Typescript (photocopy) / 132 leaves, [2] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982

Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1982. / Typescript (photocopy).

One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

Transmissible spongiform encephalopathies (TSEs) or prion diseases, are subacute neurodegenerative diseases that infect humans and animals. Many of these diseases are acquired by peripheral exposure (e.g. orally). After oral exposure prion replication within the Peyer's patches (PP) in the small intestine is necessary for the efficient spread of the disease to the brain. Within the intestine, bacteria and pathogenic microorganisms can affect the status of the gut associated lymphoid tissue (GALT). GALT consists of PP and isolated lymphoid follicles (ILF) that maintain homeostasis and protect from infections. Therefore, factors which modify GALT status, might dramatically affect oral prion disease pathogenesis by influencing the uptake of prions from the gut lumen or expanding their distribution within the host. Chronic intestinal helminth infections are common in animals and in man, and can cause significant pathology within the intestine. Little is known of the effects that intestinal helminth infections may have on oral prion diseases susceptibility. Therefore, in this study the influence that co-infection with Heligmosomoides polygyrus (a natural pathogen of the mouse small intestine) may have on oral prion disease pathogenesis and susceptibility was determined. The studies consisted of groups of 4 (for H. polygyrus characterization and for early prion detection) and 8 (for H. polygyrus-prion co-infection to terminal stage) mice infected with H. polygyrus (orally) alone or subsequently infected with ME7 scrapie prions (orally) at different time-points after parasitic infection. The effects of the H. polygyrus infection alone, and on oral prion disease pathogenesis and susceptibility were then determined. Initially the characterization of H. polygyrus infection on the host intestine revealed that this parasite caused significant pathology in the small intestine and affected the GALT microarchitecture. In the PP follicles, H. polygyrus infection increased the area of follicular dendritic cell expression, altered the positioning of mononuclear phagocytes and increased M cell density. H. polygyrus infection also reduced the number of ILF in both the small and large intestines. Additional studies in mice co-infected with a low dose of prions, revealed that these pathological changes affected the survival time and disease susceptibility. Data also show that the extent of the effects on prion disease pathogenesis and susceptibility were dependent on the stage of the helminth infection at which the mice were orally-exposed to prions. Data demonstrate that co-infection with the gastrointestinal helminth H. polygyrus can influence oral prion disease pathogenesis and susceptibility. Helminth infections can significantly modify the microarchitecture of the gut and the GALT. Data presented suggest the pathological changes that pathogens such as small intestinal helminths cause, may also influence the uptake of prions from the gut lumen after oral exposure.

Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-20Bitstream added on 2014-06-13T21:04:28Z : No. of bitstreams: 1 ramos_op_dr_arafcf.pdf: 948525 bytes, checksum: 0581866624e7c3f57ffffca838856184 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 – Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII – Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... / Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer’s patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 – Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII – Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below)

Le tropisme cellulaire des prions infectieux diffère selon lespèce animale, celui-ci est corrélé à la souche infectieuse et à des facteurs spécifiques de lhôte. Par exemple, certains prions infectieux sont lymphotropiques, notamment en cas de scrapie chez les moutons et de variant de la maladie de Creutzfeldt-Jakob (vMCJ) chez lhomme. Par opposition, certains prions se caractérisent par un neurotropisme comme observé chez des patients Creutzfeldt-Jakob atteints de la forme sporadique ou chez des bovins atteints dencéphalopathies spongiformes bovines (ESB). Lhypothèse de notre travail repose sur les observations suivantes : dans le cas du variant de la maladie de Creutzfeldt-Jakob et des encéphalopathies spongiformes bovines, lagent responsable est identique, la voie dinoculation et les lésions neurologiques le sont également, seul le tropisme de cette souche pour les organes lymphoïdes diffère. En effet, les amygdales, la rate et lappendice sont infectieux chez lhomme. Par contre, linfectiosité est surtout confinée au niveau du système nerveux chez le bovin. Lors dune inoculation expérimentale par voie orale de lagent responsable de lESB chez les bovins, les plaques de Peyer iléales sont les seuls tissus lymphoïdes infectieux. Notre hypothèse de travail est que des propriétés de lhôte interviennent dans le tropisme de lagent infectieux. Deux axes de recherche ont été envisagés afin de vérifier cette hypothèse :  Lanalyse de la distribution des fibres nerveuses au sein des tissus lymphoïdes associés aux muqueuses (MALT) des espèces bovine et humaine  Létude de lexpression de PrPc et de ses isoformes au sein des tissus lymphoïdes et nerveux des espèces bovine et humaine. Pour atteindre au mieux nos objectifs, il nous manquait un outil essentiel permettant la caractérisation spécifique des FDC bovines. En effet, aucun marqueur spécifique de ces cellules nétait commercialisé. Nous avons donc produit, en collaboration avec le Centre dEconomie Rural de Marloie, un anticorps monoclonal spécifiquement dirigé contre les cellules folliculaires dendritiques (FDC) bovines. Cet anticorps nous a permis détudier la distribution des FDC au sein des organes lymphoïdes bovins. Une attention particulière a été portée aux FDC isolées à partir des plaques de Peyer jéjunales (PPJ) et iléales (PPI). Lapparente différence dinfectivité de ces tissus lymphoïdes chez des bovins atteints expérimentalement dESB nous a conduit à comparer les capacités fonctionnelles des FDC isolées à partir de PPJ et de PPI. Ces observations sont décrites et discutées dans le chapitre 1. Dans le chapitre 2, nous avons établi une cartographie des fibres nerveuses au sein des amygdales, des plaques de Peyer iléales et jéjunales bovines de plusieurs catégories dâge et ensuite comparé ce pattern dinnervation à celui des amygdales humaines; ceci permettra de pister les voies potentielles de neuro-invasion. Une attention particulière a été portée à linterface cellules folliculaires dendritiques fibres nerveuses. En effet, les FDC matures jouent un rôle prépondérant dans la pathogenèse des maladies à prion puisquen leur absence, une infection périphérique na pas lieu. De plus, la proximité entre fibres nerveuses et FDC est un paramètre intervenant dans la neuro-invasion; nous avons dès lors aussi analysé les contacts entre les FDC et les éléments nerveux. Lexpression de la PrPc est une condition sine qua non pour la formation de PrPres. Cette protéine cellulaire sert probablement de récepteur pour son homologue infectieux mais sert surtout de substrat pour lamplification de PrPres ; toute modification au niveau de sa synthèse pourrait entraîner un changement de la cinétique dinfection et pourrait expliquer lapparente absence dinfectivité constatée au niveau du système immunitaire chez les bovins. Lexpression tissulaire et cellulaire spécifique disoformes de la PrPc représente un facteur de lhôte potentiellement capable dinfluencer le tropisme cellulaire de lagent infectieux chez lhumain et le bovin. Cette expression a été étudiée dans les systèmes MALT bovins et humains. Pour affiner notre étude, nous avons analysé, par des techniques de western-blotting, le glycopattern de la PrPc ainsi que lexpression de ses formes tronquées dans les tissus lymphoïdes humains et bovins mais également dans des populations cellulaires spécifiques, les lymphocytes et les FDC. Afin de vérifier si les isoformes de PrPc sont spécifiques aux tissus lymphoïdes, nous avons effectué une étude comparative du pattern de glycosylation et du ratio des formes clivées de PrPc, exprimés au sein de différentes régions du système nerveux central bovin et humain. Les résultats de ces travaux sont repris dans le chapitre 3.

Orientador: Beatriz Maria Machado de Medeiros / Banca: Beatriz Maria Machado de Medeiros / Banca: Maria Terezinha Serrão Peraçoli / Banca: Iracilda Zeppone Carlos / Banca: Cleni Mara Marzocchi Machado / Banca: Fernanda de Freitas Anibal / Resumo: Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 - Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII - Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer's patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 - Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII - Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below) / Doutor

8 ABSTRACT Historically pig is allocated to a group of animals which use certain parts of their small intestine to acquire a fully developed primary B cell reperoire. Development of such primary repertoire is independent on the antigen presence and resembles the primary lymphopoietic activity of avian bursa of Fabricius. However, some findings concernig the pig's alignment in the above mentioned group suggest otherwise. This graduation thesis is focused on the investigtion of lymphocyte populations and subpopulations in the small intestine of germ-free and conventional piglets. The aim is to determine whether the percentage amounts of lymphocyte populations is dependent on the intestinal colonization. Using Flow Cytometry the significant differences between individual samples were assesed allowing us to conclude which parts of the small intestine could possibly be used for the development of B cell repertoire. Moreover, the status of isotype switching of B lymphocytes isolated from different intestinal parts was determined by the means of PCR analysis. Our data suggest that the small intestine colonization has a crucial role in development of all the main lymphocyte populations as well as some of their subpopulations. The greatest influence of colonization was observed concerning B lymphocytes and their...

Crohn's disease, a chronic inflammation of the bowel, is a multi-factorial condition where uncontrolled immune responses to luminal bacteria occur in genetically predisposed individuals. The first observable clinical signs are small ulcers that form at a specialised form of epithelium, follicle-associated epithelium (FAB). The FAB covers immune inductive sites, Peyer's patches, which function primarily as sensory areas that sample the externaI gut environment. Dendritic cells are one of the key cells that are involved in sensing luminal contents and orchestrating the gut immune system. The main aim of this thesis was to determine whether the barrier of the FAB is breached in Crohn's disease and if dysfunctional immune regulators, namely dendritic cells, playaroIe in initiating and/or maintaining the chronic intestinal inflammation. Using biopsies and surgical specimens, we were able to show that in Crohn's disease, there was an increased transmucosaI transport of Escherichia coli compared to specimens from ulcerative colitis and non-inflammatory bowel disease (IBD) controIs. Dendritic cells internalised a higher percentage of bacteria that had translocated across the FAB in the Crohn's samples. Furthermore, significantly higher concentrations of TNF-u was released upon bacterial stimulation by tissues from patients with Crohn's disease than in controIs. We went on to characterise the dendritic cells present in the Peyer's patches of patients with Crohn's disease. We found an accumulation of both immature and mature dendritic cells beneath the FAB, in the sub-epithelial dome (SED). Normally, mature dendritic cells migrate towards T cell-rich areas. However, we observed mature dendritic cells accumulating in the SED because they lacked the CCR7 migratory receptor. Furthermore, they were more prone to take-up bacteria, and produced TNF-α. To study the function of mucosal dendritic cells, we performed isolation experiments and mixed Iymphocyte reactions. Dendritic cells from both the ileum and blood of patients with active Crohn's had reduced capacity for inducing T cell proliferation than non-IBD controIs. Blood dendritic cells of patients in remission had normalised function that was similar to dendritic cells from healthy controls. The SAMPl/YitFc mice, considered an appropriate murine model for Crohn's disease, had an inherent permeability defect that increased with the chronicity of intestinaI inflammation. However unlike in human Crohn's disease, dendritic cells did not seem to playaroIe in murine ileitis. This thesis highlights the accumulation of the actively surveying dendritic cells that are prone to bacterial internalisation, and points to their possible different functional roles in active versus in-active disease; thereby confirming dendritic cells as one ofthe key components in the pathogenesis ofCrohn's disease.