der unterschiedlichen Zeiten eine Verringerung der Auswachsrate zur Folge hat.
Die hergestellten Limbusepithel-Amnion-Transplantate könnten für Patienten mit Limbusstammzellinsuffizienz unterschiedlicher Genese verwendet werden.
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Role of CD4+ T cells in the regulation of the immune response against encapsulated Group B StreptococcusClarke, Damian 08 1900 (has links)
Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1.
En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène. / Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response.
In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections.
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Ex vivo Analyse antigen-spezifischer CD4+ T-Zell-Antworten im Infektionsmodell der murinen ListerioseSchiemann, Matthias 25 November 2009 (has links) (PDF)
Antigen-reaktive CD4+ T-Zellen spielen eine wichtige Rolle bei der Entwicklung schützender Immunität. So sind Kenntnisse über die Bildung und Erhaltung CD4+ T-Zellen möglicherweise bedeutend für die Entwicklung verbesserter Impfstoffe und neuer Therapieansätze bei akuten und chronischen Infektionserkrankungen. Mit dieser Arbeit wurde eine genaue Charakterisierung CD4+ T-Zell-Antworten im Vergleich zu CD8+ T-Zellen am Infektionsmodell der murinen Listeriose vorgenommen. So konnten erstmals wichtige Unterschiede zwischen den beiden T-Zell-Kompartimenten beschrieben werden, insbesondere die Beobachtung, dass - im Gegensatz zu CD8+ T-Zellen - CD4+ Effektor-Gedächtniszellen nach bakterieller Infektion kontinuierlich im gesamten Organismus über die Zeit abnehmen.
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Etude de la réponse lymphocytaire T dans l'allergie de l'enfant, au diagnostic et au cours de la désensibilisationMichaud, Bénédicte 25 October 2013 (has links) (PDF)
Les maladies allergiques sont de plus en plus fréquentent. Elles atteignent souvent l'enfant jeune chez qui l'allergie respiratoire et l'allergie alimentaire sont les principales pathologies. L'unique traitement curatif est l'immunothérapie spécifique d'antigène (ITA), largement développée dans l'allergie respiratoire et encore à ses débuts dans l'allergie alimentaire. Pour adapter au mieux la prise en charge du patient, le diagnostic précis de l'allergie est indispensable et il n'existe actuellement pas d'examen biologique totalement fiable. Seul, la présence d'IgE spécifiques permet de diagnostiquer une sensibilisation à un allergène mais pas une allergie cliniquement symptomatique. Dans une première partie, nous avons étudié l'intérêt d'un test fonctionnel, l'ELISpot (Enzyme-linked immunosorbent spot), dans le diagnostic de l'allergie aux acariens chez l'enfant asthmatique. Le nombre de lymphocytes T circulants spécifiques d'acariens sécréteur d'interleukine (IL)-4 ou d'IL-13 était associé à la présence d'une allergie symptomatique, indépendamment des IgE spécifiques. Il était plus élevé dans le cas d'une rhinite allergique sévère et plus faible dans le cas d'une rhinite allergique légère. De plus, il variait au cours de l'année en fonction des saisons avec un pic en automne et un pic en début de printemps. Dans une deuxième partie, nous avons étudié l'intérêt de l'ELISpot dans le diagnostic de l'allergie au lait de vache chez l'enfant, confirmée par un test de provocation orale en double aveugle. Nous avons décrit que le nombre de lymphocytes T spécifiques de la caséine et sécréteurs d'IL-4 et d'IL-13 était associé à l'allergie au lait de vache avec une sensibilité de 100%. Par ailleurs, le nombre de lymphocytes T spécifiques de la caséine était également associé à la dose maximale de lait tolérée par l'enfant.Enfin, dans une troisième partie, nous avons étudié la réponse lymphocytaire T au cours d'une ITA sub-linguale (SLIT) d'une part et sous-cutanée (SCIT) d'autre part, chez des enfants asthmatiques allergiques aux acariens suivis pendant une année. Nous avons décrit une diminution des lymphocytes Th2 (sécréteurs d'IL-4 et IL-13) spécifiques d'acariens après 12 mois de SLIT associée à une augmentation des cellules sécrétrices d'IL-10 (Tr1) spécifiques d'acariens après 6 mois de SLIT. De plus, les lymphocytes T régulateurs (CD4+CD25hiCD127loFoxp3+) étaient augmentés après 12 mois de SCIT. Nous n'avons pas retrouvé de production accrue d'interféron γ (IFNγ) par les lymphocytes T spécifiques d'acariens au cours de la désensibilisation.Au total, ce travail nous a permis de décrire qu'un test fonctionnel, l'ELISpot, permet de réaliser un diagnostic fiable de l'allergie aux acariens et de l'allergie au lait de vache chez l'enfant. Par ailleurs, l'ITA induit une diminution des cellules Th2 et une augmentation des cellules Tr1 par voie sub-linguale ainsi qu'une augmentation des Treg Foxp3+ par voie sous-cutanée sans immunodéviation Th2/Th1, chez l'enfant allergique aux acariens.
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Génération de progéniteurs hépatiques dérivés de cellules souches : application à l'hypercholestérolémie familialeCorbineau, Sébastien 05 October 2011 (has links) (PDF)
La transplantation d'hépatocytes représente une alternative à la transplantation hépatique pour le traitement de certaines maladies métaboliques dont l'hypercholestérolémie familiale. Les cellules souches embryonnaires (ES) et les cellules souches pluripotentes induites (iPS) humaines représentent de nouvelles sources de cellules hépatiques. Nous avons mis au point une approche de différenciation des cellules souches humaines en cellules hépatiques et généré ainsi des cellules dérivées de cellules iPS de patients atteints d'hypercholestérolémie familiale.
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Investigations on the respiratory effects of ozone in the rodent / Cornelius Jacon LotrietLotriet, Cornelius Jacob January 2010 (has links)
Ozone, being an unstable molecule, is believed to be one of the strongest oxidant
agents known to man. Rapid growth in the application of ozone — both as
disinfectant and as form of alternative medicine — led to questions about the effects
of uncontrolled ozone exposure and inhalation, whether intentional or unintentional,
on the human body.
This study specifically focussed on examining, identifying and substantiating the
respiratory effect of acute exposure (10 min or less) to considerably higher ozone
concentrations than reported on before (19.5 ± 0.5 ppm). Respiratory tissue of
rodents (Duncan–Hartley guinea pigs of both sexes and Male Wistar rats) was
subjected to ozone by utilising three distinctly diverse models of ozone introduction:
(a) in vitro exposure, (b) in vivo exposure, and (c) ex vivo by employing an isolated
lung perfusion model which allows for real–time, breath–by–breath data acquisition of
ozone’s effect on respiratory mechanics. The effect of ozone on the isolated trachea
in the presence of various drugs with well–known effects, including methacholine,
isoproterenol and ascorbic acid was also examined.
The results found in this study identified two direct effects on the isolated trachea due
to ozone exposure: (1) a definite contraction of the isolated trachea immediately after
exposure to ozone, and (2) a clearly visible and significant hyper responsiveness of
the isolated trachea to irritants, e.g. methacholine. Although ozone has a negative
effect on the trachea, it was concluded that ozone has no adverse effect on
muscarinic acetylcholine receptors. An apparent EC50 value of ozone on the trachea
was established by two different methods as (2.77 ± 0.02) x 10–3 M and (2.10 ± 0.03)
x 10–3 M, respectively. Ozone furthermore displayed an attenuation of the beneficial pharmacological
response of –sympathomimetic drugs (i.e. isoproterenol), while isoproterenol itself
has a relaxing effect on the ozone–induced contraction of the isolated trachea.
Indomethacin pre–treatment of isolated tracheal tissue significantly (77%) reduced
the ozone–induced contraction of tracheal smooth muscle, suggesting that COXproducts
of arachidonic acid play a prominent role in the development of pulmonary
function decrements consequent to acute high–dose ozone exposure. Ascorbic acid
exhibited a meaningful prophylactic effect on ozone–induced contraction of both
isolated tracheal tissue and in the isolated lung perfusion model, emphasising the
major role antioxidants play in both the epithelium lining fluid (ELF) of the respiratory
system and in plasma throughout the body in protecting against the destructive
effects of ozone.
Surprisingly, pre–treatment with ascorbic acid did not prevent hyper responsiveness
of isolated tracheal preparations to methacholine after a 10 min ozone (19.5 ± 0.5
ppm) exposure. In the lung perfusion model, the presence of ascorbic acid in the
perfusion medium did, however, significantly reduce the magnitude and rate of
decline in lung compliance after ozone exposure (46% decline with ascorbic acid
versus 96% in the control study without ascorbic acid).
Examination of a lung perfusion model exposed to ozone (19.5 ± 0.5 ppm O3; 5
seconds) presented a significant decline in lung compliance (95.6% within 2 min),
tidal volume (70%) and maximum inspiratory flow (71.2%), with an ensuing reduction
in lung elasticity and severely hampered breathing pattern.
Microscopic examination after acute high–dose inhalation studies did not display any
significant cellular damage, oedema or inflammation after acute high–dose ozone
exposure. This suggests that significant cellular injury and inflammation is possibly
not the causative factor of early breathing difficulty experienced after acute high–dose
ozone inhalation, as these symptoms and particularly the result of inflammatory
precursors, is believed to probably only set in at a later stage.
Although the potential advantages of ozone in certain fields of medicine are not
disputed, ozone, depending on its concentration and cumulative dose, can be either therapeutic or toxic. Observations in this study emphasised that even short bursts of
high–dose ozone inhalation have deleterious effects on respiratory health and care
should be taken not to jump to conclusions regarding ozone’s medical application
without relevant scientific evidence. It must be stressed that high–dose inhalation of
ozone should be avoided at all cost – especially by those with existing airway
diseases. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.
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Investigations on the respiratory effects of ozone in the rodent / Cornelius Jacon LotrietLotriet, Cornelius Jacob January 2010 (has links)
Ozone, being an unstable molecule, is believed to be one of the strongest oxidant
agents known to man. Rapid growth in the application of ozone — both as
disinfectant and as form of alternative medicine — led to questions about the effects
of uncontrolled ozone exposure and inhalation, whether intentional or unintentional,
on the human body.
This study specifically focussed on examining, identifying and substantiating the
respiratory effect of acute exposure (10 min or less) to considerably higher ozone
concentrations than reported on before (19.5 ± 0.5 ppm). Respiratory tissue of
rodents (Duncan–Hartley guinea pigs of both sexes and Male Wistar rats) was
subjected to ozone by utilising three distinctly diverse models of ozone introduction:
(a) in vitro exposure, (b) in vivo exposure, and (c) ex vivo by employing an isolated
lung perfusion model which allows for real–time, breath–by–breath data acquisition of
ozone’s effect on respiratory mechanics. The effect of ozone on the isolated trachea
in the presence of various drugs with well–known effects, including methacholine,
isoproterenol and ascorbic acid was also examined.
The results found in this study identified two direct effects on the isolated trachea due
to ozone exposure: (1) a definite contraction of the isolated trachea immediately after
exposure to ozone, and (2) a clearly visible and significant hyper responsiveness of
the isolated trachea to irritants, e.g. methacholine. Although ozone has a negative
effect on the trachea, it was concluded that ozone has no adverse effect on
muscarinic acetylcholine receptors. An apparent EC50 value of ozone on the trachea
was established by two different methods as (2.77 ± 0.02) x 10–3 M and (2.10 ± 0.03)
x 10–3 M, respectively. Ozone furthermore displayed an attenuation of the beneficial pharmacological
response of –sympathomimetic drugs (i.e. isoproterenol), while isoproterenol itself
has a relaxing effect on the ozone–induced contraction of the isolated trachea.
Indomethacin pre–treatment of isolated tracheal tissue significantly (77%) reduced
the ozone–induced contraction of tracheal smooth muscle, suggesting that COXproducts
of arachidonic acid play a prominent role in the development of pulmonary
function decrements consequent to acute high–dose ozone exposure. Ascorbic acid
exhibited a meaningful prophylactic effect on ozone–induced contraction of both
isolated tracheal tissue and in the isolated lung perfusion model, emphasising the
major role antioxidants play in both the epithelium lining fluid (ELF) of the respiratory
system and in plasma throughout the body in protecting against the destructive
effects of ozone.
Surprisingly, pre–treatment with ascorbic acid did not prevent hyper responsiveness
of isolated tracheal preparations to methacholine after a 10 min ozone (19.5 ± 0.5
ppm) exposure. In the lung perfusion model, the presence of ascorbic acid in the
perfusion medium did, however, significantly reduce the magnitude and rate of
decline in lung compliance after ozone exposure (46% decline with ascorbic acid
versus 96% in the control study without ascorbic acid).
Examination of a lung perfusion model exposed to ozone (19.5 ± 0.5 ppm O3; 5
seconds) presented a significant decline in lung compliance (95.6% within 2 min),
tidal volume (70%) and maximum inspiratory flow (71.2%), with an ensuing reduction
in lung elasticity and severely hampered breathing pattern.
Microscopic examination after acute high–dose inhalation studies did not display any
significant cellular damage, oedema or inflammation after acute high–dose ozone
exposure. This suggests that significant cellular injury and inflammation is possibly
not the causative factor of early breathing difficulty experienced after acute high–dose
ozone inhalation, as these symptoms and particularly the result of inflammatory
precursors, is believed to probably only set in at a later stage.
Although the potential advantages of ozone in certain fields of medicine are not
disputed, ozone, depending on its concentration and cumulative dose, can be either therapeutic or toxic. Observations in this study emphasised that even short bursts of
high–dose ozone inhalation have deleterious effects on respiratory health and care
should be taken not to jump to conclusions regarding ozone’s medical application
without relevant scientific evidence. It must be stressed that high–dose inhalation of
ozone should be avoided at all cost – especially by those with existing airway
diseases. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.
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Role of CD4+ T cells in the regulation of the immune response against encapsulated Group B StreptococcusClarke, Damian 08 1900 (has links)
Le Streptocoque de groupe B (GBS) est un important agent d’infection invasive pouvant mener à la mort et demeure la cause principale de septicémie néonatale à ce jour. Neuf sérotypes ont été officiellement décrits basés sur la composition de la capsule polysaccharidique (CPS). Parmi ces sérotypes, le type III est considéré le plus virulent et fréquemment associé aux maladies invasives graves, telle que la méningite. Malgré que plusieurs recherches aient été effectuées au niveau des interactions entre GBS type III et les cellules du système immunitaire innées, aucune information n’est disponible sur la régulation de la réponse immunitaire adaptative dirigée contre ce dernier. Notamment, le rôle de cellules T CD4+ dans l’immuno-pathogenèse de l’infection causée par GBS n’a jamais été étudié. Dans cet étude, trois différents modèles murins d’infection ont été développé pour évaluer l’activation et la modulation des cellules T CD4+ répondantes au GBS de type III : ex vivo, in vivo, et in vitro. Les résultats d’infections ex vivo démontrent que les splénocytes totaux répondent à l’infection en produisant des cytokines de type-1 pro-inflammatoires. Une forte production d’IL-10 accompagne cette cascade inflammatoire, probablement dans l’effort de l’hôte de maintenir l’homéostasie. Les résultats démontrent aussi que les cellules T sont activement recrutées par les cellules répondantes du système inné en produisant des facteurs chimiotactiques, tels que CXCL9, CXCL10, et CCL3. Plus spécifiquement, les résultats obtenus à partir des cellules isolées T CD4+ provenant des infections ex vivo ou in vivo démontrent que ces cellules participent à la production d’IFN-γ et de TNF-α ainsi que d’IL-2, suggérant un profil d’activation Th1. Les cellules isolées T CD4+ n’étaient pas des contributeurs majeurs d’IL-10. Ceci indique que cette cytokine immuno-régulatrice est principalement produite par les cellules de l’immunité innée de la rate de souris infectées. Le profil Th1 des cellules T CD4+ a été confirmé en utilisant un modèle in vitro. Nos résultats démontrent aussi que la CPS de GBS a une role immuno-modulateur dans le développement de la réponse Th1.
En résumé, cette étude adresse pour la première fois, la contribution des cellules T CD4+ dans la production d’IFN-γ lors d’une infection à GBS et donc, dans le développement d’une réponse de type Th1. Ces résultats renforcent d’avantage le rôle central de cette cytokine pour un control efficace des infections causées par ce pathogène. / Group B Streptococcus (GBS) is an important agent of life-threatening invasive infections and remains the leading cause of neonatal sepsis to this day. Nine serotypes have been officially described based on capsular polysaccharide (CPS) composition. Among them, capsular type III is considered one of the most virulent and frequently associated with severe invasive diseases, such as meningitis. Although extensive research has been done on the interactions between GBS type III and various cells of the innate immune system, no information is available on the regulation of the adaptive immune response against this pathogen. In particular, the role of CD4+ T cells in the immuno-pathogenesis of the infection caused by GBS has never been assessed. In this study, three different models of murine infection were developed to evaluate activation and modulation of responding CD4+ T cells against GBS type III: ex vivo, in vivo, and in vitro. Ex vivo analysis of total splenocytes showed that GBS induces the release of type-1 pro-inflammatory cytokines. A strong IL-10 production follows this inflammatory cascade, indicating the host effort to maintain homeostasis. Results also indicate that T cells were actively recruited by responding innate immune cells via the release of chemotactic factors such as CXCL9, CXCL10, and CCL3. More specifically, results obtained from isolated CD4+ T cells from ex vivo or in vivo infections showed that they actively participate in the production of IFN-γ and TNF-α, as well as IL-2, suggesting a Th1 profile of activation. On the other hand, isolated CD4+ T cells were not main sources of IL-10. This observation suggests that this immuno-regulatory cytokine is produced mainly by cells of the spleen innate immune system of infected animals. The CD4+ Th1 cell profile was confirmed using an in vitro model of infection. Our results also suggest that the GBS CPS plays an immuno-modulatory role in the development of a Th1 response.
In summary, this study addresses for this first time the contribution of CD4+ T cells in IFN-γ production during GBS infection, and thus, in the development of a Th1 response. Our data further highlight the central role of this cytokine for effective control of GBS infections.
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Diffusion tensor imaging at long diffusion timeRane, Swati 30 June 2009 (has links)
Diffusion Tensor Imaging (DTI) is a well-established magnetic resonance technique
that can non-invasively interpret tissue geometry and track neural pathways by
sampling the diffusion of water molecules in the brain tissue. However, it is currently
limited to tracking large nerve fiber bundles and fails to faithfully resolve thinner
fibers. Conventional DTI studies use a diffusion time, t[subscript diff] of 30 ms - 55 ms for
diffusion measurements. This work proposes the use of DTI at long t[subscript diff] to enhance
the sensitivity of the method towards regions of low diffusion anisotropy and improve
tracking of smaller fibers. The Stimulated Echo Acquisition Mode (STEAM) sequence
was modified to allow DTI measurements at long t[subscript diff] (approximately 200 ms), while
avoiding T2 signal loss. For comparison, DTI data was acquired using STEAM at the
shorter value of t[subscript diff] and with the standard Double Spin Echo sequence with matched
signal-to-noise ratio. This approach was tested on phantoms and fixed monkey brains
and then translated to in vivo studies in rhesus macaques. Qualitative and quantitative
comparison of the techniques was based on fractional anisotropy, diffusivity,
three-phase plots and directional entropy. Tensor-field maps and probabilistic connectivity
fronts were evaluated for all three acquisitions. Comparison of the tracked
nerve pathways showed that fibers obtained at long t[subscript diff] were much longer. Further,
the optic tract was tracked in ex vivo fixed rhesus brains for cross validation. The
optic tract, traced at long t[subscript diff], conformed to the well documented anatomical description,
thus confirming the accuracy of tract tracing at long t[subscript diff]. The benefits of
DTI at long t[subscript diff] indeed help to realize the potential of tensor based tractography
towards studying neural development and diagnosing neuro-pathologies, albeit the
improvement is more significant ex vivo than in vivo.
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Immune profiling of keloid diseaseBagabir, Rania January 2013 (has links)
Keloid disease (KD) is a benign fibroproliferative dermal disease of unknown aetiopathogenesis that occurs in genetically susceptible individuals. KD shows high heterogeneity within the lesion, harbouring different immune cell profiles, which are poorly characterised in KD at different lesional sites. Although, it has long been appreciated that chronic inflammation and dermal fibrosis is associated with other fibrotic diseases (e.g. scleroderma), this link has not, yet, been established in KD through direct evidence. Additionally, the limited availability of a simple KD animal model has hindered our understanding of the underlying pathogenesis of KD. Therefore, the main objectives were a) to identify and profile different immune cells at defined KD lesional and histological sites, b) to further characterize the potential contribution of viral particles in KD by investigating the gene and protein expression profile of toll like receptors that recognise viral particles in KD, and c) to develop an optimized long-term serum-free organ culture (OC) model for KD research as a tool for probing novel hypotheses in KD pathobiology deduced from a) and b) and to also validate the reliability and instructiveness of this novel ex vivo KD model with conventional (e.g. dexamethasone) and potential future anti-KD compounds [(-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) knock-down by siRNA]. To achieve above objectives, different cellular and molecular techniques were applied. Immune profiling of KD (chapter 2) at defined lesional and histological sites generated the first comprehensive analysis of KD-associated inflammatory cell infiltrates. This work demonstrated for the first time the presence of specific type of chronic inflammation in KD that resembles the formation of tertiary lymphoid tissues (TLTs) (in 14.7%, out of 68 KD cases). Although, these TLTs are not strictly linked to defined lesional sites within the KD, they are similar in structure to mucosa-associated lymphoid tissue (MALT). Therefore, we named this phenomenon as keloid-associated lymphoid tissue (KALT). Immunophenotyping of KD lesional sites also showed a predominance of T-cells, B-cells, M2 macrophages and OX40L+ degranulated mast cells in intralesional and perilesional sites of KD compared to normal skin and normal scar tissue. In the epidermis, Langerhans cells showed no changes, whereas the intra-epidermal T-cells were significantly increased in both the intralesional and perilesional sites of KD with an increased CD4:CD8 ratio. Intra-epidermal B-cells were only rarely found in KD. Interestingly, there was no significant statistical difference between intralesional and perilesional sites of KD immunophenotyping. These abnormal immune profiles suggest the persistence of non-resolving inflammation presence towards unknown stimuli, which require further investigation. The chronic inflammation could be followed by a reparative phase in a repetitive manner leading to KD formation. Evaluation of toll-like receptor (TLR) gene and protein expression in KD showed a significant increase in the expression of intra-epidermal TLR-6, -7 and dermal TLR-8. Since these TLRs are typically up regulated during anti-viral responses, these results further support the hypothesis that certain viruses or yet unidentified ligand may play a role in KD pathogenesis (chapter 3). A successful long-term, serum-free keloid OC model was established using a 4 mm sized punch biopsy embedded in collage matrix as air liquid interface in supplemented William’s E medium for up to 6 weeks (Chapter 4).
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