The intestinal tissue is charged with a delicate immunological task. The intestinal immune system needs to be tolerant towards nutrients and microbiota present in the intestinal lumen, while simultaneously detecting and responding to dangers such as pathogens. A single-cell layer of intestinal epithelial cells (IECs) acts as a first line of defence. There is a T cell population located between the IECs that have been named intraepithelial lymphocytes (IELs). As the main lymphoid population within the intestinal barrier, IELs are thought to have an important role in intestinal homeostasis maintenance, as well as a role in intestinal inflammatory and autoimmune diseases such as inflammatory bowel disease and celiac disease. Despite extensive research on IEL biology, there are still questions remaining in terms of the development, maintenance and activation of IELs. Furthermore, IELs survive poorly in vitro, which hinders mechanistic insights. In this thesis, a co-culture system between IELs and intestinal organoids, "mini-guts", provides an in vitro model for IELs. With this IEL-organoid co-culture system, IELs associated with the organoids survive for at least 4 days. Additional findings suggest that IELs are kept in a poised state of activation due to differences in their mitochondria compared to other T cells found in spleen, lung and skin. Upon activation or intestinal inflammation, the mitochondrial mass in IELs increases. This increase correlates with effector functions such as cytokine production and proliferation. In addition, the composition of the mitochondria-specific lipid, cardiolipins, alters drastically in IELs after activation. These data support a model of mitochondria-dependent activation of IELs. The mitochondria-dependent activation in IELs appears to have at least two pathways: one T cell receptor-dependent and one microbiota-dependent. The latter pathway suggests a model in which IELs can become activated regardless of the cause of intestinal epithelial barrier damage.

Para verificar a permissividade de células de origem hematopoiéticas à infecção por HAdV-41, foram infectados PBMC e IEL de voluntários. Os ensaios foram comparados com células HEK-293 infectadas. Foram analisadas as expressões dos genes virais E1A, E1B (55K), E3 (14K), VARNA, hexon e fibra curta (FC) e do gene celular GAPDH. O mRNA foi detectado por RT-PCR em tempo real e a produção de proteínas foi visualizada por IFI. Em HEK-293, a transcrição dos genes E1A, E1B e E3 iniciou-se às 11h p.i, hexon,às 13h pi.,VARNA e FC às 14h p.i. Em PBMC a transcrição de E1A, E1B e VARNA iniciou-se 17h p.i e a expressão dos genes hexon e fibra curta foi detectada 18h p.i e 20 h p.i. respectivamente. O nível de expressão dos genes virais em HEK-293 foi quase 200 vezes maior em relação à PBMC. Os IELs também mostraram-se permissivos à infecção pelo HAdV-41 como mostrado pela expressão dos genes virais. Essa é a primeira evidência de que este vírus possa infectar tais células. Os resultados obtidos ajudam a elucidar os mecanismos de interação do vírus com a célula-hospedeira. / In order to verify the permissiveness of hematopoietic cells to HAdV-41 infection, PBMC and IEL from volunteers were infected. The infection assays were compared with infected HEK -293 cells. We analysed the E1A, E1B (55K), E3 (14K), VARNA, hexon and short fiber (SF) viral gene expression and GAPDH cellular gene expression. The mRNA were detected by real time PCR and the viral protein synthesis were detected by IIF. In HEK-293 cells E1A, E1B and E3 gene expression were detected 11h p.i Hexon gene expression was detected at 13h p.i, while VARNA and SF were detected 14h p.i In PBMC, E1A, E1B and VARNA gene expression were detected 17h p.i and the hexon and SF were detected 18h p.i and 20h p.i, respectively. The viral gene expression level in infected HEK-293 cells was 200 fold higher than infected PBMC. The IEL also were permissive to HAdV-41 infection showed by viral gene expressions. This is the first evidence that HAdV-41 is able to infect these cells types. These results helps to understand the virus-cell interaction mechanisms.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and γδ T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and γδ T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and γδ T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

Para verificar a permissividade de células de origem hematopoiéticas à infecção por HAdV-41, foram infectados PBMC e IEL de voluntários. Os ensaios foram comparados com células HEK-293 infectadas. Foram analisadas as expressões dos genes virais E1A, E1B (55K), E3 (14K), VARNA, hexon e fibra curta (FC) e do gene celular GAPDH. O mRNA foi detectado por RT-PCR em tempo real e a produção de proteínas foi visualizada por IFI. Em HEK-293, a transcrição dos genes E1A, E1B e E3 iniciou-se às 11h p.i, hexon,às 13h pi.,VARNA e FC às 14h p.i. Em PBMC a transcrição de E1A, E1B e VARNA iniciou-se 17h p.i e a expressão dos genes hexon e fibra curta foi detectada 18h p.i e 20 h p.i. respectivamente. O nível de expressão dos genes virais em HEK-293 foi quase 200 vezes maior em relação à PBMC. Os IELs também mostraram-se permissivos à infecção pelo HAdV-41 como mostrado pela expressão dos genes virais. Essa é a primeira evidência de que este vírus possa infectar tais células. Os resultados obtidos ajudam a elucidar os mecanismos de interação do vírus com a célula-hospedeira. / In order to verify the permissiveness of hematopoietic cells to HAdV-41 infection, PBMC and IEL from volunteers were infected. The infection assays were compared with infected HEK -293 cells. We analysed the E1A, E1B (55K), E3 (14K), VARNA, hexon and short fiber (SF) viral gene expression and GAPDH cellular gene expression. The mRNA were detected by real time PCR and the viral protein synthesis were detected by IIF. In HEK-293 cells E1A, E1B and E3 gene expression were detected 11h p.i Hexon gene expression was detected at 13h p.i, while VARNA and SF were detected 14h p.i In PBMC, E1A, E1B and VARNA gene expression were detected 17h p.i and the hexon and SF were detected 18h p.i and 20h p.i, respectively. The viral gene expression level in infected HEK-293 cells was 200 fold higher than infected PBMC. The IEL also were permissive to HAdV-41 infection showed by viral gene expressions. This is the first evidence that HAdV-41 is able to infect these cells types. These results helps to understand the virus-cell interaction mechanisms.

Diversos trabajos reportan aumento en el número de linfocitos intraepiteliales (LIE), mastocitos y células enterocromafines en pacientes con Sindrome de Intestino Irritable (SII). Muchos de estos hallazgos se basan en el uso de inmunohistoquímica que son de poca disponibilidad en hospitales generales. El objetivo del presente trabajo es estudiar los hallazgos histológicos en la biopsia de colon sólo con histoquimica en pacientes con SII comparándolos con un grupo sin SII. Fueron incluidos 25 pacientes: 16 (64%), con criterios diagnósticos de SII y 9 (36%), sin SII. Se encontró un mayor número de LIE en el grupo de SII (p=0,002). Un grupo de pacientes con criterios Roma III (41,9%) presentó LIE en el rango de Colitis Linfocitica por lo que fueron excluidos de este estudio. No se encontró diferencia estadísticamente significativa en el número de mastocitos, células enterocromafines y eosinofilos. / Several studies have shown increased numbers of intraepithelial lymphocytes (IEL), mast cells, enterochromaffin cells in colonic mucosa of patients with Irritable Bowel Syndrome (IBS). Many of these findings are based is based on immunohistochemistry results, which is not available in general hospitals. Our objective is to study the histological findings observed in colon biopsies from patients with IBS compared with a group without IBS, using only histochemistry. Twenty five (25) patients were included: 16 with IBS and 9 without IBS. We found increased numbers of IEL in patients with IBS (p=0,002). A group of patients with IBS (41.9%) who fulfilled histological criteria for lymphocytic colitis were excluded. There was no significant difference in mast cells, enterochromaffin cells or eosinophils.

Orientador: Wirla Maria da Silva Cunha Tamashiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:43:40Z (GMT). No. of bitstreams: 1 Ruberti_Maristela_D.pdf: 10774061 bytes, checksum: 7afe7ee8aa8c7f97c1f80e66f0cd8bfa (MD5) Previous issue date: 2012 / Resumo: Trabalhos anteriores de nosso laboratório mostraram que camundongos transgênicos DO11.10, cuja maioria dos linfócitos T expressam TCR específico para ovalbumina (OVA) no contexto de...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Previous work from our laboratory showed that DO11.10 transgenic mice, in which the most of T lymphocytes express TCR specific for ovalbumin (OVA) in the context of...Note: The complete abstract is available with the full electronic document / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

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...

Konventionelle CD8 T-Zellen im intestinalen Epithel unterscheiden sich von T-Zellen gleicher Spezifität in anderen Organen. In dieser Arbeit sollten Mechanismen identifiziert werden, welche die Migration dieser T-Zellen in intestinale Gewebe bestimmen und/oder welche die funktionelle Adaption der T-Zellen an die mukosale Umgebung beeinflussen. Hierfür wurden Mäuse mit einem rekombinanten Listeria monocytogenes Stamm (LmOVA) infiziert, OVA-spezifische CD8 T-Zellen aus der Milz und dem intestinalem Epithel isoliert, und die mRNA-Expressionsprofile dieser Zellen mittels einer Mikroarray-Analyse verglichen. Eine Gruppe von NK-Rezeptoren zeigte auf OVA-spezifischen CD8 T-Zellen aus der intestinalen Mukosa eine geringere Expression. Dieser Unterschied war nach einer oralen Infektion wesentlich stärker ausgeprägt, als nach einer intravenösen Infektion. Die Präsenz der natürlichen Darmflora hatte nur einen sehr geringen Einfluss auf diese organspezifisch unterschiedliche NK-Rezeptor-Expression. Untersuchungen in transgenen CD4dnTGFbetaRII Mäusen wiesen auf TGFbeta als ein entscheidendes Element für die niedrigere NK-Rezeptor-Expression auf T-Zellen in der Darmmukosa hin. Die zweite Fragestellung war die, ob nach einer Lm-Infektion die primäre Aktivierung naiver CD8 T-Zellen und die Re-Aktivierung von Gedächtnis-T-Zellen auch außerhalb sekundärer lymphoider Organe stattfinden kann. Hierfür wurden Mäuse mit der immunmodulatorischen Substanz FTY720 behandelt und splenektomiert. Eine FTY720-Behandlung beeinträchtigte nach iv und nach ig Infektion weder die Ausbreitung der Lm, noch die Fähigkeit der Mäuse zu einer Elimination der Bakterien. Orale Infektionen sowie höher dosierte iv Infektionen führten auch in FTY720-behandelten und zusätzlich splenektomierten Mäusen zu einer T-Zell-Akkumulation und Proliferation in nicht-lymphoiden Organen. Nach einer niedrig dosierten iv Lm-Infektion wurden dagegen in FTY720-behandelten und splenektomierten Mäusen keine OVA-spezifischen T-Zell-Antworten ausgelöst. Insbesondere die Milz schien hier für die T-Zell Aktivierung und Proliferation erforderlich zu sein. Auch für eine Sekundärantwort nach einer iv Infektion mit LmOVA waren lymphoide Gewebe für die Ausbildung einer effektiven T-Zell-Antwort essentiell. / Conventional CD8 T cells in the intestinal epithelium differ from T cells with identical antigen specificity in other organs. One aim of this study was, to identify mechanisms, which determine gut-tropism of these T cells or which influence the functional adaptation of these cells to the mucosal environment. For this purpose, mice were infected with a recombinant Listeria monocytogenes strain (LmOVA). OVA-specific CD8 T cells were isolated from spleen and intestinal epithelium and the mRNA-expression profiles of these cells were compared in a microarray-analysis. One group of NK-receptors were substantially less expressed on Lm-specific CD8 T cells from the intestinal mucosa than on corresponding cells from the spleen. This difference was much more pronounced following oral infection than following iv infection. The presence of the natural gut-flora only slightly influenced the organ-specific NK-receptor expression in CD8 T cells. Experiments with transgenic CD4dnTGFbetaRII mice point to TGFbeta as a decisive factor for the low NK-receptor expression on T cells from the gut mucosa. In the second part of this study it was investigated, whether, following Lm infection, priming of naive CD8 T cells and re-activation of memory T cells could occur outside of secondary lymphoid organs. Mice were treated with the immunomodulatory drug FTY720 and splenectomized. Treatment with FTY720 did neither diminish bacterial dissemination into spleen, liver and MLN, nor impaired the ability of the mice to control the bacteria and to eradicate them from these organs. Oral infection and high dose iv infection led to T cell accumulation and proliferation in nonlymphoid organs of FTY720-treated and additionally splenectomized mice. Following low dose iv infection with LmOVA, no OVA-specific T cell responses were induced in FTY720-treated and additionally splenectomized mice. Especially the spleen seemed to be important for T cell activation and proliferation under these conditions. Also following a secondary iv infection with LmOVA, lymphoid tissues were essential for the generation of an effective T cell response.