Dissertations / Theses on the topic 'Immune organs'

Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies.<br>Master of Science

Dissertação(mestrado)- Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2012.<br>Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-08-29T19:19:57Z No. of bitstreams: 1 dissertao verso final-martaklosterhoff.pdf: 1924880 bytes, checksum: bc195444bdcc5176b1707b88f00b25c1 (MD5)<br>Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-09-18T03:14:20Z (GMT) No. of bitstreams: 1 dissertao verso final-martaklosterhoff.pdf: 1924880 bytes, checksum: bc195444bdcc5176b1707b88f00b25c1 (MD5)<br>Made available in DSpace on 2012-09-18T03:14:20Z (GMT). No. of bitstreams: 1 dissertao verso final-martaklosterhoff.pdf: 1924880 bytes, checksum: bc195444bdcc5176b1707b88f00b25c1 (MD5) Previous issue date: 2012<br>O bijupirá Rachycentron canadum, apresenta várias características favoráveis para a aquicultura, como alta taxa de crescimento, bons coeficientes de eficiência alimentar e carne de excelente qualidade, entre outras. No presente estudo foi realizada uma análise do sistema imune do bijupirá através de técnicas de histologia e imunohistoquímica. A ontogenia dos órgãos imunocompetentes (rim anterior, timo e baço) em larvas e juvenis de bijupirá, desde a eclosão até 53 dias após a eclosão (dae), foi realizada através da análise histológica. O rim foi o primeiro órgão linfohematopoiético a aparecer, presente no 1º dia após eclosão (dae) (3,8 ± 0,04mm), o surgimento do baço foi no 5º dae (4,8 ± 0,2mm) e no 7º dae (5,4 ± 0,2 mm) o timo; conforme análise os órgãos linfóides tornaram-se evidente com o avanço da idade do peixe. Foi possível também estabelecer a presença de receptores específicos de linfócitos através da imunomarcação com anticorpo monoclonal anti-CD3 e anti-CD4. Foi encontrado no timo os primeiros receptores linfocitários CD3 ao 7º dae com 27% mm² e 99% mm² de tecido tímico aos 53 dae (154 ± 4,6mm). A expressão fenotípica dos receptores CD3 no rim foi destacada no 8º dae (6,5 ± 0,1mm) com uma expressão de 10% mm² e 32% mm² de tecido renal aos 53 dae. A imunomarcação dos receptores linfocitários CD4 foi destacada inicialmente no timo com 7 dae, com 5% mm² e aos 53 dae com 63% mm² de linfócitos imunomarcados com anti-CD4. No Rim, a população de linfócitos T4 foi registrada primeiramente aos 13º dae (12,4 ± 0,7 mm) com 9% mm² e aos 53 dae com 28% mm² da população linfocitária CD4 do tecido renal, definindo o desenvolvimento funcional do sistema específico, associada a capacidade da memória imunológica. Também foi possível estabelecer que ocorre uma repovoação de linfócitos T no rim anterior, os linfócitos que migram do rim anterior para o timo e adquirem receptores específicos de células T, retornando ao rim anterior e mantendo suas atividades imunes. O estudo dos mecanismos do sistema imune são importantes para o sucesso de um cultivo, pois as doenças são uma das principais causas de perdas econômicas na aquicultura em todo mundo.<br>The cobia Rachycentron canadum has several desirable traits for aquaculture, most importantly a rapid growth rate, good feed conversion ratio and good flesh quality. In the present study, the immune system of cobia was evaluated through histology and immunohistochemistry. Ontogeny of immunocompetent organs (head kidney, thymus and spleen) in cobia larvae and juveniles from hatching to 53 days after hatching (dah) was histologically described. The first lymphohematopoietic organ to appear was the kidney, at 1 dah (3.8 ± 0.04 mm), followed by the spleen at 5 dah (4.8 ± 0.2 mm) and the thymus at 7 dah (5.4 ± 0.2 mm); the lymphoid organs became evident as the fish grew older. It was also possible to establish the presence of specific lymphocyte receptors through immunolabeling with the monoclonal antibodies anti-CD3 and anti-CD4. The first evidence of CD3 lymphocyte receptors was found at 7 dah with 27% mm² of thymic tissue and at 8 dah (6.5 ± 0.1 mm) in the kidney, expressed in 10% of the kidney tissue. Initially, 5% mm2 of lymphocytes with CD4 lymphocyte receptors were initially immunolabeled in the thymus. In the kidney, T4 lymphocyte population was registered to be present at 13 dah (12.4 ± 0.7 mm) with 9% mm², defining the functional development of the specific system, associated to immunological memory capacity It was also possible to establish a repopulation of T lymphocytes in the head kidney; lymphocytes migrate from the head kidney to the thymus and acquire specific T-cell receptors, returning to the head kidney and maintaining their immune activities. The knowledge about the immune system mechanisms is important for farming activities, as diseases are the major causes of economic losses in global aquaculture.

L'opinion générale est que les ulcères sont rares pendant l'enfance, les lésions provoquées par Helicobacter pylori (H. pylori) ne se produisant que des décennies après l'acquisition de l'infection. L’infection par cette bactérie est en outre moins fréquente chez les enfants dans les pays développés par rapport aux adultes. Par ailleurs, l’usage chronique de médicaments gastro-toxiques est peu fréquent dans cette tranche d’âge. Cependant, plusieurs études ont montré qu’environ 1/10 des enfants référés pour des symptômes de dyspepsie en Europe et infectés par H. pylori présentent un ulcère gastrique ou duodénal, mais aussi que la fréquence de ces lésions chez les enfants non infectés n’est pas nulle.<p>Afin de déterminer la fréquence des ulcères gastriques et duodénaux et des érosions, nous avons commencé par réaliser une étude prospective avec la participation de 19 centres répartis dans 14 pays d'Europe. Tous les enfants référés pour une endoscopie haute ont été recrutés durant une brève période de 1 mois. Parmi les 694 enfants inclus, 56 (8,1%) avaient soit des ulcères (ulcère gastrique 17/56, 30% - ulcère duodénal 7/56, 13%) soit des érosions (érosions gastriques 21/56, 37% - érosions duodénales 9/56, 16% - érosions gastriques et duodénales 2/56, 4%). Cette étude a permis de confirmer que la fréquence des lésions augmente avec l’âge, les enfants atteints de lésions étant significativement plus âgés que les témoins. En effet, les lésions ont surtout été observées chez les enfants dans la deuxième décade de vie. Une infection par H. pylori était présente seulement chez 15 des 56 enfants (27%), un médicament gastro-toxique avait été utilisé chez 13/56 (23%), une maladie inflammatoire chronique de l’intestin était présente chez 7/56 (13%) et une polyarthrite juvénile chez 2/56 (4%, plus d'un facteur de risque présent dans la plupart des cas). Aucun facteur de risque n’a pu être démontré chez 24/56 enfants (43%), une proportion beaucoup plus élevée que celle initialement attendue.<p>Nous avons ensuite réalisé une étude cas-témoins prospective et multicentrique (12 centres participants). Tous les patients avec une lésion érosive ou ulcérée de la muqueuse gastroduodénale ont été inclus avec deux témoins appariés pour l’âge, le centre et la période. Sept cent trente-deux patients (244 cas dont 153 avec seulement des érosions et 91 avec un ou des ulcères, 488 témoins) ont été inclus. Les enfants qui avaient reçu un antibiotique, un inhibiteur de la pompe à proton ou un anti-H2 durant les 4 semaines précédant l’endoscopie ont été exclus de l’analyse statistique parce que ces médicaments influencent la détermination<p>7<p>du statut H. pylori et la gravité des lésions (42 cas et 98 témoins). Nos résultats montrent que, chez les enfants, l'infection à H. pylori est un facteur de risque pour les ulcères duodénaux et les érosions duodénales, mais pas pour les lésions gastriques. Le sexe masculin, la consommation d'AINS, les maladies rénales chroniques et le tabagisme sont d'autres facteurs de risque indépendants de lésions érosives ou d’ulcères gastroduodénaux. Cependant, aucun facteur de risque identifiable n’a été retrouvé dans une grande proportion d'enfants (97/202, 48.0%) ce qui confirme les résultats de notre première étude.<p>Chez les adultes également la proportion d’ulcères sans infection à H. pylori et sans prise d’AINS est en augmentation ces dernières années tout en restant plus faible que chez l’enfant. La fréquence des ulcères gastriques et duodénaux avec un diamètre d’au moins 5 mm a été comparée, dans notre centre et dans un centre d’endoscopie adulte situé dans la même région de Bruxelles, sur une période de deux ans. Ces données montrent que les ulcères sont moins fréquents chez les enfants que chez les adultes (20/1279 enfants avec endoscopie haute - 1,6% vs adultes 58/1010 - 5,7%, OR 0,30, 95%CI 0,10-0.86, p = 0,02) et surtout moins fréquemment associés à une infection par H. pylori (8/20 vs 40/58, OR 0,26, 95%CI 0,16- 0.78, p <0,0001).<p>Comme l’activation de la réponse immunitaire locale est inefficace pour éliminer l’infection par H. pylori et serait plutôt impliquée dans la pathogenèse des lésions de la muqueuse, nous avons comparé la réponse immunitaire muqueuse des lymphocytes T et les réponses naïves chez les enfants et chez les adultes infectés par H. pylori ainsi que chez des témoins non infectés appariés pour l’âge.<p>Dans une première étude, nous avons obtenu des biopsies de la muqueuse antrale chez 43 patients dyspeptiques (12 enfants, 31 adultes). Les concentrations de cytokines libérées dans le milieu de culture et la densité de cellules CD3+, CD25+ et CD69+ ont été évaluées par cytométrie en flux. Le nombre de cellules sécrétant de l’interféron-γ (IFN-γ), de l’interleukine-4 (IL-4) et de l’IL-10 a été mesuré par ELISPOT. Les données obtenues montrent que l’augmentation de la sécrétion d'IFN-γ et l’élévation du nombre de cellules secrétant de l’IFN-γ au niveau de la muqueuse antrale lors d’une infection par H. pylori sont plus faibles chez les enfants que chez les adultes.<p>8<p>Dans une seconde étude, nous avons comparé l’infiltrat inflammatoire de la muqueuse antrale dans différents groupes d’âge (moins de 8 ans, 8 à 17 ans, 18 à 55 ans) de patients successifs infectés par H. pylori et des témoins appariés pour l’âge. Nous avons montré une corrélation entre l'âge et la densité de neutrophiles, de cellules CD3+ et de CD8+, mais pas de cellules CD20+. Le recrutement des neutrophiles dans la muqueuse antrale est plus faible chez les enfants et apparaît corrélé avec une plus faible activation du facteur de transcription NF-kB (déterminé par immunohistochimie et par EMSA) dans cette même muqueuse. L’infiltrat inflammatoire et l’activation du NF-kB sont légèrement (mais non significativement) plus intenses en cas d’infection par une souche plus virulente (facteur de virulence cagA). Ces souches cagA+ sont retrouvées en proportion équivalente dans les différents groupes d’âge. Par contre, la charge bactérienne, mesurée par un score semi-quantitatif en histologie, n’influence pas l’intensité de l’infiltrat inflammatoire.<p>En conclusion :H. pylori reste un facteur étiologique majeur pour les ulcères et les érosions duodénales chez l’enfant, mais pas pour les lésions gastriques dans les pays à faible prévalence de l'infection et la proportion de lésions associées à une infection est plus faible que chez les adultes. Aucun facteur d’exposition connu ne peut être associé aux lésions endoscopiques dans la moitié des cas, ce qui justifiera des études ultérieures pour identifier d’autres causes exogènes ou endogènes à ces lésions.<p>La réponse immunitaire de l’hôte est impliquée dans la pathogenèse des lésions gastroduodénales associées à une infection par H. pylori. Or il a été démontré dans les travaux faisant l’objet de cette thèse que cette réponse immunitaire est plus faible chez l’enfant que chez l’adulte pour certains facteurs (cytokines Th1, immunité humorale, recrutement des polynucléaires et des lymphocytes au niveau muqueux, activation du facteur de transcription NF-κB). D’autres études confirment la plus faible réponse humorale et Th1, mais également Th17 ainsi qu’une activation plus intense des Treg. Les cytokines ou les voies de signalisation responsables de cette réponse immunitaire plus faible restent inconnues, ce qui ouvre la voie à d’autres investigations.<br>Doctorat en Sciences médicales<br>info:eu-repo/semantics/nonPublished

Multiple myeloma is characterized by the malignant proliferation of clonal plasma cells producing monoclonal paraproteins, leading to multi-organ damage. On the other hand monoclonal B-cell lymphocytosis (MBCL) is characterized by the malignant proliferation of clonal B-lymphocytes, with potential to develop into chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). CLL/SLL can result in visceromegaly, anemia, thrombocytopenia, fevers, night sweats and unintentional weight loss. Literature review demonstrates these two malignant clonal bone marrow disorders are most frequently seen independently in patients; however, we report one rare diagnostic challenge where both clonal disorders were identified in a single patient concurrently. A 64-year-old man initially presented with worsening back pain. Thoracic spine x-ray revealed a T11 compression fracture, confirmed by magnetic resonance imaging. Complete blood count revealed a white blood cell count of 7.3 K/uL with 54% lymphocyte predominance and peripheral smear demonstrated a population of small lymphocytes with round nuclei and an atypical chromatin pattern suggestive of CLL/MBCL. Flow cytometry revealed a monoclonal B-cell CD5 positive, CD23 positive, CD10 negative population with an absolute count of 1.6 K/uL. Due to the instability and pain associated with the spinal fracture, patient had kyphoplasty performed and intraoperative bone biopsies were taken from both T11 and T12 vertebrae. Interestingly each bone biopsy revealed involvement by both a kappa-light chain restricted plasma cell neoplasm, ranging from 15% to 30% cellularity, as well as a CD5-positive B-cell lymphocyte population. It suggested two concurrent but pathologically distinct pathologies including plasma cell myeloma and a separate B-cell lymphoproliferative disorder with immunophenotypic features suggestive of CLL/MBCL. Bone marrow biopsy was performed for definitive evaluation and confirmed multiple myeloma with 15-20% kappa-restricted plasma cells identified, and also confirmed concurrent MBCL with CD5 and CD23-positive, kappa-restricted B-cells identified on bone marrow flow cytometry. Adding an additional layer of complexity, bone marrow molecular genetics revealed presence of a MYD88 mutation, raising concern for possible lymphoplasmacytic lymphoma (LPL). However, secondary pathologic review ruled out LPL, as the immunophenotypic pattern of the clonal B-cells was not consistent with that of LPL, and although the MYD88 mutation is predominantly seen in LPL, it has also been seen in a small percentage of CLL/SLL cases and exceedingly rarely described in MM as well. Serum protein electrophoresis with immunofixation, serum quantitative immunoglobulins and serum quantitative free light chain assay revealed findings consistent with IgG kappa multiple myeloma and systemic CT imaging was negative for any lymphadenopathy, confirming MBCL. Patient was started on first-line multiple myeloma systemic therapy for transplant eligible patients and has demonstrated an excellent response to treatment thus far. This patient case serves to demonstrate the importance of maintaining a broad differential when approaching hematological problems; It also underlines the necessity for a complete diagnostic evaluation to identify rare clinical conundrums such as with our patient, allowing for proper and timely treatment. While we use “Occam’s razor” to explain multiple problems with a single unifying diagnosis the rare possibility of divergent diagnosis is to be always entertained.

Crohn’s disease [CD] is a chronic inflammatory disease of the intestinal tract with considerable heterogeneity among affected patients in terms of disease phenotype and therapeutic responses. Despite the increase in the number of drugs approved for the management of CD, a significant percentage of patients remain unresponsive or lose response over time to treatments, and eventually require surgery to control disease activity and/or complications. Nonetheless, in a fraction of these refractory patients, intestinal resection may not be possible due to disease location, extension or previous surgeries. For such patients, autologous hematopoietic stem cell transplantation [HSCT] represents a potential salvage therapy despite the risks associated with this procedure. Stem cell transplantation is an accepted therapy for hematological disorders, aplastic anemia and immunodeficiencies. In the context of autoimmune diseases, the serendipitous benefits of transplantation were initially reported in patients suffering from both immune-mediated diseases and hematological disorders. This led to trials that have shown the efficacy of autologous HSCT in treating an array of autoimmune diseases including refractory severe multiple sclerosis, systemic lupus erythematosus, juvenile idiopathic arthritis, rheumatoid arthritis and, more recently, CD. The benefit of HSCT in autoimmunity is thought to originate from the ability of intense immune depletion to eliminate auto-reactive cells regardless of their specificity. This would lead to de novo generation of immune cells that could re-establish tolerance, although no objective evidence of this ‘resetting’ has been reported thus far. To explore this hypothesis, we group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumor necrosis factor [TNF]-α were included for comparison. Severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T- cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content. HSCT dramatically renewed the pre-existing T-cell receptor [TCR] repertoire; however, persistent high-frequency TCR clones were detected in all patients in both blood and biopsy throughout the first year of follow-up. The number of persistent resident T cell clonotypes in tissue after treatment decreased in patients that achieve endoscopic remission. Furthermore, we found that low TCR peripheral diversity at baseline, and up to 1 year after HSCT, were associated with a lack of response to the immunoablative protocol. Peripheral blood immune remodeling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα.<br>Se ha demostrado la eficacia del trasplante hematopoyético de células madre (THCM) en pacientes con enfermedad de Crohn severamente refractaria. La hipótesis aceptada es que el proceso de THCM elimina células auto-reactivas, devolviendo así la tolerancia. No obstante, no existen estudios específicos y exhaustivos sobre la reconstitución inmune en pacientes con enfermedad de Crohn tratados con THCM. En esta tesis, monitorizamos una cohorte de 18 pacientes con enfermedad de Crohn refractaria que recibieron trasplante autólogo de células madre hematopoyéticas, de los cuales el 50% consiguieron la remisión endoscópica sin tratamiento de mantenimiento. Para elucidar los mecanismos que determinan la eficacia, monitorizamos los cambios después de THCM en la composición de células inmunes tanto en sangre como en tejido intestinal. Como resultado, observamos que la ablación inmune producida durante el TCMH induce cambios dramáticos en las células T y B en sangre en todos los pacientes, independientemente de la eficacia del tratamiento. La remisión endoscópica en la semana 52 posterior al TCMH se asoció con cambios transcripcionales intestinales significativos. Una comparación de la transcripción en remisión con la de la remisión inducida por fármaco identificó genes comunes y únicos en la respuesta inducida por el TCMH. A través de un análisis de deconvolución de los datos del transcriptoma de la biopsia intestinal, mostramos que la respuesta al TCMH, pero no a fármaco inmuno- modulador, se asocia con una expansión de las células B naïve y una disminución en el contenido de células T memoria. Como se esperaba, la remisión endoscópica, en respuesta a tanto a TCMH como a fármaco, condujo a una reducción significativa en el contenido de neutrófilos intestinales y macrófagos M1 La remodelación inmune de la sangre periférica después del TCMH no predice la eficacia. No obstante, la depleción de las células T intestinales mantenida un año después del trasplante se asocia con la curación mucosa después del TCMH.

Le rôle des neutrophiles dans la réponse immunitaire innée est bien connu. Ils résident dans le sang et ont une durée de vie limitée à quelques heures. Suite à une infection, ils quittent le flux sanguin et se dirigent vers les sites inflammatoires en réponse à des chimiokines produites par des cellules endothéliales et des fibroblastes. Au niveau du site d’inflammation, les neutrophiles phagocytent et éliminent les pathogènes extracellulaires, et produisent des cytokines inflammatoires. <p><p>Des travaux récents montrent que les neutrophiles peuvent également jouer un rôle dans l’immunité adaptative. En effet, ils ont la capacité de transporter des antigènes vers les ganglions lymphatiques, d’induire la différenciation des lymphocytes et d’influencer la réponse immune adaptative par la production de cytokines. <p><p>La fonction des neutrophiles dans l’induction et/ou la régulation de la réponse adaptative requiert l’interaction entre ceux-ci et d’autres populations cellulaires, telles que les cellules dendritiques et les lymphocytes. <p>Nous avons donc examiné la localisation des neutrophiles au niveau de la rate dans des conditions basales ou inflammatoires. D’une manière générale, nos résultats montrent que, en cas d’infection, les neutrophiles migrent vers la pulpe blanche de la rate et se localisent en contact étroit avec les lymphocytes T. Ce phénomène de migration est dépendant des molécules CD14 et MyD88 et corrèle avec l’augmentation de l’expression des chimiokines CXCL1 et 2, ainsi qu’avec la diminution de l’expression du récepteur CXCR2 à la surface des neutrophiles. <p>Cependant, au niveau de la cavité péritonéale, le recrutement des neutrophiles est augmenté en absence de la molécule CD14. Nos résultats montrent que la migration des neutrophiles, dans les organes lymphoïdes et non lymphoïdes, est dirigée par des mécanismes différents. <p><br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished

Kidney transplantation is the only therapy for patients with end-stage kidney failure. There is a shortage of suitable organs from living donors and heart-beating donors (HBDs) leading to an increased use of marginal organs, including those from non- heart-beating donors (NHBD) to expand the donor pool. However, such kidneys are exposed to periods of warm and cold ischaemia plus reperfusion (IR) injury which can be associated with acute tubular necrosis leading to primary non function (PNF), delayed graft failure (DGF) with an increased risk of acute and chronic allograft rejection. Therefore, minimising ischaemic injury will be beneficial to improve immediate allograft function and reduce the incidence of PNF and DGF. Perfusate solutions can ameliorate the damage caused by IR injury. This study examined the possible protective effect of heparinoids, low molecular weight (LMW) sodium heparin (HS-3400) and sodium pentosan polysulfate (NaP PS), as supplements in perfusate solutions. An in vivo rodent surgical model of NHB kidney donation was used to investigate the effects of heparinoids as immune modulators during kidney preservation. It was demonstrated that heparinoids significantly reduced oedema but did not have any effects on pressure, intra-renal vascular resistance (I RVR) or osmolarity of the perfusate solution. Microarray and qPCR analysis revealed that many genes involved in IR injury were differentially expressed after warm oxygenated perfusion. Network analysis revealed that the pro-inflammatory cytokine TNF-a was the common denominator and a potential key regulator of IR injury. Data obtained strongly demonstrate that these networks were removed by the addition of heparinoids. In vitro biological assays investigated the effects of heparinoids on class 11 MHC antigen expression on primary endothelial cells (ECs) treated with IFN-y. It was found that NaPPS and the majority of its derivatives were more efficient in antagonising IFN-y activity. In addition, NaPPS and its derivatives significantly inhibited transendothelial migration of leukocytes across an IFN-y-activated endothelial monolayer. The anti-inflammatory properties of heparinoids can be exploited for better preservation of NHBD organs to improve short and long-term allograft survival and function. Significantly, this is the first study to reveal the potential of heparinoids as TNF-a antagonists and additives to perfusate solutions.

Transplantation is the treatment of choice for end stage organ failure however current immunosuppressive therapies whilst effective at preventing acute allograft rejection, fail to prevent late graft loss due to chronic rejection and are associated with an increased risk of infection and malignancy. Therefore there is a clear unmet clinical need for improved strategies to prevent allograft rejection. OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40L pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. Therefore the aim of this thesis was to determine the impact of OX40 blockade on conventional and regulatory T cell responses to allografts. We found that activation of CD4+ and CD8+ naïve and memory T cells resulted in the induction of OX40 expression and that blockade of OX40-OX40L interactions partially inhibited the response of alloreactive T cells in vitro and prevented skin allograft rejection but did not result in the induction of tolerance. OX40 blockade was found to have no effect on the activation and proliferation of T cells but rather effector T cells failed to accumulate and migrate to skin allografts. This was shown to be the result of an enhanced degree of cell death amongst proliferating effector cells. In addition, blockade of OX40-OX40L interactions at a time of exposure to alloantigen resulted in a pool of Treg with an enhanced ability to suppress T cell responses to alloantigen in vitro and in vivo. Counter-intuitively, OX40 blockade was found to increase the potency of alloreactive Treg by promoting survival following re-activation. Finally, although OX40 blockade impacted both conventional and regulatory T cell responses, anti-OX40 administration did not promote skin or heart allograft survival in immunocompetent recipients and failed to synergise with blockade of other costimulatory molecules to prevent allograft rejection. In conclusion, these data demonstrate that blockade of OX40-OX40L interactions can attenuate naïve and memory T cell responses to alloantigen whilst promoting the survival of alloreactive Treg. Therefore, we propose that anti-OX40 would be a worthwhile adjunct to pre-existing strategies to induce tolerance.

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).

Persistent organic pollutants (POPs), bisphenol A (BPA) and pthalates may increase the risk of respiratory infections and allergic diseases in infants and the effects might last until, at least, adolescence. Cytokines and biomarkers of inflammation can provide information of the mechanisms behind such associations. Data from the “Infancia y Medioambiente” (INMA) population-based birth cohort project and from six other existing European birth cohort studies have been used in the present thesis, which also includes a systematic review. Results of the present work, which includes five scientific publications, suggest that prenatal exposure to POPs affects the immune and respiratory health of children, that the effects are observed even at low levels of exposure and that these may last until adolescence. Biological mechanisms behind such effects were not possible to describe in the present thesis, however, it provided information of a potential biomarker (interleukin 10) of chronic immunotoxic effects of POPs. Results also indicate potential effects of prenatal exposure to BPA and phthalates on the development and functioning of the immune and respiratory systems of infants and children. In the present thesis we highlight the limitations of existing studies in the field and provide recommendations for future research.<br>Els compostos orgànics persistents (COPs), el bisfenol A (BPA) i els ftalats podrien estar relacionats amb un increment del risc de patir infeccions respiratòries i símptomes relacionats amb l’al•lèrgia en infants i fins, com a mínim, l’adolescència. Les citoquines i els marcadors d’inflamació poden aportar informació dels mecanismes que hi ha darrera d’aquestes associacions. En la present tesis s’han utilitzat dades de la cohort de naixement “Infancia y Medioambiente” (INMA) i de sis cohorts de naixement Europees. La tesis també inclou una revisió sistemàtica. Els resultats d’aquest treball, que inclou cinc publicacions científiques, suggereixen que l’exposició prenatal a COPs afecta els sistemes immunitari i respiratori dels infants i nens, que aquests efectes es donen inclús a exposicions relativament baixes i que aquests efectes poden perdurar fins a l’adolescència. Els mecanismes biològics que podrien explicar els efectes observats no s’han pogut descriure en el present treball, tot i així, hem aportat informació d’un possible biomarcador (interleuquina 10) dels efectes immunotòxics crònics dels COPs. Els resultats també mostren efectes potencials de l’exposició prenatal a BPA i ftalats sobre el desenvolupament i funcionament dels sistemes immunitari i respiratori dels infants i nens. A la present tesis es remarquen les principals limitacions dels estudis existents en aquest camp i es proposen recomanacions de millora per a futurs estudis. Mentrestant, es recomana revisar la legislació actual per tal de reduir l'ús d'aquells compostos que encara estan al mercat i que s'utilitzen àmpliament.

no<br>There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

У дисертаційній роботі вирішене важливе завдання – покращення диференційної діагностики запальних процесів органів черевної порожнини, шляхом виявлення імунних механізмів патогенезу гострого і хронічного запалення. Було обстежено 226 хворих на гострий неспецифічний запальний процес органів черевної порожнини (гострий апендицит, гострий калькульозний холецистит, гострий мезаденіт) і 60 осіб хворих на абдомінальний туберкульоз, який представляє специфічне хронічне запалення. Встановлено, що гостре запалення супроводжується нейтрофільним типом гемограми, активацією гуморальної та кілерної ланок імунітету на тлі Т-клітинного імунодефіциту. Встановлено підвищення експресії мембранних активаційних антигенів CD25, CD95, CD23 на лімфоцитах крові. При гострлому запаленні, яке перебігає без ускладнень, переважають процеси активації апоптозу над проліферацією, що підтверджено зниженням співвідношення CD25/CD95. Ускладнені форми запалення (гнійно-септичним, деструктивним процесом) супроводжується більш вираженим зниженням показника CD25/CD95 порівняно із неускладненими, Встановлена значна активація макрофагально-моноцитарної системи, що проявляється зростанням рівнів IL-1β, IL-6, IL-8, TNF-α. Ключова роль у патогенезі гангренозного запалення встановлена для TNF-α. Хронічне запалення (туберкульозне) визначається лімфоцитарним типом гемограми, Т-клітинним імунодефіцитом, активацією гуморальної і кілерної ланок імунітету, проявами гіперчутливості I, IV типів, помірно підвищеною експресією активаційних антигенів на лімфоцитах, підвищеним рівнем IL-1β, IL-6, IL-8, TNF-α, IL-2, IL-4, IL-10. Встановлено підвищення вмісту гуморальних імунних факторів та доведена інформативність індексу активності запалення, як інтегрального показника, що характеризує неспецифічну гуморальну імунну відповідь; зниження фагоцитарної активності нейтрофілів з одночасним посиленням кисневого метаболізму при усіх досліджуваних станах; активацію NO-синтази за рахунок індуцибельної ізоформи та збільшення рівня стабільних метаболітів оксиду азоту найбільш виражену при деструктивних формах гострого запалення; вмісту у крові хворих маркерів ендогенної метаблічної та імунної інтоксикації; доказано інформативність застосування індексу імуно-метаболічної інтоксикації для оцінки характеру інтоксикації. При хронічному запаленні органів черевної порожнини кількість сильних кореляційних зв'язків є більшою порівнянно з гострим запальним процесом. Допомогою факторного аналізу виділено "Фактори", які об’єднують показники клітинного імунітету та цитокіни, що дозволяє диференційовано охарактеризувати механізми імунної відповіді за умов гострого та хронічного запалення органів черевної порожнини.<br>В диссертационной работе решена важная задача - улучшение дифференциальной диагностики воспалительных процессов органов брюшной полости, путем выявления иммунных механизмов патогенеза острого и хронического воспаления. Было обследовано 226 больных острым неспецифический воспалительный процесс органов брюшной полости (острый аппендицит, острый калькулезный холецистит, острый мезаденит) и 60 человек больных абдоминальный туберкулез, представляющий специфическое хроническое воспаление. Установлено, что острое воспаление сопровождается нейтрофильным типу гемограммы, активацией гуморального и киллерной звеньев иммунитета на фоне Т-клеточного иммунодефицита. Установлено повышение экспрессии мембранных активационных антигенов CD25, CD95, CD23 на лимфоцитах крови. При остром воспалении, которое протекает без осложнений, преобладают процессы активации апоптоза над пролиферацией, что подтверждено снижением соотношения CD25 / CD95. Осложненные формы воспаления (гнойно-септическим, деструктивным процессом) сопровождается более выраженным снижением показателя CD25 / CD95 по сравнению с неосложненными, выявлена значительная активация макрофагально-моноцитарной системы, проявляется увеличением уровней IL-1β, IL-6, IL-8, TNF-α . Ключевая роль в патогенезе гангренозного воспаления установлена для TNF-α. Хроническое воспаление (туберкулезное) определяется лимфоцитарним типом гемограммы, Т-клеточным иммунодефицитом, активацией гуморального и киллерной звеньев иммунитета, проявлениями гиперчувствительности I, IV типов, умеренно повышенной экспрессией активационных антигенов на лимфоцитах, повышенным уровнем IL-1β, IL-6, IL-8 , TNF-α, IL-2, IL-4, IL-10. Установлено повышение содержания гуморальных иммунных факторов и доказана информативность индекса активности воспаления, как интегрального показателя, характеризующего неспецифическую гуморальный иммунный ответ; снижение фагоцитарной активности нейтрофилов с усилением кислородного метаболизма при всех исследуемых состояниях; активацию NO-синтазы за счет индуцибельной изоформы и увеличение уровня стабильных метаболитов оксида азота наиболее выраженную при деструктивных формах острого воспаления; содержания в крови больных маркеров эндогенной метабличнои и иммунной интоксикации; доказано информативность применения индекса иммуно-метаболической интоксикации для оценки характера интоксикации. При хроническом воспалении органов брюшной полости количество сильных корреляционных связей больше по сравнению с острым воспалением. С помощью факторного анализа выделены "Факторы", которые объединяют показатели клеточного иммунитета и цитокины, позволяет дифференцированно охарактеризовать механизмы иммунного ответа в условиях острого и хронического воспаления органов брюшной полости.<br>The dissertation is devoted to the study of immune mechanisms in the pathogenesis of various forms of inflammation of the organs of the abdominal cavity. 226 people were examined with acute non-specific inflammatory process of the organs of the abdominal cavity (acute appendicitis, acute calculous cholecystitis, acute mesadenitis) and 60 persons with abdominal tuberculosis, which represents a specific chronic inflammation. The study was conducted to identify specific signs of various forms of inflammation of the organs of the abdominal cavity for further implementation in the diagnostic and therapeutic process. It has been established that the development of acute inflammation in the abdominal cavity is determined by profound changes in the immune system. This is evidenced by the neutrophilic type of hemogram, the activation of the humoral and killer immune system against the background of the T-cell immunodeficiency state. Increased expression of membrane activation antigens of CD25, CD95, CD23 on blood lymphocytes was revealed and differences in their correlation with different inflammation forms were revealed. It has been proved that in acute inflammatory processes that run smoothly, processes of activation of apoptosis over proliferation prevail, which is confirmed by a decrease in the ratio of CD25/CD95. Complicated forms of inflammation (purulent-septic, destructive process) are accompanied by a more pronounced decrease in the CD25/CD95 compared to the uncomplicated ones. Significant activation of the macrophage monocytic system is established, which is manifested by the growth of levels of IL-1β, IL-6, IL-8, TNF-α. A key role in the pathogenesis of gangrenous inflammation is established for TNF-α. An increase in the content of humoral immune factors has been established and informativeness of the index of inflammatory activity as an integral indicator characterizing a non-specific humoral immune response has been proved. For the first time, it has been shown that the development of chronic inflammatory process in the abdominal cavity is determined by various changes in the immune system, such as lymphocytic hemogram type, T cell immunodeficiency, activation of humoral and killer immunity, manifestations of hypersensitivity I, IV types, expression of activation antigens on lymphocytes is moderate. Moderate activation of the macrophage monocyte system: elevated levels of IL-1β, IL-6, IL-8, TNF-α, IL-2, IL-4, IL-10. It was first proved that the profile of secreted by the macrophage monocytic system of cytokines in the examined groups of patients determines the form of the inflammatory process and its course, which allows differentiating acute and chronic inflammation. An increase in the content of humoral immune factors was established, and the informativeness of the inflammatory activity index was proved as an integral indicator characterizing a non-specific humoral immune response. A decrease in the phagocytic activity of neutrophils, together with an increase in oxygen metabolism in all the conditions studied, indicates a pro-inflammatory polarization of neutrophils. The activation of NO synthase due to the inducible isoform and the increase in the level of stable metabolites of nitric oxide, which is most pronounced in destructive forms of acute inflammation of the abdominal cavity organs, is a consequence of the activation of pro-inflammatory cytokines. An increase in the blood content markers of endogenous metabolic (medium-weight molecules, bilirubin) and immune (circulating immune complexes) intoxication was established, which does not depend on etiological factors, but depends on the severity of the pathological process and the informativeness of the use of the immuno-metabolic intoxication index for character assessment intoxication. Analyzing the correlations found between the levels of interleukins, the parameters of cellular and humoral immunity, we noted that during the chronic inflammatory process of the abdominal organs, the number of strong correlation bonds is increased, compared with the acute inflammation process. The "Factors" that combine the indicators of cellular immunity and cytokines are distinguished, which makes it possible to differentially characterize the mechanisms of the immune response in conditions of acute and chronic inflammation of the abdominal organs. In the dissertation the important task is solved - improvement of differential diagnostics of inflammatory processes of abdominal organs, by revealing pathogenetic mechanisms of development of acute and chronic inflammation.

Le facteur de transcription Ikaros est un régulateur essentiel de la lymphopoïèse. Ikaros est nécessaire à la différenciation des lymphocytes B et joue aussi un rôle important dans la suppression des LAL-T. Contrairement aux souris mutantes nulles pour Ikaros, les souris mutantes hypomorphes IkL/L développent des lymphocytes B matures après la naissance. Avec l'âge, toutes les souris IkL/L développent des leucémies T Notch dépendantes avec des mutations similaires à celles trouvées chez les patients atteints de LAL-T. La souris IkL/L est donc un excellent modèle pour étudier l'activation des cellules B matures et la pathogenèsedes LAL-T. Nous avons montré que la délétion spécifique du promoteur et de l'exon 1 de Notch1 dans les cellules T conduit à l'activation de promoteurs cryptiques dans la région 3' du gène, qui génèrent des transcrits codant pour des protéines Notch1 constitutivement actives qui accélèrent la leucémogenèse dans la souris IkL/L. De plus, nous mettrons en évidence l'existence de cellules initiatrices de leucémie dans les tumeurs IkL/L puisque nous avons trouvé que des cellules ayant la capacité de s'auto-renouveler représentent 1 sur 500. Enfin, nous avons montré que les cellules B IkL/L ont une activation excessive d'ERK et dep38 après la stimulation du BCR, ce qui résulte en une hyper-prolifération et une production d'autoanticorps liés au lupus systémique érythémateux. Nos résultats suggèrent qu'Ikaros est un régulateur négatif de l'activation des lymphocytes B.

Le système immunitaire nécessite le recrutement et la différenciation coordonnée de nombreux types cellulaires. Or, les facteurs transcriptionnels déterminent en grande partie le phénotype et le degré de différenciation des cellules. Parmi ces facteurs transcriptionnels, la famille Rel/NF-kappa B est sans doute importante. Les protéines Rel/NF-kappa constituent une famille de 5 facteurs transcriptionnels ubiquitaires, p50, p52, p65, c-Rel et RelB. Ces protéines sont caractérisées par l'existence d'un domaine d'homologie commun appelé domaine Rel, et sont fonctionnelles à l'état d'homo ou d'hétérodimères, formant les complexes Rel/NF-kappa B. Ces complexes participent à la régulation positive de la transcription des gènes ayant un site spécifique, appelé site kappa B, dans leur région régulatrice. Les sites kappa B sont retrouvés au sein des régions régulatrices de nombreux gènes de la réponse immune. Les complexes Rel/NF-kappa B contenant les protéines c-Rel ou p65 sont retenus dans le cytoplasme par interaction avec des inhibiteurs spécifiques, les protéines I-kappa B. L'activation NF-kappa B consiste en la dissociation de ces complexes d'avec ses inhibiteurs I-kappa B suivi de leur translocation dans le noyau, où leur activité transcriptionnelle s'exerce. La diversité de signaux activateurs et le nombre de gènes cibles potentiels posent la question du rôle effectif des protéines Rel/NF-kappa B dans la réponse immune in vivo. In vitro, l'activation NF-kappa B peut être obtenue dans tous les tous les types cellulaires, quelle que soit l'origine embryologique, par des agents de nature très variée. Ainsi, il est très difficile d'extrapoler les résultats expérimentaux obtenus in vitro pour déduire les rôles réels des protéines Rel/NF-kappa B in vivo dans le système immunitaire. C'est pourquoi, afin d'aborder ce problème, nous avons analysé l'expression des protéines Rel/NF-kappa B in situ dans le thymus et les organes lymphoïdes secondaires. En effet il existe une relation étroite entre la différenciation cellulaire et la microanatomie dans ces organes lymphoïdes. De plus, il est possible de reconnaître individuellement les cellules dans ces tissus, soit morphologiquement, soit à l'aide d'immunomarquages. Enfin, nous avons analysé l'activité NF-kappa B in situ dans les organes lymphoïdes de souris transgéniques ayant comme transgène le gène de la (3galactosidase sous contrôle des sites kappa B. Nos résultats montre que, i) la localisation nucléaire de p50, p52 et RelB n'est constatée que pour les cellules accessoires de l'immunité avec p50 pour les cellules épithéliales du thymus, p52 et RelB pour les cellules accessoires de l'immunité, p50, p52 et RelB pour les cellules présentatrices de l'antigène, suggérant un rôle majeur de ces protéines dans cette catégorie de cellules, ii) Dans le thymus, c-Rel est absent des thymocytes corticaux, mais présent dans les thymocytes médullaires et, dans les organes lymphoïdes secondaires, c-Rel prédomine dans les lymphocytes B vierges, suggérant que c-Rel joue un rôle dans la différenciation thymocytaire et dans la différenciation lymphocytaire B et, iii) p65 est retrouvée de façon dispersée dans les organes lymphoïdes secondaires et dans le thymus; cependant, dans le thymus, p65 prédomine dans les thymocytes médullaires. Ces résultats suggèrent donc que, dans le système immunitaire, la localisation nucléaire des protéines Rel/NF-kappa B est associée à l'accomplissement d'un programme génétique particulier en rapport avec la fonction et ou la différenciation de la cellule. La nature exacte du(des) complexe(s) déterminerait(aient) alors l'ensemble des gènes ciblés pour la réalisation de ce programme génétique.

2005/2006<br>Tissue loss or end-stage organ failure caused by injury or other types of damage is one of the most devastating and costly problems in human health care. Although surgical strategies have been developed to deal with these problems, and significant advances have been achieved in organ transplantation, it is extremely limited by a critical donor shortage and the necessity of lifelong immunosuppression and its serious complications. In the USA, more than 6,000 people die each year as a result of shortage of donor organs. Tissue engineering [TE] is seen by many as the only way to address this shortage. TE is an interdisciplinary field that draws from materials science, cell biology, biotechnology and chemistry, and strives to offer a new solution to tissue loss or organ failure through the use of synthetic, hybrid, or natural materials that have been designed and fabricated into a 3-dimensional scaffolds that provide support and allow cell attachment, proliferation, differentiation and function. However, Skin and cartilage are the only two tissues grown under laboratory conditions that have achieved successful clinical application. The main reason for this is that cartilage doesn’t require blood vessels or nerves and skin is sustained by nutrients that diffuse through the thickness of the cells that make up the graft. Attempts to grow more biologically challenging tissue and organs have been had mixed results. The obstacles and challenges that have to be overcome include: 1. Graft loss/failure due to at the cyto-incompatibility at the graft-biomaterial interface and bio-incompatibility host-biomaterial interface. 2. Inadequate neovascularization and nutrient channels to support cell survival deep in the interior of the scaffolds. 3. Immuno-rejection of allogenic graft. 4. Lack of healthy easily accessible cells for use in tissue engineering To this end we have developed “smart” biomaterials with nano-scale architecture to elicit desirable cell response (cytocompatibility) at the cell-biomaterial interface and desirable host response (biocompatibility) at the host biomaterial interface. We then designed and microfabricated an original scaffold that incorporated the “smart” architecture and microfluidic network to permit the flow of nutrient-rich media deep in the interior. The scaffold consists of two microporous hemi-membranes that are superimposed and aligned in such a way that the micropores are laterally offset. Sandwiched between these hemi-membranes is a microfluidic channel network that runs perpendicular to the micropore axis and permits interconnectivity between the laterally offset micropores. By decreasing the size of the channels from the micro- to the nano- scale the scaffold acquires a another “smart” characteristic as a immuno-isolating membrane. To examine the scaffold’s potential for tissue regeneration, muscle myoblast cells (mouse C2C12 cell-line), neuroblastomas (mouse PC12 cell-line) and embryonic stem cells (mouse TBV-2 cell-line) were seeded and cultured on the scaffolds. Biocompatibility was evaluated by subcutaneous implantation of the scaffold in mice. Results show that myoblast and neuroblastomas attached, proliferated and differentiated. The exponential cell proliferation associated with in vitro embryonic stem cell culture was controlled. In vivo studies demonstrated scaffold-host integration as evidenced by vascular colonisation of the scaffold.. By developing the ability to construct and control the following scaffold parameters; microporous architecture; microfluidic interconnectivity and canal size; the external and internal shape of the scaffold and it’s multi-scaled surface architectures, the “smart” scaffold developed in our laboratories have great potential as an ideal scaffold for tissue engineering.<br>Nella cura del benessere degli uomini, tra i problemi di maggior impatto e costo si trovano la perdita di tessuti e di funzionalità degli organi, causati da ferite o incidenti di altro genere. Sebbene siano state sviluppate efficaci strategie chirurgiche per ovviare a tali problemi, e nonostante i significativi avanzamenti tecnici raggiunti nel campo del trapianto degli organi, il ricorso a tale soluzione è fortemente limitato dal basso numero di donatori di organi e dalla necessità di durature terapie immuno-soppressive, con il loro portato di serie complicazioni. Basti pensare che a causa della mancanza di un adeguato numero di donatori, solo negli USA ogni anno muoiono più di 6000 persone. Secondo il parere di molti, la strada più promettente per affrontare questo problema è la Ingegneria dei Tessuti Biologici (ovvero Tissue Engineering [TE], dall’acronimo dell’equivalente inglese). Si tratta di un approccio interdisciplinare, che combinando conoscenze e tecnologie dai campi della Scienza dei Materiali, Biologia Cellulare, Biotecnologia e Chimica offre la possibilità, in principio, di trovare soluzioni innovative per la sostituzione di tessuti o organi non più funzionali; la strada maestra in questo campo è la progettazione e costruzione di strutture tri-dimensionali (scaffold) capaci di provvedere al supporto funzionale di cellule cresciute in vitro, integrando materiali sintetici, naturali e ibridi. Tuttavia, al momento soltanto pelle e cartilagini sono state cresciute in condizioni controllate da laboratorio e hanno raggiunto lo stadio della applicazione clinica. Il motivo fondamentale per tale successo, e per la limitazione a questi due soli casi, è che la cartilagine non necessita di vascolarizzazione o innervazione per sostentarsi, e la pelle ottiene i suoi nutrienti per mezzo della diffusione attraverso gli strati di cellule che la sostengono e ne garantiscono l’attecchimento. Tentativi per crescere altri tipi di tessuti e organi hanno incontrato gravi difficoltà; 1. perdita di adesione a causa della cito-incompatibilità all’interfaccia di connessione e della bio-incompatibilità tra i materiali dell’impianto e il corpo ospitante 2. scarsa o inadeguata vascolarizzazione dell’impianto, tale per cui le cellule all’interno della struttura 3-D non vengono raggiunte dai nutrienti necessari per una sopravvivenza a lungo termine 3. rigetto immunologico del tessuto trapianto 4. mancanza di facile accesso alle cellule necessarie per la costruzione degli impianti Con lo scopo di rispondere a queste richieste, abbiamo sviluppato bio-materiali “smart” contraddistinti da una architettura a livello nano-metrico, capaci di stimolare la desiderata risposta cellulare (compatibilità citologica) all’interfaccia con il bio-materiale stesso, e capace di promuovere la bio-compatibilità con il corpo ospitante. Usando le tecniche della micro-fabbricazione, abbiamo quindi progettato e realizzato una struttura 3-D originale (lo scaffold) che incorpora tale architettura “smart” e il sistema micro-fluidico che garantisce l’apporto del flusso dei nutrienti al suo interno. Tale struttura consiste da membrane sovrapposte ed allineate in modo che lateralmente si aprano dei pori di comunicazione con dimensioni micrometriche. Compreso tra queste membrane si trova il sistema microfluidico, capace di garantire l’interconnessione trai pori con un sistema di canali che scorre perpendicolarmente all’asse dei pori stessi. Una ulteriore caratteristica di questo sistema è che riducendo la scala dei canali microfluidici al livello di scala nano-metrica, lo “scaffold” acquisisce proprietà immuno-isolanti. Per lo studio della sue potenzialità nel campo della rigenerazione tissutale, abbiamo cresciuto nello “scaffold” mioblasti muscolari (linea cellulare C2C12 del topo), neuroblastomi (linea cellulare PC12 del topo) e cellule staminali embrionali (TBV-2 del topo). La biocompatibilità è stata valutata impiantando lo “scaffold” in topi da laboratorio. I risultati mostrano come i mioblastomi e i neuroblastomi aderiscono allo “scaffold”, proliferano e si differenziano. Abbiamo controllato la proliferazione esponenziale associata con la cultura in vitro delle cellule staminali embrionali e lo studio delle condizioni in vivo dimostra la integrazione dello “scaffold” nel corpo dell’ospite, come risultato della riuscita vascolarizzazione della sua struttura. Con il controllo della architettura micro-porosa; interconnettività microfluidica e dimensione dei canali; della geometria esterna ed interna e della sua struttura e conformazione superficiale a scala nano-metrica, lo “scaffold” risultante, sviluppato nei nostri laboratori, mostra enormi potenzialità come struttura ideale per la Ingegneria dei Tessuti Biologici.<br>XIX Ciclo<br>1967

La recherche clinique et bibliographique présente s’articule sur trois points et constitue l’entreprise tripartite de cette thèse doctorale. Le premier concerne l’étude proche de la théorie freudienne et de l'enseignement lacanien et du concept de phénomène psychosomatique, afin d’aborder la maladie organique auto-immune et idiopathique, la Sclérose par Plaques et la maladie de Crohn, sur deux sujets souffrants au sein d'une cure analytique. Le deuxième concerne l’investigation des objets de construction dans leur discours et de leur place occasionnée, et le troisième l’investigation de savoir s’il y a eu pause du symptôme et ratage de l’induction signifiante, c’est-à-dire échec de la métaphore subjective, ainsi que de savoir si la maladie des sujets souffrants remplit les critères du phénomène psychosomatique et si la métaphore subjective est prise en charge par l’organisme. L’analyse qualitative de l’énoncé et de l’énonciation des sujets à travers les séances se réalise avec une analyse de discours structurelle critique et une analyse de leur parole durant une période de cure dépassant les quatre ans, en faisant une comparaison avec des recherches antérieures. Nous en concluons que la maladie remplit les critères du phénomène psychosomatique et que la prise en charge de la métaphore subjective a fonctionné, confirmant ainsi nos hypothèses. Nous aboutissons également à des conclusions qui sont principalement en accord avec des recherches préalables. Enfin, nous notons l'amélioration de l'état de la santé des sujets et la stabilisation ou la disparition des récidives durant leur analyse, en parallèle avec leur traitement médical, ce qui n’avait pas été le cas auparavant, et l'appropriation ses points de la douleur et la souffrance de leur histoire de façon qu'il ne conduise pas à la voie des pathologiques pulsionnelles actes et à la décharge par l’organisme<br>The present clinical and bibliographical research is articulated on three points and constitutes the tripartite undertaking of this doctoral thesis. The first relates to the study closely the Freudian theory and the Lacanian teaching and the concept of phenomenon psychosomatic, in order to approach the auto-immune and idiopathic organic disease, the Multiple Sclerosis and the disease of Crohn, on two suffering subjects within an analytical cure. The second relates to the investigation of the objects of construction in their discourses and their caused positions, and the third the investigation of knowing if exists pause of symptom and failure of signifying induction, i. E. Failure of the subjective metaphor, as to know if the disease of the suffering subjects fills the criteria of the phenomenon psychosomatic and if is held assumption of subjective metaphor by the organism. The qualitative analysis of the enunciate and the enunciation of the subjects through the sessions is carried out with an analysis of discourses structural criticism and an analysis of their speech during one period of cure exceeding the four years, by making a comparison with former research. We conclude that the disease fills the criteria of the phenomenon psychosomatic and that the assumption of the subjective metaphor it is held by the organism, thus confirming our hypothesis. We also arrive at conclusions which are mainly in agreement with preliminary research. Finally, we note the improvement of the health of the subjects and the stabilization or the disappearance of the repetitions during their analysis, in parallel with their medical care, which that had not been observed before, and the appropriation of the points of the pain and the suffering of their history so that it does not lead to the way of pathological pulsing’s acts and the discharge by the organism

Afin d'étudier la distribution des lymphocytes b-like et t-like dans les organes lymphoïdes rein, rate, thymus et le tissu lymphoïde associe à l'intestin du turbot et de la truite arc-en-ciel, des ac polyclonaux de lapin dirigés spécifiquement contre les immunoglobulines (ig) et les thymocytes de chaque espèce ont été produits. Un manque de spécificité des sérums antithymocytes n'a pas permis la détection des lymphocytes t-like. Seuls les anticorps anti-ig ont donc été utilisés dans cette étude menée sur des poissons, indemnes de toute maladie. Quels que soient les organes considérés, un plus grand nombre de cellules ig + est observé chez la truite. En ce qui concerne l'intestin des différences majeures sont notées entre les deux espèces : les cellules ig + sont présentes à la fois dans la lamina propria et la lamina epithelialis chez la truite alors qu'elles ne sont détectées que dans la lamina propria chez le turbot. Dans un second temps la mise au point de techniques de prélèvement du mucus intestinal, et d'isolement des cellules mononuclées présentes dans l'intestin postérieur du turbot, avec la possibilité d'obtenir séparément les cellules issues de la lamina epithelialis et de la lamina propria, a permis d'appréhender les mécanismes de déclenchement de la réponse immune au niveau intestinal a la suite de vaccinations orales (vo) contre la vibriose à vibrio anguillarum. L’absorption de l'ag et la réponse immune systémique ont également été étudiées. Différentes formulations vaccinales ont été testées : des vaccins constitués de corps bactériens entiers, gastroproteges (vog) ou non (voe), et des vaccins constitués d'ag solubles (vos). La voie intraperitoneale a toujours été testée en parallèle car utilisée comme référence. A la suite des vaccinations par vo, l'ag est detecte dans la lamina propria et dans les organes systémiques, où il est transporte jusqu'aux cellules immunocompetentes. En réponse a cette stimulation antigénique des cellules b-like et/ou des plasmocytes apparaissent dans l'intestin au niveau de la lamina propria, puis dans la rate et le rein. Contrairement aux cellules intestinales issues de la lamina propria et aux cellules rénales qui possèdent une aptitude a la phagocytose et sont capables de sécréter des ac, aucune activité comparable n'est mise en évidence sur les cellules issues de la lamina epithelialis. Les réponses observées dans l'intestin restent modérées. A la suite d'une vaccination par vo, une réponse immunitaire locale et générale existe donc et ceci malgré l'absence d'ac anti-vibrio anguillarum, dans le mucus intestinal et dans le sérum. L’injection parentérale, a montré quant à elle que l'ag atteint les organes systémiques mais parvient également dans la lamina propria, dans laquelle une lymphoprolifération de cellules b-like est observée. De plus des ac sont détectés dans le plasma et le mucus indiquant que le système immunitaire général et intestinal sont conjointement sollicites. Enfin, l'étude d'un transfert passif d'immunité, démontre l'existence d'une réaction immunitaire humorale consécutive aux vaccinations par ip mais aussi par vo avec l'ag soluble, sans que la protection induite soit réellement attribuable aux ig.

Organic semiconductors (OSCs) have already been shown to have great potential to play an important role in the future of clean energy generation (organic solar cells) and provide energy efficient lighting (organic light-emitting diodes, OLED). Prior research has found that the light-emission efficiency of OLED is severely limited by the magnetic state (technically the spin-configuration) of the light-emission process. In this thesis, we work on the processes using external magnetic fields that can overcome these magnetic limitations. A major focus of this research is to enhance the performance of OLED, while at the same time to unravel the scientific mechanisms by which magnetic fields act on OSCs devices. Thermally activated delayed fluorescence (TADF) is a next-generation OLED emission technology which enables nearly 100% light-emission efficiency without using heavy precious metals. TADF characteristics depend on the probability of reverse intersystem crossing (RISC) from the triplet excited states (T1) to singlet excited states (S1). The conversion (T1 to S1) process depends strongly on spin dynamics, thus we predict a dramatic magnetic field effects (MFEs) in such TADF OLED devices. In subsequent experiments we observed that changes in TADF devices due to various forms of electrical stress can lead to enormous increases in magnetic field effects (MFEs) on the current (> 1400%) and electroluminescence (> 4000%). Our work provides a flexible and inexpensive pathway towards magnetic functionality and field sensitivity in current organic devices. Such OLED pave the way for novel magnetic sensitive OSCs devices with integrated optical, electronic and magnetic characteristics. Organic magnetoresistance (OMAR) has been observed to alter the current and efficiency of OLED without any ferromagnetic components. Here we utilizes slight alterations to the device properties, the addition of a radical-doped functional layer, in which the spin-relaxing effects of localized nuclear spins and electronic spins interfere, to address the assumption about the importance of the hyperfine interaction and to attempt to differentiate between the different models for OMAR. A feature where the magnitude of OMAR exhibits a plateau over a wide range of doping fraction was observed at all temperatures investigated. This phenomenon is well explained by a theory in which a single dopant spin strongly interacts, by exchange, with one of the bottleneck sites. A similar can be used to explain the efficiency increases observed in organic solar cells for certain doping fractions.

Essais d'induction de tolerance aux haptenes de type alpha-methylene gamma butyrolactones (amgbl) responsables de nombreuses allergies de contact. Synthese de differents tolerogenes potentiels hydrosolubles de la forme lactone-derives lysinyl et lactone-derives cysteinyl. Etude de l'induction asymetrique observee lors de l'addition de michael d'un dipeptide (voc-cys-ala-ome) a differentes amgbl. Observation d'un parallelisme entre cette induction asymetrique et l'enantioselectivite de la reponse immunitaire dans le phenomene de l'allergie croisee

Toll like receptors (TLRs) are found on B cells, macrophages, monocytes, and dendritic cells, and these cells belong to the innate immune system that recognizes antigens and induces multiple cell responses through the release of cytokines. TLR1, TLR2 and TLR6 function as heterodimers, either as TLR1/TLR2 or TLR2/TLR6 to recognize lipopeptides. TLR1/2 dimer activation releases inflammatory cytokines, while TLR2/TLR6 dimer activation releases immunomodulatory cytokines. Based on the size of the binding pocket between TLR2 and TLR6, it was hypothesized that lipopeptides, such as FSL1, could be simplified while keeping overall activity. FSL1 is a lipopeptide first isolated from Mycoplasma salivarum that activates macrophages at picomolar concentrations. It is expected that synthetic lipopeptides mimicking immunostimulatory molecules such as FSL1 will allow development of better ways to stimulate or modulate the immune system. Therefore, novel synthetic TLR2/6 ligands were synthesized replacing the polylysine chain with a polyamine chain showing activation of the immune cells in a manner like FSL1. Natural killer T-cell (NKT) antigens, such as α-galactosylceramide (α-GalCer), are carried through the body by lipid transfer proteins before they interact with the NKT cells. Not all the proteins involved in glycolipid transportation have been characterized. The synthesis of an α-GalCer analogue, termed CD1d-Triceps was designed to help find additional proteins involved in glycolipid trafficking. CD1d-Triceps has three functionalities: the first is the α-GalCer structure, and the other two are on C6 of the sugar: biotin, which helps tag the molecule for its purification, and a photoactive tag that, upon UV light activation, will cross-link with neighboring proteins. Antibiotic-resistant strains of Staphylococcus aureus (SA) are a growing health problem worldwide. Serotype 5 and 8 are the most common SA pathogens. Loading the serotype 5 or 8 disaccharides onto Qβ-particles that are linked to an NKT cell activator yield a vaccine that is expected to trigger adaptive immunity to the disaccharide. Previous similar studies showed production of antibodies with high affinity against Streptococcus pyogenes oligosaccharides in a similar vaccine.

Adaptive immunity requires T cell responses to foreign pathogens to be counterbalanced with the need to limit collateral destruction of the host’s own tissues. Further, the presence of a substantial pool of lymphocytes capable of recognizing selfantigen in the periphery poses a threat to the maintenance of peripheral tolerance and prevention of autoimmunity. Regulatory T cells (Treg) that can suppress potentially self-reactive T cells are critical regulators of peripheral tolerance as well as initiation of immune responses. Treg cells employ several context-dependent mechanisms to establish regulation. In this thesis, we describe two distinct pathways of regulation used by Treg cells involving negative costimulation by CTLA-4 and immunomodulation by the morphogen, TGFβ. CTLA-4 is a co-inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. CTLA-4 expression is induced in conventional T cells following activation, whereas it is constitutively expressed in regulatory FOXP3+CD4+ regulatory T cells. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both, is not known. We present evidence here that in addition to a critical CTLA-4 function in regulatory T cells, CTLA-4 in conventional T cells is also necessary for controlling the consequences of abnormal T cell activation. CTLA-4 expression in activated conventional T cells only in vivois unable to compensate for the impaired function of CTLA-4-less regulatory T cells that results in systemic lymphoproliferation, but it can prevent the aberrantly activated T cells from infiltrating and fatally damaging non-lymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell homeostasis: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells can prevent the harmful accumulation of inappropriately activated pathogenic T cells in vital organs. In addition, we have identified Disabled-2 (Dab2), a TGFβ signaling intermediate, as a FOXP3 target gene that is expressed exclusively in Treg cells and is critical for in vitro and in vivo regulation by Treg cells. During T cell development, DAB2 is also expressed in a Foxp3-independent manner in thymic precursor cells, and acts as a sensor of TGFβ signals that is required for programming normal TGFβ responsiveness in T cell progenies. Naïve CD4+ T cells that differentiate from Dab2-deficient precursors favor Th17 cell generation at the expense of FOXP3+ Treg cells as a result of altered sensitivity to TGFβ. Importantly, retinoic acid can restore TGFβ signaling capacity of naïve CD4+ T cells generated from Dab2-deficient precursors, emphasizing the cooperative nature of retinoic acid and TGFβ signaling pathways in promoting Treg cell development and maintenance.

Les infections et l’activation du système immunitaire stimulent l’hématopoïèse. L’activation des récepteurs Toll-like (TLRs) des cellules souches hématopoïétiques, par leur reconnaissance de motifs moléculaires portés par des agents infectieux, en oriente la différenciation vers les voies myéloïdes, renforçant la capacité de notre organisme à lutter contre les infections. Ici, nous avons étudié si les agonistes TLRs peuvent, au contraire, induire au sein de la moelle osseuse l’émergence de progéniteurs hématopoïétiques présentant des propriétés immunorégulatrices. Nous montrons que l’incubation de moelle osseuse de souris en présence de l’agoniste TRL-9, CpG-B, entraîne l’émergence d’une population de progéniteurs au stade pro-B (appelée CpG-proBs). Le transfert adoptif de seulement 60,000 CpG-proBs par receveur, à l’apparition des premiers signes cliniques, confère une protection à long terme dans deux modèles expérimentaux de maladies auto-immunes, le Diabète de Type I (T1D) et l’Encéphalomyélite Auto-immune Expérimentale (EAE). La migration, la différenciation, et les mécanismes cellulaires et moléculaires de cette population protectrice sont décrits et comparés entre ces deux modèles. Dans les deux modèles, les CpG-proBs migrent vers le tissu cible de la réponse auto-immune et se différencient en cellules B matures régulatrices. Dans le T1D, l’interféron-γ (IFN-γ) produit par les cellules T s’avère essentiel pour induire la surexpression de FasL à la surface des CpG-proBs, entraînant l’apoptose des cellules T effectrices. De plus, l’IFN-γ produit par les CpG-proBs réduit la production par les cellules T de l’IL-21, une cytokine pathogène majeure dans le T1D. La descendance des CpG-proBs est composée de précurseurs transitionnels B, de cellules B de la zone marginale et de cellules B folliculaires, exprimant de forts niveaux de FasL et toujours capables d’induire l’apoptose des cellules T, prolongeant ainsi le contrôle des cellules effectrices T auto-immunes in vivo. Dans l’EAE, l’IFNγ est indirectement responsable de la rétention des cellules T, par l’internalisation de CCR7, au sein des ganglions lymphatiques, inhibant ainsi leur migration au système nerveux central (SNC). Dans la moelle épinière, tissu cible de l’EAE, les CpG-proBs se différencient en cellules B220+CD5+CD1dhiCD11b+, secrétant la cytokine anti-inflammatoire IL-10. Enfin, la mobilisation des progéniteurs hématopoïétiques par un cocktail de facteurs hématopoïétiques confère à une sous-population multipotente au stade MPP2 la propriété d’augmenter l’expansion des Foxp3+ Tregs et de prévenir la survenue du diabète de type 1. Nous montrons que les MPP2 mobilisés s’avèrent également capables d’exercer un effet protecteur envers l’EAE. Leur capacité à induire l’expansion de Treg Foxp3+ au sein du SNC et à la périphérie joue un rôle essentiel dans la protection des souris envers l’EAE, puisque la déplétion des Treg abolit la protection déjà établie. Pour conclure, nous avons mis en évidence que diverses stimulations de l’hématopoïèse induisent l’émergence de nouvelles populations de progéniteurs hématopoïétiques qui présentent des propriétés immunorégulatrices et constituent de nouveaux outils de thérapie cellulaire des maladies auto-immunes<br>It is well known today that various infectious events or other stimuli of the immune system can trigger hematopoiesis. The hematopoeitic stem and/or progenitor cells express on their cell surface Toll-like receptors which can recognize molecular motifs of infectious agents. The stimulation of TLRs on hematopoietic stem cells favors their differentiation into myeloid lineages, reinforcing the capacity of our body to fight against the pathogens. Herein, we have investigated whether the stimulation of TLRs can induce, instead, the emergence within the bone marrow of selective progenitor cells with immunoregulatory properties. We show that incubation of bone marrow cells with the TLR-9 ligand CpG-B can induce a pro-B cell population (named CpG-proBs) whose adoptive transfer at low numbers of 60,000 cells provided long-lasting protection in two models of autoimmune diseases, Type I Diabetes (TID) and Experimental Autoimmune Encephalomyelitis (EAE) at the onset of clinical signs. The migration, differentiation and molecular mechanism of action of this protective population is described and compared between these two models. In both models, the CpG-proBs migrate to the target tissue of autoimmune responses and differentiate into more mature regulatory B cells. In TID, IFN-γ produced by both T and CpG-proB cells is essential for the upregulation of FasL at the surface of CpG-proBs, inducing the apoptosis of the effector T cells. In addition, IFN-γ reduced the T-cell production of IL-21, a major pathogenic cytokine in TID. The progeny of the adoptively transferred CpG-proBs, including transitional precursors B cells, marginal zone and follicular B cells, display high expression of FasL, promote apoptosis of effector T cells and prolong the control of autoimmune effector T cells in vivo. In EAE, IFN-γ was responsible for the restriction of T cells to the lymph nodes, inhibiting their homing to the CNS. IFN-γ indirectly induced the internalization of CCR7, a receptor required for the migration across the blood-brain barrier. In the spinal cord (target tissue in EAE), CpG-proBs differentiated into B220+CD5+CD1dhiCD11b+ cells secreting the anti-inflammatory cytokine IL-10. Finally, hematopoietic progenitor populations mobilized to the periphery by a cocktail of G-CSF and Flt3l, at the stage of MPP2, have already been shown to protect against TID by expanding the Foxp3+ Tregs. We evaluated them in the EAE model, showing that the ability of these mobilized progenitor cells to trigger the expansion of Foxp3+ Treg within the CNS and the periphery was necessary for providing protection to EAE mice since Treg depletion abrogated the protection once established. In conclusion, we provide evidence for the emergence of new populations of hematopoietic progenitor cells which can display immunoregulatory properties and might be used for cell therapy of autoimmune diseases

Neurogenin 3 and its downstream target NeuroD are basic helix-loop-helix transcription factors which promote endocrine differentiation in the gastrointestinal tract. However, mice lacking Ngn3 still produce several hormones in the stomach. Lineage tracing mouse models demonstrated that a majority of hormone cells in the corpus region of the stomach did not express Ngn3 or NeuroD during differentiation. Serotonin and histamine cells were entirely NeuroD-independently derived, and serotonin cells were additionally entirely Ngn3-independently derived. In this study, we isolated serotonin and histamine cells from the gastric corpus of transgenic mice expressing the fluorescent marker CFP. Serotonin cells expressed multiple mast cell markers by RT-PCR, and were found to be nearly absent in a mast cell-deficient mouse model. Labeled bone marrow transplant mice showed all serotonin cells derived from bone marrow. Histamine-expressing ECL cells, while lacking NeuroD, did not appear to express granulocyte or mast cell markers by analytical flow cytometry and RT-PCR, and resemble other enteroendocrine cell populations. Mouse gastric corpus serotonin cells, but not antral serotonin cells, are bone marrow-derived mast cells.

Chez les espèces aviaires, le stockage des spermatozoïdes s’étend sur plusieurs semaines principalement au niveau du réservoir de la jonction utéro-vaginale, contenant les tubules de stockage des spermatozoïdes (SST). Les mécanismes impliqués dans ce processus restent indéterminés. L’effet de l’insémination artificielle (IA) a été évalué sur le protéome du fluide utérin (FU), des protéines cibles et des glycanes dans les SST, provenant de poules possédant une longue (F+) ou courte (F-) durée de stockage. La longue durée de stockage est associée à une abondance relative dans le FU après IA des protéines exosomales (ANXA4, ANXA5), des protéoglycanes (TSKU), des protéines liant les protéoglycanes (HAPLN3, FN1, VTN), des transporteurs de lipides (VTG1, VTG2, APOA1, APOA4, APOH), et des protéines matricielles de la coquille (OCX32). Au contraire, la faible capacité de stockage est associée à la régulation après IA des protéines immunitaires (PIGR, immunoglobulines) ou pro-inflammatoire (LTA4H), des protéases (XPNPEP1), des chaperones (HSPA8), des mucines (MUC5AC, MUC5B), et de l’ovalbumine (OVALY). Au niveau des SST, les protéines matricielles de la coquille (OC-116, OCX36, OC-17) ont été identifiées dans l’épithélium et la lumière. La longue durée de stockage est associée à la sécrétion luminale de résidus Glc/GlcNAc, à la mobilisation apicale de protéines exosomales (ANXA4), et la non-activation des voies métaboliques impliquant les protéines PIGR, HSPA8, et ANXA5 dans les SST. En conclusion, la composition protéique du FU et des SST requièrent des régulations spécifiques après IA certainement pour garantir le stockage des spermatozoïdes<br>In avian species, the sperm storage mainly takes place in uterovaginal sperm storage tubules (SST) during several weeks. Mechanisms implied in this process are not fully understood. The effect of artificial insemination (AI) has been evaluated on the uterine fluid (UF) proteomic composition, and on SST candidate proteins, from hens exhibiting long (F+) or short (F-) sperm storage duration. Long sperm storage duration was associated with the relative abundance in UF after AI of proteoglycans (TSKU), proteoglycan binding proteins (HAPLN3, FN1, VTN), lipid transporters (VTG1, VTG2, APOA1, APOA4, APOH), and eggshell matrix proteins (OCX32). In contrast, poor sperm storage ability was associated with the regulation of immune factors (PIGR, immunoglobulins), pro-inflammatory factors (LTA4H), proteases (XPNPEP1), chaperone (HSPA8), mucins (MUC5AC, MUC5B), and ovalbumin related protein Y (OVALY). At the level of SST, eggshell matrix proteins (OC-116, OCX36, OC-17) were identified in SST cells and lumen. Long sperm storage duration was associated in SST with the luminal secretion of Glc/GlcNAc residues, ANXA4 apical mobilization, and non-activation of metabolic pathway implying PIGR, HSPA8, and ANXA5. In conclusion, the proteomic composition of UF and SST require specific regulation after insemination, most probably to guarantee the success of sperm storage process

In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).

博士<br>國防醫學院<br>生命科學研究所<br>94<br>In order to understand the immune system of economical species, Epinephelus malabaricu, the histological observation of immune relative organs in one-year-old juveniles and development was investigated, and the immune relative genes, rag1 and ikaros was cloned in present study. The histological architecture of major immune organs in the Malabar grouper Epinephelus malabaricus was investigated. The novel characteristics such as melanomacrophage centers (MMCs) appeared in the thymus and lymphopoietic tissue formed as foci in the head kidney. Leukocyte distribution in organs was identified by enzyme histochemistry. b-glucuronidase (BG) reactive cells in the cortex region of the thymus were botryoidally aggregated. Both acid phosphatase (AcP) and BG reactive cells concentrated within the specialized lymphopoietic foci in the head kidney, suggesting that the foci might be functional. Primitive histological characters of immunity were observed in the spleen. Although leukocyte aggregation was demonstrated in the spleen, additional enzyme histochemistry indicated that the aggregate might not be the equivalent of white pulp found in other vertebrates. The histological evidence did not support intestinal involvement in the immune system: there was no demonstrable gut associated lymphoid tissue. The limited distribution in the cortex and medulla boundary and the condensed format of the lymphocytes suggests a functional role for the MMC in the thymus of E. malabaricus. The MMC appearance in the thymus of a teleost was unusual. Ikaros and rag1 gene expressed early in vertebrate development, the appearance pointed lymphopoiesis was preceded. The thymus is a central immune organ in fish and is considered as the first lymphopoiesis organ. Partical ikaros and rag1 gene was cloned and the expression profiles comparing to histogenesis study was utilized to understand the thymus development in Epinephelus malabaricus. Histologically, a bud-like thymus behind the ear cavity was observed at 9 days post-hatch (dph), lymphopoietic cells appeared at 19 dph, and additional development, such as delineation of the cortex/medulla and development of lymphocytes occurred by 21 dph Trabecula development was detected at 41 dph, at which time the thymus was histological mature. In development, both ikaros and rag1 expressed as early as 3 dph, earlier than thymic organogenesis. The interested result indicated the phenomenon of extrathymically lymphopoiesis in malabar grouper could be illustrated by rag1 expression before thymus development.

博士<br>國立臺灣大學<br>獸醫學研究所<br>100<br>Porcine circovirus type 2 (PCV2) is a small, non-enveloped, single-stranded, circular DNA virus, and the infection of PCV2 is distributed in swine population worldwide. Experimental and field studies indicate that PCV2 is a required but insufficient causative agent of postweaning multisystemic wasting syndrome (PMWS), a disease that has a significant impact on swine production. The characteristic feature of PMWS is the presence of high levels of PCV2 antigens and nucleic acid in the lymphoid system along with variable lymphoid depletion and granulomatous inflammation. Until now, the precise mechanism regarding the replication of PCV2 in immune cells and the pathogenesis of PMWS are remained to be elucidated. Therefore, serial in vitro (Chapters II and III) and in vivo (Chapters IV, V and VI) models were performed in the present study to clarify above issues. In the first portion of the present study, it is hypothesized that immune activation induces PCV2 replication in activated lymphocytes. Therefore, concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) was used as an in vitro model to clarify the susceptibility of swine lymphocytes to PCV2 (Chapter II). Subsequently, this model was further expanded by the addition of monocyte-derived dendritic cells (MoDCs) and recombinant cytokines, including interleukin 2 (IL-2), IL-4, and interferon γ, to determine the correlations between lymphocyte activation and PCV2 replication (Chapter III). The evidences of increase in cellular PCV2 antigen- and/or nucleic acid-containing rates, PCV2 genome copy number, and titer of infectious PCV2 in mitogen-stimulated, PCV2-infected PBLs support that PCV2 infects and replicates in activated swine lymphocytes. By phenotyping the PCV2 antigen-positive cells, it was revealed that T and B lymphocytes as well as monocytes were susceptible to PCV2 infection; however, IgM-positive B lymphocytes appeared to have a relatively higher PCV2-positive rate (Chapter II). While lymphocytic cells served as a major site of PCV2 replication, MoDCs harbored a significant amount of PCV2 antigens but did not actively sustain the replication of PCV2. Further study has demonstrated that IL-2 and the accessory cell function of DCs were key factors required for cell proliferation and PCV2 replication in PCV2-infected lymphocytes (Chapter III). In the second portion of the present study, serials in vivo studies were conducted to characterize the changes in inguinal LNs in the naturally PCV2-infected pigs (Chapters IV and V) and to infer the possible mechanism of PCV2-associated lymphoid lesion developed (Chapter V and VI). It is speculated that limited by their sensitivity, conventional in situ hybridization (ISH) and/or immunohistochemical (IHC) staining may not be sufficient to detect few copies of PCV2 contained in a portion of immune cells. Therefore, a more sensitive method, indirect in situ polymerase chain reaction PCR (ISPCR), was developed through the combination of PCR and ISH procedure (Chapter IV). Using the indirect ISPCR, signals of PCV2 nucleic acid, especially those in germinal centers, could be more effectively detected. In addition, a detailed grading system was proposed to categorize the pattern of natural PCV2 infection in lymphoid follicles (Chapter IV) and used as criteria for sample selection in the following studies (Chapters V and IV). To obtain tissue-based information effectively, a new model of high-throughput tissue microarray (TMA) in conjunction with semi-quantified ISH/IHC staining and statistic analysis was established (Chapter V). The results of multiple regression analysis showed that each of PCV2, porcine respiratory and reproductive syndrome virus (PRRSV), and porcine parvovirus (PPV) had its own contributions on the development of lymphoid lesions in PMWS with PCV2 as the major causative agent. B lymphocyte depletion and macrophage proliferation and infiltration are the two hallmarks of PCV2-associated lymphoid lesions. Apart from blood recruitment, local T cell and macrophage proliferation may play a crucial role on the development of granulomatous inflammation (Chapter V). Furthermore, the expressions of 92 selected immune genes in 7 and 35 inguinal LNs obtained from healthy subclinically PCV2-infected and PMWS-affected pigs were assessed by the integration of several quantitative reverse transcription polymerase chain reaction experiments (Chapter IV). Using hierarchical cluster analysis, the gene expression profiles in these PCV2-infected LNs were generally compatible with the divergent functions of different immune cell populations. Aberrant immune activation and imbalanced Th1/Th2 orientation are considered to contribute to the development of PCV2 infection-associated lymphoid lesions. Principle component analysis of the expression profile of the 92 selected immune genes in the 42 above mentioned LNs revealed that 52.23% of the total data variants could be explained by the top-3 principle components, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors (Chapter VI). In conclusion, evidences from the present study suggest that lymphocytes are indeed susceptible to PCV2 (Chapters II, III and IV). Immune activation modulated by DCs and cytokines triggers the replication of PCV2 in lymphocytes (Chapter III). Subsequently, the perturbation of immune regulation by PCV2 facilitates the co-infection of other pathogens. Due to the persistent or repeated episodes of antigenic stimulation, the balance between immune suppression and activation is further perturbed (Chapter VI). As the progression of PMWS development, lymphoid depletion and granulomatous inflammation occur in the LNs bearing heavy PCV2 load (Chapter V).