Dissertations / Theses on the topic 'Intestinal mucosal immune system'

Wir nutzten in dieser Studie den apikomplexen Parasiten Eimeria falciformis als Modell. Unsere Ergebnisse zeigen, dass das in infizierten Wildtypmäusen dominierende Zytokin IFN-γ für Immunschutz und für die Entwicklung der Darmpathologie entbehrlich war. E. falciformis-infizierte IFN-γR-/- and IFN-γ-/- Mäuse zeigten extremen Körpergewichtsverlust und starke Pathologie im Darm. Die Entwicklung des Parasiten in diesen Mäusen war überraschenderweise reduziert. Diese Beobachtungen gingen mit einer drastisch erhöhten Produktion von parasiten-spezifischem IL-17A und IL-22 durch CD4+ T Zellen einher. Gleichzeitige Neutralisierung von IL-17A und IL-22 in E. falciformis-infizierten IFN-γR-/- Mäusen verringerte den Körpergewichtsverlust und die Darmpathologie, und führte zu einer erhöhten Ausscheidung von Parasiten. Die Behandlung einer E. falciformis-infizierten intestinalen Epithelzelllinie mit IL-17A oder IL-22 führte zu einer signifikant reduzierten Entwicklung von E. falciformis in vitro. Diese Daten demonstrieren erstmalig einen anti-parasitären Effekt von IL-22 im Darm und deuten auf redundante Rollen von IL-17A und IL-22 im Hinblick auf die Förderung von Darmpathologie in Abwesenheit von IFN-γ hin. Um E. falciformis als Modellsystem weiter zu entwickeln, haben wir die Transfektion von E. falciformis Sporozoiten mit verschiedenen Plasmiden die den Reporter YFP und den Resistenzmarker DHTS enthalten etabliert. Rektal in Mäuse injizierte Sporozoiten entwickelten sich erfolgreich zu Oocysten, wenn auch mit geringerer Effizienz im Vergleich zur oralen Infektion mit Oozysten. Wiederholte in vivo Selektion YFP-exprimierender Oozysten führte zu Populationen mit maximal 34 % YFP-exprimierenden Parasiten. Wir demonstrieren in dieser Arbeit zum ersten Mal die Transfektion von E. falciformis und zeigen Perspektiven im Hinblick auf die Etablierung einer stabil transgenen Parasitenlinie auf.
The roles of Th1 and Th17 responses as mediators of host protection and pathology in the intestine are the subjects of intense research. Here we investigated a model of intestinal inflammation driven by the intracellular apicomplexan parasite Eimeria falciformis. Although IFN-γ was the predominant cytokine during E. falciformis infection in wild type mice, it was found to be dispensable for host defence and the development of infection-driven intestinal inflammation. E. falciformis-infected IFN-γR-/- and IFN-γ-/- mice developed dramatically exacerbated body weight loss and intestinal pathology, but surprisingly harboured fewer parasites. This was associated with a striking increase in parasite-specific IL-17A and IL-22 production in the mesenteric lymph nodes and at the site of infection. Concurrent neutralisation of IL-17A and IL-22 in E. falciformis infected IFN-γR-/- mice resulted in a reduction in infection induced body weight loss and inflammation and significantly increased parasite shedding. Taken together these data demonstrate for the first time an anti-parasitic effect of IL-22 during an intestinal infection and suggest that IL-17A and IL-22 have redundant roles in driving intestinal pathology in the absence of IFN-γ signalling. To further develop E. falciformis as a model system, we established transfection of E. falciformis sporozoites using various plasmids that contain the fluorescent reporter YFP and the resistance marker DHTS. Sporozoites applied rectally to mice were shown to complete their life cycle, albeit with a lower efficiency in comparison to oral infection with oocysts. Repeated in vivo selection using pyrimethamine and/or FACS and manual sorting led to a maximum percentage of 34 % YFP-expressing oocysts. Taken together, we demonstrate for the first time transfection of E. falciformis and provide perspectives for further work on the establishment of a stable transgenic parasite line.

An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages; contains 171 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Nov. 10.

The gastrointestinal tract of mammals is inhabited by thousands of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. It has previously been shown that these gut microbes play an important role in modulating host immune responses. On the other hand, commensals can also contribute to pathology in the context of acute infection. For instance, oral infections with Toxoplasma gondii in certain inbred strains of mice lead to an exacerbated intestinal inflammation that is accompanied by a loss of diversity within the gut flora. Furthermore, the microbiota was shown to aggravate the immunopathology of the disease. The mechanisms underlying this phenomenon still remain poorly understood. In order to study how the recognition of innocuous microbes can influence immune responses and pathological consequences during acute mucosal infections we treated mice a cocktail of antibiotics. Treated mice showed decreased inflammatory responses and lower parasite load. Germfree mice infected with T. gondii displayed less severe disorder with reduced parasite burden and lower levels of liver enzymes. Systemic translocation of gut bacteria was observed at the peak of infection in T. gondii-infected mice as well as temporal changes in diversity of the gut microbial community. Three different bacteria that were abundant in the gut of T. gondii-infected mice at the peak of infection were isolated and used for investigation of specific immune responses against commensal bacteria. T. gondii acute infection induced specific antibody responses towards antigens from the microbiota and adaptive cellular responses indicated by a strong DTH reaction against antigens from one of the isolated bacteria. Moreover, Moreover, CBir 1 TCR Tg cells that only respond to a specific peptide from flagellin become activated after oral infection with T. gondii. We also showed that antibody responses against the microbiota occur also after a less intense inflammatory response induced by Microsporidia infection or after Citrobacter rodentium colitis. Furthermore, vaccination of mice against E. coli led to a more efficient clearance of T. gondii parasites but did not worsen the immunopathology. All together our findings suggest that mucosal acute infections can trigger an adaptive immune response against gut commensals that in turn contributes to the protection of the host against subsequent infections.
O trato gastrointestinal de mamíferos é colonizado por uma diversa comunidade microbiana que co-existe mutualisticamente com seu hospedeiro. Estudos recentes têm demonstrado o papel importante destes microrganismos na modulação de respostas imunes. Por outro lado, bactéria da microbiota podem também contribuir para a patologia no contexto de infecções agudas. Por exemplo, infecções orais com Toxoplasma gondii em certas linhagens murinas levam a uma inflamação intestinal exacerbada que é acompanhada por perda de diversidade da microbiota. Além disso, a microbiota agrava a imunopatologia da toxoplasmose. Os mecanismos que explicam este fenômeno ainda não são completamente compreendidos. No presente estudo utilizamos de camundongos tratados com antibióticos para estudar como o reconhecimento de microrganismos da microbiota pode influenciar respostas imunes e a patologia de infecções agudas de mucosa. Camundongos tratados mostraram menor resposta inflamatória e menor quantificação de parasita após infecção com T. gondii. Camundongos germfree também infectados com T. gondii têm menor carga parasitária e níveis reduzidos de enzimas do fígado. Translocação sistêmica de bactérias intestinais foi observada no pico da infecção assim como mudanças temporais de diversidade dentro da comunidade microbiana intestinal. Três bactérias mais encontradas no intestine de camundongos infectados com T. gondii foram usadas para o estudo de respostas específicas contra a microbiota. A infecção com T. gondii foi capaz de induzir respostas humorais específicas contra antígenos da microbiota e respostas adaptativas celulares indicadas por uma forte reação de DTH contra uma das bactérias isoladas. Além disso, células CBir TCR transgênicas que somente respondem a um peptídeo de flagelina se tornam ativadas após a infecção oral com T. gondii. O presente estudo também demonstrou que uma infecção menos intensa, com Microsporidia, também induz anticorpos contra antígenos microbiamos assim como a colite induzida pela bactéria Citrobacter rodentium. A vacinação de camundongos contra uma bacteria commensal levou a um controle mais eficiente de T. gondii mas não agravou a imunopatologia da doença. Em conjunto, nossos resultados sugerem que infecções agudas de mucosa podem ativar respostas adaptativas imunes contra bactérias da microbiota intestinal que por sua vez contribuem para a proteção contra infecções subsequentes.

Five experiments were conducted comparing differential intestinal immune responses to two isolates of Eimeria acervulina (EA), EA1 and EA2. In three experiments, broiler chicks were divided into control (non-challenged), EA1, or EA2 challenged (14 days of age) groups. On day 6 post-challenge (PC), changes in body weight were determined, intestinal lesions were scored, and duodenal tissue was evaluated for morphometric alterations and mucosal mast cell responses. EA1 produced duodenal lesions and reduced villus height to crypt depth ratios when compared to controls; however, no differences were found in mast cell counts. EA2 produced differing results, and observed data were suggestive of an intestinal secretory response when compared to EA1 or controls. In Experiment 4, tissues were analyzed from day 2 through day 6 PC. Villus atrophy and crypt hyperplasia were heightened on day 5 PC in both challenged groups. Mast cell counts were significantly greater on days 3 and 4 PC in EA1 birds. In Experiment 5, EA2 oocysts were cleaned with 5.25% sodium hypochlorite to evaluate the possibility of a bacterial contaminant contributing to the pathogenesis of intestinal alterations. Weight gains were decreased by challenge and villus heights and crypt depths were significantly altered in challenged birds, resulting in lower villus to crypt ratios, however, there were no differences in mast cell number. These data are indicative of differential host response and immunovariability between different isolates of the same Eimeria species and are suggestive of mast cell involvement in coccidial immunity in broiler chickens.
Master of Science

La Síndrome de l’Intestí Irritable (SII) és un trastorn gastrointestinal crònic i prevalent que cursa amb alteracions a la motilitat intestinal i amb dolor abdominal. La SII constitueix un trastorn recurrent i potencialment incapacitant sense un marcador de diagnòstic específic i únicament hi ha disponibles tractaments pal·liatius. L’absència d’una patofisiologia ben establerta destaca la necessitat d’identificar les causes orgàniques subjacents a les alteracions intestinal i la generació de símptomes. A la mucosa intestinals d’aquests pacients s’ha identificat un cert grau d’inflamació així com un increment de la permeabilitat intestinal i una major activitat de la resposta immunitària. Estudis previs del nostre grup van revelar un de la activitat humoral a la mucosa intestinal dels pacients SII amb predomini de diarrea (SII-D), associats a símptomes més severs. Per una altra banda, els pacients que pateixen SII sovint presenten ansietat i depressió recolzant que una disfunció de l’eix cervell-intestí podria estar involucrada en l’aparició i el desenvolupament dels símptomes. Considerant l’alta innervació de l’intestí i la modulació bidireccional descrita entre el sistema nerviós i el sistema digestiu, l’objectiu d’aquesta tesi doctoral és caracteritzar l’activació de la resposta humoral per mecanismes neuro-immunològics a la mucosa intestinal dels pacients amb SII-D. Per assolir el nostre objectiu, aquest projecte s’ha dividit en tres capítols. Al capítol 1, vam obtenir biòpsies jejunals, sang i femta de pacients amb SII-D i voluntaris sans. Vam quantificar les immunoglobulines (Igs) a les mostres de femta i es va observar que els nivells de IgG són més alts al grup SII-D, concretament IgG2 i IgG3, aquesta última sense diferències significatives. La quantitat de IgG total correlaciona de forma positiva amb la intensitat del dolor abdominal reportada pels pacients. Vam realitzar una anàlisis fenotípica a les biòpsies de l’expressió dels marcadors de cèl·lula plasmàtica CD38/CD138, el marcador neuronal PGP9.5 i TACR1 (receptor de substància P), involucrat a la senyalització nociceptiva. Vam observar que les cèl·lules plasmàtiques i las terminacions nervioses es troben en proximitat i en realitzar la quantificació d’aquesta distància mitjançant microscòpia electrònica de transmissió, els resultats van mostrar que les cèl·lules plasmàtiques estan significativament més properes a les terminacions nervioses al grup SII-D. Aquesta distància correlaciona amb els símptomes d’estrès agut i els nivells de depressió indicats pels pacients, l’últim sense assolir la significació estadística. Al capítol 2, vam realitzar una anàlisi de RNA-seq amb RNA extret de biòpsies jejunals de pacients amb SII-D i voluntaris sans. Vam dur a terme en aquestes mostres, una anàlisi d’enriquiment del conjunt de gens, vam observar que el fenotip immunològic de la resposta humoral està enriquit i 60% dels gens amb un nivell major d’enriquiment estan involucrats a l’estructura de les Ig. Les vies associades amb la funció barrera a l’intestí també es troben sobrerepresentades a la SII-D. Finalment, al capítol 3, es van avaluar diferents fonts d’obtenció de cèl·lula B com a model in vitro per estudiar l’efecte dels neuropèptids a l’activitat immunològica, més específicament la substància P, a l’activació/diferenciació de la cèl·lula B i la producció de Ig. Vam comparar cèl·lules de cultiu primari (cèl·lules B i cèl·lules plasmàtiques aïllades de la mucosa intestinal i sang) i una línia de cèl·lula B immortalitzada (126BLCL). També es va fer una anàlisis cèl·lules plasmàtiques diferenciades in vitro obtingudes a partir de les cèl·lules aïllades de sang. Rere una detallada caracterització fenotípica, vam concloure que les cèl·lules B de sang són el model in vitro més adequat i factible.
El Síndrome del Intestino Irritable (SII) es un trastorno gastrointestinal crónico y prevalente que cursa con alteraciones en la motilidad intestinal y dolor abdominal. Constituye un trastorno recurrente y potencialmente incapacitante para el cual no existe un marcador específico diagnóstico ni tratamientos especificos. La ausencia de una patofisiología bien establecida subraya la necesidad identificar las causas orgánicas subyacentes a las alteraciones intestinales y a la generación de síntomas. En la mucosa intestinal de estos pacientes, se ha identificado un cierto grado de inflamación, un incremento de la permeabilidad intestinal y una mayor actividad de la respuesta inmunológica. Estudios previos de nuestro grupo revelaron un incremento de la actividad humoral en la mucosa intestinal de los pacientes con SII con predominio de diarrea (SII-D), asociados a síntomas más severos. Por otra parte, los pacientes que sufren de SII a menudo presentan ansiedad y depresión, sustentando que una disfunción del eje cerebro-intestino podría estar involucrada. Teniendo en cuenta la alta inervación de la mucosa intestinal, juntamente con la modulación bidireccional entre sistema nervioso e inmunitario, el objetivo de esta tesis doctoral es caracterizar la activación de la respuesta humoral por mecanismos neuro-inmunológicos en la mucosa intestinal de los pacientes con SII-D. Para lograr nuestro objetivo, este proyecto se ha divido en tres capítulos. En el capítulo 1, obtuvimos biopsias yeyunales, sangre y heces de pacientes con SII-D y voluntarios sanos. Cuantificamos las Ig en muestras de heces y observamos que los niveles de inmunoglobulina (Ig) G son más altos en el grupo SII-D, concretamente IgG2 e IgG3, esta última sin alcanzar diferencias significativas. La cantidad de IgG total correlaciona de forma positiva con la intensidad del dolor abdominal reportada por los pacientes. Llevamos a cabo un análisis fenotípico en las biopsias de la expresión de los marcadores de célula plasmática CD38/CD138, el marcador neuronal PGP9.5 y la expresión de TACR1 (receptor de sustancia P), involucrado en la señalización nociceptiva. Observamos que las células plasmáticas y las terminaciones nerviosas se encuentran en proximidad y, cuando realizamos la cuantificación de esta distancia mediante microscopía electrónica de transmisión, los resultados mostraron que las células plasmáticas están significativamente más cerca de las terminaciones nerviosas en SII-D. Esta distancia correlaciona con los síntomas de estrés agudo y los niveles de depresión, el segundo sin llegar a alcanzar la significación estadística. En el capítulo 2, realizamos un análisis de RNA-seq con RNA extraído de biopsias yeyunales de pacientes con SII-D y voluntarios sanos. En estas muestras llevamos a cabo un análisis de enriquecimiento del conjunto de genes observamos que el fenotipo inmunológico de la respuesta humoral está enriquecido y 60% de los genes con mayor nivel de enriquecimiento están involucrados en la estructura de las Igs. Las vías asociadas con la función barrera en el intestino también se encuentran sobrerrepresentadas en el SII-D. Finalmente, en el capítulo 3, se evaluaron diferentes fuentes de obtención de célula B como modelo in vitro para estudiar el efecto de los neuropéptidos sobre la actividad inmunológica, más específicamente la sustancia P, en la activación/diferenciación de la célula B y la producción de Igs. Comparamos células de cultivo primario (células B y células plasmáticas aisladas de la mucosa intestinal y sangre) y una línea de célula B inmortalizada (126BLCL). También analizamos células plasmáticas diferenciadas in vitro obtenidas de las células aisladas de sangre. Tras realizar una detallada caracterización fenotípica, concluimos que las células B de sangre son el modelo in vitro más adecuado y factible para alcanzar nuestro objetivo.
Irritable Bowel Syndrome (IBS) is a chronic and prevalent gastrointestinal disorder which curses with intestinal motility alterations and abdominal pain. IBS constitutes a relapsing and potentially disabling disorder and, currently, there is no specific diagnosis biomarker and only palliative treatments are available. The absence of a well-established pathophysiology highlights the need of identifying the underlying organic causes of motility alterations and the onset of symptoms. In the intestinal mucosa of these patients a certain degree of inflammation has been identified together with an increased intestinal permeability and a higher activity of the immune response. Previous studies from our group showed an increased humoral activity in the intestinal mucosa of diarrhea-predominant IBS (IBS-D) patients, associated with more severity of the symptoms. IBS patients often present anxiety and depression, and dysfunction of the gut-brain axis features IBS onset and outcome. Considering intestinal mucosa is highly innervated and the existence of a bidirectional modulation between nervous and immune systems, the main objective of this thesis was to characterize the activation humoral response by neuro-immune mechanisms in the intestinal mucosa of IBS-D patients. To achieve our purpose, this project has been divided into three chapters. In chapter 1, we collected jejunal biopsies, blood and feces from IBS-D patients and healthy volunteers. We quantified immunoglobulins (Igs) in stool and observed higher levels of IgG in IBS-D group, more specifically IgG2 and IgG3, despite this last one did not reach statistical significance. The amount of total IgG positively correlated with the intensity of the abdominal pain reported by the patients. We conducted a phenotypical analysis in jejunal biopsies for the expression of CD38 and CD138 plasma cell marker, PGP9.5 neural marker and TACR1 (substance P receptor) expression, involved in nociceptive signaling. We observed that plasma cells and nerve endings are found in proximity and, when we performed a quantification of this distance by transmission electron microscopy (TEM), results showed plasma cells are significantly close to nerve endings in the IBS-D group. This distance inversely correlates with acute stress symptoms and depression score reported by patients, the later not reaching significance. In Chapter 2, an RNA-seq analysis was conducted in RNA extracted from IBS-D patients and healthy volunteer jejunal biopsies. We performed a Gene Set Enrichment Analysis with all the genes in these samples; we observed the humoral response immunological phenotype is enriched and 60% of the genes with a highest enrichment score are involved in Ig structure. Pathways associated to intestinal barrier function are also overrepresented in IBS-D. Finally, in Chapter 3, we evaluated several sources to obtain a B cell in vitro model to study the effect of neuropeptides on immune activity, more specifically substance P, in B cell activation/differentiation and Ig production. We confronted primary culture cells (B and plasma cells isolated from intestinal mucosa and blood) and an established B cell line (126BLCL). In vitro differentiated plasma cells obtained from blood were also analyzed. After conducting a deep phenotypic characterization, we concluded blood isolated B cells are the most suitable and feasible in vitro model for our purpose. The results of this thesis reinforce the hypothesis of the neuro-immune crosstalk in the intestinal as playing a crucial role in IBS pathophysiology.

Previous studies of the ontogeny of the mucosal immune system have shown a significant increase in salivary Immunoglobulin A levels occurring at about five years of age. This study has monitored a group of 3 and 4 year old children during one year of attendance at Pre-School to examine whether such an increase could be linked to increased antigenic exposure associated with moving into a school like environment. Saliva samples were collected at regular intervals and analysed for immunoglobulin and total protein levels. Daily health records were maintained for each child, and a detailed social and medical history was collected for each child at the beginning of the study. The elevated mucosal immune response observed in previous studies involving children in day care centres and attending school was not seen in this study. No significant difference was observed between children who had previously attended Pre-School or child care centres and those who were attending for the first time. However, a marked seasonal increase in mean salivary IgA during the winter months was observed and this increase correlated with an increase in respiratory infections. Hence, in studies of developmental aspects of mucosal immune response it is essential that modifiers such as season and infection be recorded.

It is now accepted that gut microbiota are crucial for the development of the mucosal immune system. It has been suggested that differences in early life microbial colonisation may be associated with variable predisposition to allergic, auto immune, and inflammatory diseases. The exact underlying mechanisms are difficult to study in human infants, however, the similarities in physiology and nutritional requirements between pigs and humans suggest that piglets can be good models to elucidate the pathways involved. The aim of this PhD project was to investigate how differences in microbiota, early on in life, could affect immune regulation in telms of regulatory T cells (Tregs), a cell type fundamental for immune homeostasis. Manipulation of gut microbiota was attempted using different housing conditions [specific pathogen-free (SPF) facility and farm], birth environment (indoor and outdoor fmm) as well as with administration of antibiotics and specific microbiota inocula (with simple and complex composition). Furthermore, the effect of nutritional interventions, (inulin, starch and medium-chain triglycerides) with the potential to manipulate gut microbiota, was also studied. The effect of these manipulations on small intestinal Tregs was examined using fluorescent immunohistology. Furthermore, activation-induced Foxp3 expressions on gut and blood CD4 T cells was also investigated using flow cytometry. It was observed that indoor-born piglets were more susceptible to a reduction in Tregs when transfened to an SPF facility than outdoor bom piglets, but treatment with antibiotics reduced gut Tregs of outdoor-bom piglets at the level of those born indoor. On the other hand, colonisation of new-bom piglets with a complex microbiota inoculum reduced gut Tregs in comparison to the simple microbiota inoculum. However, none of the nutritional interventions had a significant effect on Tregs. Furthermore, no activation induced expression ofFoxp3 was observed in either gut or blood CD4 T cells of 5-month old piglets. The results of this Thesis suggest that both environment and direct manipulation of gut microbiota can affect levels of small intestinal Tregs, whereas the effect of nutrition is less clear. A more detailed analysis of small-intestinal microbiota is necessary to confirm that these observations are a result of differences in the microbiome between the groups or whether other possible factors are also involved.

The mucosal system, which forms a barrier between internal organ systems and the external environment, is frequently exposed to pathogenic microorganisms. Immunoglobulin A (IgA) antibody secreting cells (ASCs) localize in the lamina propria, and produce IgA antibodies which help protect mucosal tissues. The concept of a common mucosal immune system in which IgA ASCs have the ability to populate any mucosal site has been proposed (1, 2). However, recent research has suggested that IgA ASCs primed in different mucosal sites might possess different sets of chemokine receptors, and therefore migrate specifically to particular mucosal locations (3). In this study, the specific compartmentalization of IgA ASCs in two mouse salivary glands: sublingual gland (SLG), and submandibular gland (SMG) was studied. It was observed that SLG had 12 times more IgA ASCs per gram of gland than that of SMG (p<0.01). This suggested that IgA ASCs migrated to the two salivary glands with different efficiencies. Since the migration of lymphocytes is mediated by interactions between tissue specific chemokines and chemokine receptors, I hypothesized that the specific compartmentalization of IgA ASCs in the SLG and SMG was mediated by the differential expression of IgA ASC attracting chemokines. Quantitative PCR was used and showed that SLG expressed high levels of CCL28 and its receptor CCR10, which correlated to the distribution of IgA ASCs in the two salivary glands. In agreement with QPCR data, reduced levels of IgA ASCs were found in the SLG of CCR10 deficient mice when compared to wild type (WT) mice. Adoptive transfer of CCR10 deficient mice with WT spleen cells reconstituted the WT phenotype. It was therefore concluded that the interaction between CCL28 and CCR10 play an important role in mediating the migration of IgA ASCs into SLG. These results suggested that the accumulation of IgA ASC to distinct salivary glands is a highly selective process. These data also suggested that homing within mucosal sites is not common but rather a highly regulated process with specific subsets of cells homing to different tissues within the mucosal immune system.

Mice lacking the transcription factor T-bet in the innate immune system (Tbx21-/- x Rag2-/- Ulcerative Colitis, or TRUC) develop microbiota-dependent inflammatory bowel disease (IBD). The innate immune mechanisms responsible for causing disease are still to be resolved, yet potentially offer novel insights into the pathogenesis of IBD, and how T-bet might influence inflammation in the gut. In this thesis it was shown that chronic IBD in TRUC mice was dependent on interleukin-17A (IL-17A) producing IL-7R+ CD90+ innate lymphoid cells (ILCs). Depletion of ILCs or IL-17A neutralization cured disease. Cytokines responsible for driving innate IL-17A included IL-23, IL-6 and TL1A. IL-23 and IL-6 played functionally important roles in TRUC disease, since neutralisation reduced innate IL-17A production and attenuated disease. TNFα, predominantly produced by CD11chigh class II+ CD11b+ CD103- dendritic cells (DCs), synergised with IL-23 to drive IL-17A production by ILCs. Helicobacter typhlonius (HT) was identified as the key colitigenic bacterium driving TRUC disease. Four-five-four ribosomal RNA gene sequencing demonstrated that HT was consistently present in the faeces of all TRUC mice, but absent from a newly-derived colony of disease-free Tbx21-/- x Rag2-/- mice. Inoculation of pure HT cultures was sufficient to reinstate disease into this new colony of healthy mice. T-bet impacted on colitis in several ways. In the absence of T-bet, ILCs selectively produced IL-17A and were poor producers of interferon-γ. Second, T-bet deficient DCs produced excess TNFα in comparison with T-bet sufficient DCs. Finally, T-bet was a transcriptional repressor of the Il7ra locus. IL-7R was expressed by ILCs and signalling through this receptor was critical for TRUC disease. Selective IL-7R blockade attenuated disease. Together these data provide new insights into TRUC disease and demonstrate at least some of the mechanisms by which T-bet regulates the interplay between mucosal innate immune cells and the intestinal microbiota.

In title, [beta] is represented by the Greek letter. Copies of author's previously published articles inserted. Errata pages pasted onto back end-paper. Bibliography: leaves 104-137. Studies milk TGF-[beta] and its receptors in the post-natal gut using a rat model to investigate a link between milk TGF-[beta] and the development of the infant gut and gut mucosal immune system. Finds maternal milk may be a major source of TGF-[beta] to the immature gut and may react with receptors on the cells of the mucosal immune system along the gastro-intestinal tract, modulating infant mucosal immune responses in the transition to the post-natal enteral feeding.

The intestinal tract of salmonids provides a dynamic interface that not only mediates nutrient uptake but also functions as the first line of defence against ingested pathogens. Exposure of the immune system to beneficial microorganisms and different dietary immunostimulants via the intestine has been shown to prime the immune system and help in the development of immune competence. Furthermore, the morphology and function of teleostean intestines are known to respond to feed components and to ingested and resident bacterial communities. Histological appraisal is still generally considered to be the gold standard for sensitive assessment of the effects of such dietary modulation. The aim of the present study was to improve understanding of salmonid intestinal function, structure and dynamics and to use the knowledge gained to develop a model for analysis, which would allow intestinal health to be assessed with respect to different intestinal communities and feed components. Virtual histology, the process of assessing digital images of histological slides, is gaining momentum as an approach to supplement traditional histological evaluation methodologies and at the same time, image analysis of digitised histological sections provides a practical means for quantifiable assessment of structural and functional changes in tissues, being both objective and reproducible. This project focused on the development of a rapid, practical analytical methodology based on advanced image analysis, that was able to measure and characterise a range of features of the intestinal histology of Atlantic salmon in a quantitative manner. In the first research chapter, the development of a novel histological assessment system based upon advanced image analysis was described, this being developed with the help of a soybean feed model known to induce enteropathy in Atlantic salmon. This tool targeted the evaluation of the extent of morphological changes occurring in the distal intestine of Atlantic salmon following dietary modulation. The final analytical methodology arrived at, could be conducted with minimal user-interaction, allowing rapid and objective assessment of 12 continuous variables per histological frame analysed. The processing time required for each histological frame was roughly 20-25 min, which greatly improved the efficiency of conducting such a quantitative assessment with respect to the time taken for a subjective semi-quantitative alternative approach. Significant agreement between the fully automated and the manual morphometric image segmentation was achieved, however, the strength of this quantitative approach was enhanced by the employment of interactive procedures, which enabled the operator / observer to rectify preceding automated segmentation steps, and account for the specimen’s variations. Results indicated that image analysis provided a viable alternative to a pathologist’s manual scoring, being more practical and time-efficient. In the second research chapter, feeding Atlantic salmon a high inclusion level of unrefined SBM (25 %) produced an inflammatory response in the distal intestine as previously described by other authors. The model feed trial successfully generated differentiable states, although these were not, for the most part, systemically differentiable through the majority of standard immunological procedures used, being only detectable morphologically. Quantitation of morphometric parameters associated with histological sections using the newly developed image analysis tool successfully allowed identification of major morphological changes. Image analysis was thus shown to provide a powerful tool for describing the histomorphological structure of Atlantic salmon distal intestine. In turn, the semi-automated image analysis methods were able to distinguish normal intestinal mucosa from those affected by enteritis. While individual parameters were less discriminatory, use of multivariate techniques allowed better discrimination of states and is likely to prove the most productive approach in further studies. Work described in the third research chapter sought to validate the semi-automated image analysis system to establish that it was measuring the parameters it was purported to be measuring, and to provide reassurance that it could reliably measure pre-determined features. This study, using the same sections for semi-quantitative and quantitative analyses, demonstrated that the quantitative indices performed well when compared to analogous semi-quantitative descriptive parameters of assessment for enteritis prognosis. The excellent reproducibility and accuracy performance levels indicated that the image analysis system was a useful and reliable morphometric method for the quantification of SB-induced enteritis in salmon. Other characteristics such as rapidity, simplicity and adaptability favour this method for image analysis, and are particularly useful where less experienced interpreters are performing the analysis. The work described in the fourth research chapter characterised changes in the morphology of the intestinal epithelial cells occurring as a result of dietary modulation and aspects of inflammatory infiltration, using a selected panel of enzyme and IHC markers. To accomplish this, image analysis techniques were used to evaluate and systematically optimise a quantitative immunolabelling assessment protocol. Digital computer-assisted quantification of labelling for cell proliferation and regeneration; programmed cell death or apoptosis; EGCs and t-cell like infiltrates; mobilisation of stress-related protein regenerative processes and facilitation of nutrient uptake and ion transport provided encouraging results. Through the description of the intestinal cellular responses at a molecular level, such IHC expression profiling further characterised the inflammatory reaction generated by the enteropathic diet. In addition, a number of potential diagnostic parameters were described for fish intestinal health e.g. the relative levels of antigenicity and the spatial distribution of antigens in tissues. Work described in the final research chapter focused on detailed characterisation of intestinal MCs / EGCs in order to try to elucidate their functional role in the intestinal immune responses. Through an understanding of their distribution, composition and ultrastructure, the intention was to better characterise these cells and their functional properties. The general morphology, histochemical characteristics and tissue distribution of these cells were explored in detail using histochemical, IHC and immunogold staining / labelling, visualised using light, confocal and TEM microscopy. Despite these extensive investigations, their physiological function and the content of their granules still remain somewhat obscure, although a role as immunodulatory cells reacting to various exogeneous signals through a finely regulated process and comparable to that causing the degranulation of mammalian MCs is suggested. The histochemical staining properties demonstrated for salmonid MCs / EGCs seem to resemble those of mammalian mucosal mast cells, with both acidophilic and basophilic components in their granules, and a granule content containing neuromodulator / neurotransmitter-peptides such as serotonin, met-enkephalin and substance-p. Consequently, distinguishable bio-chromogenic markers have been identified that are of utility in generating a discriminatory profile for image analysis of such cells.

Helicobacter pylori causes chronic gastritis, peptic ulceration and gastric cancer. This bacterium is one of the most prevalent in the world, but affects mostly the populations with a lower socioeconomical status. While it causes gastric and duodenal ulcers in only 20% of infected patients, less then 1% will develop gastric adenocarcinoma. In fact, H. pylori is the most important risk factor in developing gastric cancer. Epidemiological studies have shown that 80% of gastric cancer patients are H. pylori positive. The outcome of the infection with this bacterium depends on bacterial factors, diet, genetic background of the host, and coinfection with other microorganisms. The most important cofactor in H. pylori induced disease is the host immune response, even though the exact mechanism of how the bacterium is causing disease is unknown. The structural complexity of Helicobacter bacteria makes us believe that different bacterial factors interact with different components of the innate immunity. However, as a whole bacterium it may need mainly the TLR2 receptor to trigger an immune response. The type of adaptive immunity developed in response to Helicobacter is crucial in determining the consequences of infection. It is now known for decades that a susceptible host will follow the infection with a strong Th1 immune response. IFNγ, IL-12, IL-1β and TNF-α are the key components of a strong adaptive Th1 response. This is further supported by our work, where deficient T-bet (a master regulator for Th1 response) mice were protected against gastric cancer, despite maintaining an infection at similar levels to wild type mice. On the other hand, a host that is resistant to Helicobacter develops an infection that is followed by a Th2 response sparing the mucosa from severe inflammation. Human studies looking at single nucleotide polymorphism of cytokines, like IL-1β, IL-10 and TNF-α have clearly demonstrated how genotypes that result in high levels of IL-1β and TNF-α, but low IL-10 expression may confer a 50-fold higher risk in developing gastric cancer. The outcome of Helicobacter infection clearly relies on the immune response and genetic background, however the coinfection of the host with other pathogens should not be ignored as this may result in modulation of the adaptive immunity. In studying this, we took advantage of the Balb/C mice that are known to be protected against Helicobacter induced inflammation by mounting a strong Th2 polarization. We were able to switch their adaptive immunity to Th1 by coinfected them with a T. gondii infection (a well known Th1 infection in mice). The dual infected mice developed severe gastritis, parietal cell loss and metaplastic changes. These experiments have clearly shown how unrelated pathogens may interact and result in different clinical outcomes of the infected host. A strong immune response that results in severe inflammation will also cause a cascade of apoptotic changes in the mucosa. A strict balance between proliferation and apoptosis is needed, as its disruption may result in uncontrolled proliferation, transformation and metaplasia. The Fas Ag pathway is the leading cause of apoptosis in the Helicobacter-induced inflammation. One mechanism for escaping Fas mediating apoptosis is upregulation of MHCII receptor. Fas Ag and MHCII receptor interaction inhibits Fas mediated apoptosis by an impairment of the Fas Ag receptor aggregation when stimulated by Fas ligand. Because H. pylori infection is associated with an upregulation of the MHCII levels on gastric epithelial cells, this indeed may be one mechanism by which cells escape apoptosis. The link between chronic inflammation and cancer is well known since the past century. Helicobacter infection is a prime example how a chronic inflammatory state is causing uncontrolled cell proliferation that results in cancer. The cell biology of “cancer” is regarded not as an accumulation of cells that divide without any control, but rather as an organ formed of cancer stem cells, tumor stromal support cells, myofibroblasts and endothelial cells, which function as a group. The properties of the cancer stem cells are to self-renew and differentiate into tumor cells thus maintaining the tumor grow, emphasizing that a striking similarity exists between cancer stem cells and tissue stem cells. We looked into what role would BMDCs play in chronic inflammation that causes cancer. Using the mouse model of Helicobacter induced adenocarcinoma we discovered that gastric cancer originates from a mesenchymal stem cell coming from bone marrow. We believe that chronic inflammation, in our case of the stomach, sets up the perfect stage for bone marrow stem cells to migrate to the stomach where they are exposed to inflammatory stimuli and transform into cancer stem cells. One of the mechanism by which the MSC migrate to the inflammation site is the CXCR4/SDF-1 axis. Our work sheds new light on Helicobacter induced gastric cancer pathogenesis. I hope that our findings will promote the development of new therapies in the fight against this deadly disease.

The intestine is the largest lymphoid organ in the body, challenged constantly by an enonnous quantity and diversity of antigens. Distinct from peripheral lymphocytes, intestinal lymphocytes have evolved unique mechanisms of tolerance and appear to govern mucosal processes such as "chronic physiologic inflammation" and oral tolerance. Failure of mucosal tolerance has been implicated in the pathogenesis of several diseases, including inflammatory bowel disease, celiac disease, and even autoimmune diabetes. One population of intestinal lymphocytes, intraepithelial lymphocytes (IELs), exists within the intestinal epithelium itself and remains poorly characterized. IELs respond to unique activation signals and appear to be in part responsible for the maintenance of epithelial integrity and mucosal tolerance. Type 1 diabetes is one of the most common chronic childhood illnesses and causes significant morbidity and mortality. Type 1 diabetes mellitus is an autoimmune disease that results from immune-mediated destruction of insulin-producing pancreatic beta cells and is characterized by an absolute insulin deficiency. Several animal models are used to study the immunopathogenesis of type 1 diabetes, including the BB rat and NOD mouse. BBDP rats spontaneously develop autoimmune diabetes mellitus and are severely deficient in peripheral T cells. BBDR rats do not spontaneously develop autoimmune diabetes, have nonnal numbers of peripheral T cells, and can be induced to become diabetic by injections of a cytotoxic anti-ART2a mAb and low doses of poly I:C. The cause of autoimmune diabetes in BB rats and humans is still unknown, but both genetic and environmental factors appear to participate. I hypothesize that one important class of environmental factors--diet and enteromicrobial agents--participates in this pathogenic process through the mediation of the gut immune system. In this dissertation, I report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The yield of rat IELs using this method is 5-10 fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrate that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyer's patch lymphocytes. Phenotypic analysis reveals 5 major subsets of IELs, including populations of γδ T and natural killer (NK) cells present at levels not previously detected. I also report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, IENK cells differ from splenic NK cells phenotypically, and a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-γ. I conclude that rat IELs harbor a large population of NKR-P1A+ CD3-cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive intraepithelial NK cells may participate in the regulation of mucosal immunity. I next demonstrate that, prior to diabetes, both BBDP and ART2a-depleted BBDR rats have a reduced total number of IELs and exhibit a selective deficiency of IENK cell number and function as compared to control BBDR rats. The deficiency of BBDP rat IELs can be corrected by engraftment of bone marrow from histocompatible WF donors. These results suggest 1) that the peripheral lymphopenia in BBDP rats extends to the IEL compartment, particularly to IENK cells, 2) that in BBDR rats the diabetes-inducing treatment depletes IELs, particularly IENK cells, and 3) that the defect in BBDP rat IELs is intrinsic to hematopoietic cells, not intestinal stromal cells. I also establish that, unlike BBDR and WF rats, BBDP rats are also deficient in γδTCR+IELs, a population of T cells that may play a role in normal mucosal tolerance. In addition, I report preliminary data supporting the hypothesis that systemic autoreactivity may be initiated in the intestine; peripheral autoreactive lymphocyte populations appear to emanate first from mesenteric lymph nodes that drain the intestine, and such cells may initiate a type 2 autoimmune phenomenon driven by IL-4. Collectively, my findings support the hypothesis that a failure of mucosal tolerance in BBDP rats, perhaps secondary to deficiencies in one or more IEL subpopulations, participates in the pathogenesis of autoimmune diabetes in these animals by activating peripheral autoreactive T cells. The nature of the autoimmune response in BB rats (driven by IL-4) appears to be distinct from that of NOD mice. Despite the differences between these two well-accepted animal models of autoimmune diabetes, until more is known about the pathogenesis of type 1 DM in humans, lessons learned from both the BB rat and NOD mouse continue to be of tremendous benefit to our understanding of human disease.

Citocinas geradas durante a I/R-i promovem lesão pulmonar aguda e podem causar imunossupressão. Investigamos se a inflamação pulmonar após dias de reperfusão intestinal está relacionada com alterações no sistema imune. Ratos machos Wistar foram submetidos a oclusão da artéria mesentérica superior por 45 min e a 2 h, 3, 5 ou 20 dias de reperfusão. Observamos que após 5 dias de reperfusão intestinal enterobactérias estão presentes nos linfonodos mesentéricos e que os neutrófilos circulantes estão mais ativos no combate a patógenos, enquanto leucócitos da cavidade peritoneal não. Notamos aumento de IL-1b, TNF-a, IL-17, IL-4 e diminuição de IL-10 no explante pulmonar e aumento de IL-1b, IFN-g e LDH no soro. Já os níveis de nitritos, corticosterona, HMGB-1, ácido úrico, AMPc e a mecânica respiratória não se alteraram. Os resultados apontam para a I/R-i como promotora de modificação temporais no organismo. Alterações nos mecanismos regulatórios da inflamação e do sistema imune contribuem para a inflamação pulmonar e sistêmica, sem alterar a função mecânica pulmonar
Cytokines generated during intestinal I/ R promote acute lung injury and may cause immunosuppression. We investigate if the pulmonary inflammation observed after several days of reperfusion is related to changes in the immune system. Male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min and reperfused for 2 h , 3, 5 or 20 days. We observed that after 5 days of reperfusion enterobacteraceae are present in mesenteric lymph nodes and that circulating neutrophils are more effective against pathogens, whereas leukocytes from the peritoneal cavity are not. We noted increase of IL-1b, TNF-a, IL-17, IL -4 and decrease of IL-10 in lung explant and increase of IL-1b, IFN-g and LDH in serum. Furthermore, the levels of nitrites, corticosterone, HMGB -1, uric acid, cAMP, as well as respiratory function, did not change. Based on the foregoing, we observed that alterations in regulatory mechanisms of inflammation and immune system after contribute to pulmonary and systemic inflammation, but do not alter pulmonary mechanical function.

Aunque el uso de altos niveles de fuentes de proteína vegetal en piensos para doradas de engorde se ha alcanzado con éxito en cuanto al crecimiento, estas dietas todavía están asociadas a efectos negativos en la eficiencia nutricional y en la capacidad inmunitaria. El intestino es el órgano donde se produce la primera interacción entre el pez, los nutrientes y las bacterias del medio, y desarrolla un papel crucial en la digestión de los nutrientes y la respuesta infamatoria e inmune. Esta tesis doctoral se centra en el impacto de distintas dietas con altos niveles de proteína vegetal, y especialmente, en la evaluación del estatus intestinal de las doradas de engorde alimentadas con altos niveles de sustitución de la harina de pescado durante un periodo largo de tiempo. Los cambios observados en el intestino se caracterizaron mediante el uso de distintas estrategias, como el análisis de la digestibilidad y la retención de amino ácidos, de la excreción de amonio, de la actividad de enzimas digestivos, de los cambios histológico o de la expresión de genes relacionados con la función y el mantenimiento de la arquitectura intestinal, así como técnicas ómicas para el análisis del proteoma y de la microbiota intestinal. Se ensayaron distintos niveles de sustitución de harina de pescado, pero el impacto de las dietas con una sustitución completa, bien complementada con subproductos de origen marino o suplementada con aminoácidos libres sintéticos, recibió mayor atención. La sustitución completa de la harina de pescado provocó una reducción, aunque ligera, del crecimiento y de la eficiencia digestiva y nutritiva de la dorada de engorde, aunque el impacto sobre el crecimiento era mayor cuando los peces eran alimentados desde la época de juveniles con estas dietas. La digestibilidad y el nivel de síntesis de proteína se vio alterada, aunque no se observaron diferencias significativas en la actividad enzimática digestiva. No obstante, el impacto de las fuentes vegetales cuando no había fuentes de proteína marina en la dieta era especialmente crítico para la supervivencia de los peces. En el intestino de estos peces solo se observaron diferencias menores relacionadas con la inflamación a nivel histológico, pero también se observó una disminución en la expresión génica de genes involucrados en la inflamación y la respuesta inmune. El análisis de la microbiota intestinal reveló cambios significativos en la composición de su composición, especialmente en el intestino posterior, sugiriendo una posible falta de capacidad de regular la respuesta inmune y de modular la colonización de bacterias patógenas tras un largo periodo de alimentación con esta dieta. Por otro lado, el análisis del proteoma de la mucosa intestinal también mostró un claro impacto sobre distintos procesos biológicos relacionados con el mantenimiento del homeostasis intestinal y de la integridad epitelial. Por el contrario, no se observó un impacto de la sustitución de la harina de pescado a nivel de expresión génica o del proteoma cuando se incorporaba a la dieta una fuente de proteína marina complementaria, aunque sí que se observaron algunos signos menores de inflamación. Por último, se desarrolló un sistema ex vivo para estudiar la respuesta inflamatoria e inmune de la mucosa intestinal a la presencia de distintas bacterias, y se realizó un ensayo preliminar en dorada para evaluar el efecto de la dieta sobre esta respuesta. En resumen, en este trabajo se ha realizado una evaluación extensa y detallada de los efectos a nivel intestinal de la inclusión de altos niveles de proteína vegetal en la dieta para doradas de engorde. Los resultados indican que las alteraciones en la capacidad inmune, la homeostasis y la microbiota intestinal aparecían solo cuando la proteína procedía exclusivamente de fuentes vegetales, y podrían explicar la mayor mortalidad registrada con esta dieta.
Malgrat que la utilització d'alts nivells de proteïna vegetal en pinsos per a dorades en la fase d'engreixament s'ha aconseguit amb èxit en quan al creixement, aquestes dietes encara s'associen amb freqüència amb efectes negatius en l'eficiència nutricional i la capacitat immunitària. L'intestí és l'òrgan on es produeix la primera interacció entre el peix, els nutrients de la dieta i les bactèries de l'ambient, i juga un paper fonamental en la digestió dels nutrients i en la resposta inflamatòria i immune. Aquesta tesi doctoral es centra en l'impacte de diferents dietes experimentals amb un alt nivell de proteïna vegetal, i especialment, en l'avaluació de l'estat de l'intestí de les dorades d'engreixament alimentades durant un llarg període amb alts nivells de substitució de farina de peix. Els distints canvis observats a nivell intestinal es van descriure mitjançant l'ús de distintes estratègies, com l'anàlisi de la digestibilitat i la retenció dels aminoàcids, de l'excreció d'amoni i de l'activitat enzimàtica, dels canvis histològic o de l'expressió de gens relacionats amb la funció i el manteniment de l'estructura intestinal, així com tècniques òmiques per a l'anàlisi del proteoma i de la microbiota intestinal. Es van assatjar diferents nivells de substitució de farina de peix, però l'impacte de les dietes amb substitució completa, bé complementada amb subproductes d'origen marí o suplementada amb aminoàcids lliures sintètics, va rebre major atenció. La substitució completa de la farina de peix va tenir un efecte lleugerament negatiu sobre el creixement i l'eficiència digestiva i nutritiva de la dorada d'engreixament, encara que l'impacte era major quan els peixos eren alimentats des de la fase de juvenils amb aquesta dieta. La digestibilitat i el nivell de síntesis de proteïna es va veure alterada, encara que no s'observaren diferències significatives en l'activitat dels enzims digestius. No obstant, l'impacte de les fonts vegetals quan s'eliminaven per complet les fonts de proteïna marina de la dieta era especialment crític en la supervivència dels peixos. En l'intestí d'aquests peixos sols s'observaren xicotets indicis d'inflamació a nivell histològic, però també es va observar una disminució l'expressió de gens involucrats amb el procés inflamatori i la resposta immune. L'estudi de la microbiota intestinal va revelar canvis significatius en la composició, especialment a l'intestí posterior, suggerint una possible falta de capacitat de regular la resposta immunitària i de modular la colonització per part de patògens després d'un llarg període d'alimentació amb aquesta dieta. D'altra banda, l'anàlisi del proteoma de la mucosa intestinal també va mostrar un impacte clar sobre diferents processos biològics relacionats amb el manteniment de l'homeòstasi intestinal i de la integritat de l'epiteli. Per contra, no es van observar un impacte de la substitució de la farina de peix a nivell d'expressió gènica o proteoma quan s'incloïa a la dieta una font complementària de proteïna d'origen marí, encara que sí que s'observaven alguns signes d'inflamació. Per últim, es va desenvolupar un sistema ex vivo per avaluar la resposta inflamatòria i immune de la mucosa intestinal davant la presència de diferents bactèries, i es va realitzar un assaig preliminar per determinar l'efecte de la dieta sobre aquesta resposta. En resum, en aquest treball s'ha realitzat una avaluació extensa i detallada dels efectes a nivell intestinal de la inclusió d'alts nivells de fonts de proteïna vegetal a les dieta per a les dorades d'engreixament. Els resultats indiquen que les alteracions en la capacitat immunitària, l'homeòstasi i la microbiota intestinal eren observades solament quan la proteïna era exclusivament obtinguda de fonts vegetals, i podrien explicar la major mortalitat observada amb aquesta dieta.
Although the inclusion of plant protein sources at high levels in aquafeeds for on-growing gilthead seabream has been successfully achieved on gilthead seabream in terms of growth, these diets are still associated to detrimental effects in feed efficiency and immune capacity. The intestine is the organ where takes place the first interaction of the host with dietary antigen or environmental bacteria, and plays a major role in the digestion of nutrients and the inflammatory and the immune response. The present PhD thesis focus on the impact of classical formulated high plant protein diets on fish performance, but especially, on evaluation of the intestinal status in on-growing fish long-term fed with high levels of fishmeal replacement. Changes at intestinal level were characterized by using different approaches, including protein and amino acid digestibility and retention and ammonia excretion, digestive enzyme activity, histology, expression of genes related with inflammation, immunity, structure and digestion, but also using whole tissue-level techniques for the analysis of the impact on proteome and gut microbiota. Different levels of fishmeal replacement were assayed, although the impact of diets with total replacement, complemented by inclusion of alternative marine by-products or supplemented by free amino acids, received greater attention. Total fish replacement produced a negative but minor impact on the growth and nutritive and digestive performance of on-growing gilthead seabream. Nevertheless, when fish were fed from juvenile stage with plant protein based diets, a higher negative impact in growth terms was noticed. Digestibility and metabolic use of amino acids was altered, but no differences were observed in the digestive enzyme activities. Nonetheless, feeding fish with total dietary fishmeal replacement by plant protein without any marine protein source was especially critical for survival rate. In these fish, gut histological assessment only revealed minor alterations related with an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response. Moreover, a drastic change in the microbiota composition was observed, especially at the hindgut, revealing a possible lack of capacity to regulate a defensive response and to face with pathogen colonisation after a long-term coupling with these diet. Likewise, gut mucosa proteome analysis also suggests an impact on biological processes related with the maintenance of gut homeostasis and the epithelial integrity. In contrast, total fishmeal replacement did not induce alterations at transcript or proteomic level when diet was complemented with marine ingredients, although some minor inflammatory signs were reported. On the other hand, an ex vivo system to study the inflammatory and immune response of the gut mucosa to the presence of different bacteria was developed, and a preliminary assay evaluating the impact of the diet on this response was performed. To sum up, present works represents a wide assessment at intestinal level of the effects of including plant protein sources at high levels in aqua feeds for on-growing gilthead seabream. Results indicate that alterations in the immune capacity, the gut homeostasis and the microbiota were observed when protein was exclusively provided by plant sources, and could explain the higher mortality reported with this diet.
Estruch Cucarella, G. (2018). ASSESSMENT OF THE LONG-TERM IMPACT OF HIGH PLANT PROTEIN DIETS ON THE INTESTINAL STATUS OF THE ON-GROWING GILTHEAD SEA BREAM (Sparus aurata, L.) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113063
TESIS

En els últims anys ha crescut considerablement l’interès per descobrir aliments naturals funcionals amb propietats beneficioses per a l’hoste. En aquest sentit, el cacau ha passat a ser un dels principals subjectes d’estudi pel seu contingut en flavonoides. Són molts els estudis que associen la ingesta de cacau amb efectes beneficiosos sobre la salut. A més, se li han atribuït propietats immunomoduladores en rata. En base a aquests efectes descrits, l’objectiu d’aquesta Tesi Doctoral va ser establir l’efecte de dietes enriquides amb cacau, flavonoides del cacau i fibra de cacau sobre la microbiota intestinal així com sobre la funció immunitària intestinals. Per tal d’assolir aquests objectius, s’han portat a terme estudis preclínics en rata amb una dieta enriquida amb cacau convencional al 10%, dietes elaborades a partir de dos extractes de cacau no fermentats i una dieta amb fibra de cacau. Pel que fa als resultats de microbiota, es va observar diferent patró de composició després de la intervenció nutricional amb les dietes enriquides amb flavonoides del cacau però únicament la dieta de fibra de cacau va mostrar un efecte prebiòtic al promoure el creixement dels gèneres Bifidobacterium i Lactobacillus. A més, la dieta de fibra de cacau va comportar els canvis més pronunciats en la producció d’AGCC en femtes i contingut cecal. Particularment, va augmentar la concentració a nivell fecal i cecal dels àcids acètic, propiònic i butíric. A més, les dietes de cacau i fibra de cacau van modular de forma diferencial l’expressió gènica de TLR en còlon. Quant a les immunoglobulines en el compartiment mucosal, totes les dietes enriquides amb polifenols del cacau van modular la secreció d’IgA intestinal, tot i que de forma no proporcional al seu contingut en flavonoides. La fibra de cacau, depenent del compartiment intestinal estudiat, va exercir un efecte o un altre. Pel que fa al compartiment extraintestinal, tot i que la dieta de fibra de cacau va mostrar el mateix efecte atenuador de la síntesi d’IgA i d’IgM que la dieta de cacau, el mecanisme d’acció va ser diferent. A més, totes les dietes enriquides amb flavonoides del cacau, van disminuir la seva proporció de bacteris fecals units a IgA independentment del seu contingut en flavonoides, mentre que aquest percentatge va incrementar amb la dieta de fibra de cacau. Únicament la dieta al 10% de cacau provoca un alentiment en la corba ponderal. Aquest efecte es correlaciona amb els canvis produïts en la microbiota i es pot associar amb la modificació de l’expressió en còlon dels gens implicats en el metabolisme lipídic. Pel que fa al perfil metabòlic en orina, les dietes de cacau i fibra de cacau van provocar patrons diferencials, els quals poden ser usats com a marcadors d’ingesta. El perfil metabòlic es correlacionen amb els efectes del cacau sobre el pes corporal, amb les hormones metabòliques, amb la immunitat intestinal i amb la composició de la microbiota. A més, aquestes variables també mostren correlació entre si. Els efectes del cacau són el resultat de la suma del efectes del components bioactius present en el cacau: polifenols i fibra de cacau, els quals exerceixen efectes sinèrgics o bé contraris depenent de la variable estudiada. Altres components del cacau també estan involucrats en aquests efectes.
In the last few years, cocoa has become one of the main subjects of study due to its high content in flavonoids. Several studies have associated the cocoa intake with health benefits. Moreover, immunomodulatory properties in rats have been also attributed to cocoa. On this basis, the aim of the present thesis was to establish the impact of diets enriched with cocoa, cocoa flavonoids or cocoa fiber on the fecal microbiota composition and its activity as well as on the immune function in the gut. To achieve this objective, preclinical studies were carried out in rats fed a 10% conventional cocoa-enriched diet, diets elaborated with different amounts of non- fermented cocoa extracts and cocoa fiber diet. Regarding microbiota results, differential composition pattern was observed after all the experimental diets intake but only the cocoa fiber diet increased the Bifidobacterium spp. and Lactobacillus spp. proportion. In addition, the cocoa fiber diet was the one which caused the most pronounced changes in the short chain fatty acids (SCFA) production. Particularly, it increased the cecal and fecal concentration of acetic, propionic and butyric acids. Moreover, both the 10%-cocoa diet and cocoa fiber diets differentially modulated the TLR gene expression in the colon. Concerning the mucosal immunoglobulin production, all cocoa polyphenol-enriched diets modulated the intestinal IgA secretion although this effect was not proportional to their flavonoid content. The cocoa fiber diet also exerted an effect on intestinal IgA secretion but in a different way depending on the compartment. Focusing on the extraintestinal compartment, although the cocoa fiber diet showed the same down- modulatory effect on IgA and IgM secretion as the cocoa diet, its mechanisms were different. In addition, all cocoa flavonoid-enriched diets decreased IgA-coated bacteria proportion in a non-dose dependent manner whereas this percentage increased by the cocoa fiber intake The 10% cocoa diet was the only one that caused a slower body weight gain. This effect is correlated with the microbiota modulation. The change induced by cocoa diet on the expression of genes involved in the lipid metabolism in the colon could be also involved. Regarding the urinary metabolites, the cocoa and the coca fiber diets caused differential metabolic profile that can be used as consumption marker. The metabolic fingerprint correlated well with the body weight, the metabolic hormones, the intestinal immunity and the microbiota composition. Moreover, all these variables showed also an association between them. Therefore, the effects produced by cocoa intake are due to the differential effects caused by each one of its main bioactive compounds - polyphenols and fiber - which act in a synergistic or opposite manner depending on the variable. Other cocoa compounds are also involved in such effects.

Tesis por compendio de publicaciones
Introducción: La lactancia materna es la forma de alimentación recomendada, siempre que sea posible, al menos durante los primeros seis meses de vida. Si esto no es posible, existen fórmulas artificiales con las que poder cubrir los requerimientos nutricionales de los recién nacidos. La exposición a determinados factores ambientales, incluidos factores nutricionales, durante el periodo intrauterino y los meses posteriores al nacimiento podría determinar la susceptibilidad a determinadas enfermedades en etapas posteriores de la vida. Se ha demostrado una menor susceptibilidad a determinadas enfermedades en niños alimentados con leche materna frente a aquellos que lo han sido con fórmulas infantiles, incluyendo menor riesgo de enfermedades gastrointestinales y respiratorias, y menor riesgo de obesidad y diabetes en la edad adulta. El hecho de que estos cambios se prolonguen hasta la edad adulta sugiere que la exposición temprana a factores nutricionales podría estar asociada a cambios epigenéticos. Las poliaminas son policationes orgánicos presentes en todas las células de mamíferos. Su interés es debido a su función, siendo esenciales para la proliferación y la diferenciación celular. Su presencia ha sido demostrada en la leche humana siendo las poliaminas más abundantes la espermidina y la espermina. Objetivos: El objetivo de la presente Tesis ha sido evaluar si la adición de poliaminas tras el procesado podría mejorar el desarrollo del sistema inmune y producir un patrón de colonización microbiana similar al obtenido mediante lactancia materna utilizando como modelo ratones BALB/cOlaHsd. Métodos: 60 crías de ratón de 14 días de edad fueron distribuidas aleatoriamente en cuatro grupos: 1) ratones sin destetar con lactancia normal; 2) ratones destetados precozmente alimentados con fórmulas infantiles; y 3) tres grupos distintos de ratones destetados precozmente alimentado con una fórmula infantil suplementada con concentraciones crecientes de poliaminas. Se analizó la microbiota intestinal mediante fluorescent in situ hybridization (FISH) y detección por citometría de flujo y PCR cuantitativa. Además, se analizaron poblaciones linfocitarias mediante citometría de flujo y la expresión de genes relacionados con la activación, proliferación y diferenciación de linfocitos T y B. Resultados y Conclusiones: En relación a la microbiota, nuestros resultado muestran, por primera vez que nosotros sepamos, que la suplementación de las fórmulas infantiles con poliaminas modula las poblaciones bacterianas de Bacteroides-Prevotella, Bifidobacterium spp., Lactobacillus spp. y bacterias similares a Akkermansia, incluida A. muciniphila, hasta niveles parecidos a los del grupo alimentado con lactancia normal en un efecto que parece ser dependiente de la dosis de poliaminas administrada. De forma similar a lo que ocurría con la microbiota intestinal, la suplementación de la fórmula infantil con poliaminas induce cambios en las poblaciones linfocitarias y la expresión génica, incrementándose el porcentaje de linfocitos B en sangre, y linfocitos CD4+, CD8+ y B en bazo, en una proporción dependiente de la dosis de poliaminas; y reduce las diferencias en la expresión de los genes Cd1d1, Cd40, Hdac5, Hdac7, Clcf1 y Tlr4 cuando se compara con lactancia normal. Los resultados obtenidos de este estudio ayudan a comprender la compleja interrelación entre la nutrición, el sistema inmune y la microbiota intestinal durante el periodo de lactancia. Los resultados obtenidos deberían ser estudiados en humanos, ya que de demostrarse efectos similares, la suplementación de las fórmulas infantiles con poliaminas podría contribuir a un mejor desarrollo, con un efecto beneficioso sobre la salud, de niños alimentados con fórmulas.
Introduction: As has been recommended by a large number of health or breastfeeding organisations, whenever feasible, infants should be fully breastfed for at least six months. If full breastfeeding is not possible, safe and suitable infant formula should be used. According to the early programming theory, environmental exposure, including nutritional exposure during the intrauterine stage and during the perinatal months of life, might make children more susceptible to some diseases later in life. Indeed, it has been demonstrated that infants who have been breastfed have a lower susceptibility to some diseases than infants who were fed with artificial manufactured formulas. The fact that these changes are extended into adult life suggests than early exposure to nutritional factors are associated with epigenetic changes. Polyamines are organic polycations that are present in all mammalian cells. They have significant interest due to their reported biological roles in eukaryotic cells, because they are essential to cell proliferation and differentiation. Their presence has been demonstrated in human milk as being the most abundant spermidine and spermine polyamines. Objectives: the main objective of the present thesis was to evaluate whether the addition of polyamines after processing could improve immune system development and the microbial colonization pattern in a similar way that breast feeding do. Methods: A total of 60 mice pups (14-days old) were randomly assigned to four-day intervention groups as follows: 1) breastfed unweaned pups; 2) early weaned pups fed with infant formula; and 3) three different groups of early weaned pups fed with infant formula supplemented with increasing levels of polyamines. After a four-day diet intervention, samples were obtained. Microbiota composition was analysed by fluorescent in situ hybridization (FISH) coupled with flow cytometry detection, and by quantitative PCR targeted at 14 bacteria genus and species. The lymphocyte populations in the blood, spleen and mesenteric lymph nodes were analysed by fluorescence activated cell sorting (FACS); moreover, the expression of genes encoding T-cell and B-cell activation, proliferation and differentiation, as well as Toll-like receptors (TLRs), in the small intestinal tissues was assessed using a 96-well RT-PCR arrays. Results and Conclusions: Regarding the microbiota composition, independently of the analysis methods, our results showed, for the first time to our knowledge, that supplementation of infant formula with polyamines modulates Bacteroides-Prevotella, Bifidobacterium spp., Lactobacillus spp. and Akkermansia-like bacteria groups, including A. muciniphila, to levels closely related to normal lactation group, in a dose-dependent manner and immune system parameters in infant formula feeding compared to mice with normal lactation. Similar to microbiota, the supplementation of manufactured infant formula in polyamines induces changes in lymphocyte populations and gene expression, increasing the percentage of B-cells in blood and CD4+, CD8+ and B-cells in the spleen in a dose-dependent manner, and it reduces differences in the expression of Cd1d1, Cd40, Hdac5, Hdac7, Clcf1 and Tlr4 genes compared with normal lactation. The results obtained from this study highlight the complex interplay between nutrition, the immune system and microbial colonization patterns during lactation. Such an effect requires further investigation in human infants because supplementation of an infant formula in polyamines might contribute to healthy gastrointestinal tract development in children fed with commercial formula.

碩士
國立臺灣大學
漁業科學研究所
101
Mushroom polysaccharides are distinguished as important immunostimulant in animal body. Administrate glucan to animal inducing different isotype immunoglobulin secretion. Immunoglobulin A is the major antibody in the intestinal mucus, and transcytosis of IgA across epithelia is mediated by the poly-Ig receptor. Neutralization is important protection mechanism against antigen by IgA in gut. In the present study simulate acting intestinal mucosal immune system. Further to study the effects of mushroom polysaccharide on immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM) concentration in serum and small intestine washing fluid (SIWF) and intestine tissue poly-immunoglobulin receptor (poly-Ig receptor) mRNA expression. Feeding mice polysaccharide IgA and IgG in SIWF are increase significantly, and IgG and IgM in serum are also increase significantly. The results suggest that the effect of polysaccharides was induced at intestinal mucosa firstly, and induced body circulation immune response further. Poly-Ig receptor mRNA expression increase significantly, too. Our study highlights the efficacious effect of mushroom polysaccharides increasing immunoglobulin concentration in intestinal tract and serum immunoglobulin concentration, and increase poly-Ig receptor mRNA expression in intestine tissue. Mushroom polysaccharide may stimulate intestinal mucosal immune system to protect the intestinal tract from being damaged by the bacterial over-population.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.

90% of Human Immunodeficiency Virus (HIV) infections are acquired across the genital or gastrointestinal mucosa, and infection leads to profound depletion of CD4+ lymphocytes. Antiretroviral therapy can restore blood CD4+ T cells. However, immune dysfunction and defects in mucosal antimicrobial defence persist. Some CD4+ T-subsets, particularly antimicrobial Th17 cells, show enhanced susceptibility to HIV infection and are also preferentially depleted in the course of HIV infection; the latter may allow microbial translocation into the bloodstream. Genital infections have been shown to have direct mucosal immune effects and to increase susceptibility to HIV; however, the effect of systemic infections, such as Malaria (which is holo-endemic in some HIV prevalent regions) is unknown. Understanding the relationship between HIV, highly susceptible immune cells, immune activation and malaria infection on mucosal tissues has been the main focus of my thesis. In HIV-infected individuals, I explored whether HIV antiretroviral therapy restores gut Th17 populations and improves gut antimicrobial defences. Therapy restored gut Th17 populations in some, but not all individuals, but antimicrobial defence remained impaired. I then piloted a novel mucosal-optimized PCR assay to measure cervical immune gene responses, as standard mucosal assays are inadequate. I succeeded in measuring mitogen-induced, but not HIV-specific, cervical immune responses in HIV-infected individuals. Next, using this PCR platform I examined mitogen-induced cervical immune responses in individuals demonstrating reduced susceptibility to HIV, and found that they had reduced production of both Th17-associated and pro-inflammatory cytokines from cervical cells. Finally, in a murine model I found that malaria caused genital and gastrointestinal mucosal immune activation, and increased both the expression of mucosal HIV susceptibility immune markers, and mucosal T cell immune activation. In summary, insufficient gastrointestinal Th17 cells restoration does not underlie persistent mucosal immune activation and microbial translocation in HIV-infected people on therapy. A reduced frequency of highly susceptible Th17 cells in the cervix of HIV-exposed but uninfected individuals was identified as a correlate of reduced HIV susceptibility. Malaria, a common systemic infection in HIV-endemic countries, may enhance susceptibility to HIV through increasing putative immune markers of HIV susceptibility and immune activation in potential mucosal sites of HIV exposure.

碩士
國立陽明大學
微生物暨免疫學研究所
89
Oral tolerance is the obstacle for inducing immunity by oral route. There were some powerful mucosal adjuvants, such as CT and LT that had been studied for their capacity in immune potentiation. Due to their toxicity per se, their usage was carefully considered. In the previous study from our laboratory, it was shown that Ling-zhi polysaccharides isolated from Ganoderma lucidum had the effects on anti-tumor and modulating the immune system through oral administration, though their absorption by intestine and metabolism were unclear. This implied that Ling-zhi polysaccharides, in some way, might act on the intestinal immune system. We tried to identify the posibility of using Ling-zhi polysaccharides orally as an immunomodulator or a mucosal adjuvant. We used two different antigens ovalbumin entrapped in PLGA (OVA particles) and Pneumovax 23 vaccine to induce humoral immune response in mice by subcutaneous immunization or by oral administration. Additionally, Ling-zhi polysaccharides were supplemented in daily drink for mice as an immunomodulator or mixed with antigens given orally as a mucosal adjuvant. For an immunomodulator, Ling-zhi polysaccharides enhanced sera antibody production induced by subcutaneous immunization with OVA particles or Pneumovax 23 vaccine, and enhanced pulmonary IgA production induced by oral inoculation with OVA particles and Pneumovax 23 vaccine, but inhibited sera IgG production induced by oral inoculation with the same antigens. Marvelously, using Ling-zhi polysaccharides as an immunomodulator in 20-week-old mice, they broke oral tolerance and induced significant sera IgG production induced by oral administration with ovalbumin protein directly. For a mucosal adjuvant, Ling-zhi polysaccharides inhibited sera IgG production but enhanced pulmonary and intestinal IgA secrection induced by oral administration with OVA particles. Additionally, it enhanced sera IgM production but had no influence on mucosal IgA secrection by oral inoculation with Pneumovax 23 vaccine. In another way, we used reverse transcription and polymerase-chain-reaction (RT-PCR) to analyze the expression of some cytokines in mice fed with Ling-zhi polysaccharides for three days. In all the tissues we analyzed, including spleen、intestinal epithelium and lamina propria, IL-1beta was enhanced and its expression might be related to the induction of natural killer cell’s activity. Otherwise, the expressions of IFN-gamma and TGF-beta in spleen were inhibited with the increasing dose of Ling-zhi polysaccharides, while those in Peyer’s patch were opposite. Taken together, Ling-zhi polysaccharides did have their effects on intestinal immune system possibly through cytokine regulation.

Inflammatory bowel disease causes structural and functional alterations in the enteric nervous system (ENS). Since the onset of intestinal inflammation involves the activation of resident immune cells as well as rapid influx of infiltrating cells, we proposed that changes in the ENS are a result of the release of toxic inflammatory factors. We hypothesized that early damage to the ENS in inflammation is caused by harmful levels of nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) found in immune cells. This was assessed in the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-model of colitis in rats. Large increases in infiltrating granulocytes, particularly neutrophils and blood-derived monocytes were found in the muscularis layers adjacent to the ENS. A rapid increase in iNOS immunoreactivity in the muscularis regions during early stages of inflammation (6 – 24 hr) was observed. Whether high NO levels generated by chemical donors could be toxic to neurons was tested in a co-culture model of myenteric neurons, smooth muscle and glia enzymatically isolated from neonatal rats. Exposure of co-cultures to NO for 48 hr resulted in significant, concentration dependent decrease in neuron survival. We then developed a model that permitted the direct study of immune cell interactions with myenteric neurons. Myenteric neurons were co-cultured with activated peritoneal immune cells that expressed iNOS and generated high NO levels (49 + 6.2µM) for 48 hr. This caused significant neuronal death, reducing neuron number by 19 + 5%, and disruption of axons. Pre-treatment of immune cells with a selective iNOS-inhibitor, L-NIL resulted in neuron numbers that were not significantly different from control (96 + 2%) suggesting that NO played a central role in mediating the damaging effects of immune cells. Lastly, when direct contact between immune cells and neurons was prevented in the previous experiment through use of trans-wells, unanticipated neurotrophic effects were observed. Increased axon outgrowth (282 + 57%) was detected in addition to loss of the neurotoxic effects in spite of similar experimental conditions. We concluded that proximity and contact plays an important role in determining the nature of immune cell mediated alterations in enteric neurons.
Thesis (Master, Physiology) -- Queen's University, 2009-11-30 10:09:38.384

碩士
中原大學
生物科技研究所
103
A gene delivery system carrying the gene segment of the antigen protein into the host body to express in non-viral way to stimulate mucosal and systemic immune responses has been developed. The current study used the ternary complex（DNA-CLPEI-HA）composed of plasmid DNA, disulfide-crosslinked low molecular linear polyetheleneimine(CLPEI) ,and hyaluronic acid (HA), which exhibited high gene transfection efficiency and low toxicity in cell culture systems. The examination by electron microscopes showed the physical properties of DNA-CLPEI-HA complex. The stability of DNA in the complex after oral delivery was determined by DNA electrophoresis and the digestions of restriction enzyme. The transfection efficiency of green fluorescence protein (GFP) gene delivered by DNA-CLPEI-HA complex in the intestinal epithelial cells in cell culture systems and the cell viability of the complex were examined in vitro. The expression of GFP and the immune responses induced in mice was evaluated by the levels of anti-GFP IgG antibody. The DNA-CLPEI-HA complex had a particle size of about 300 ~ 400nm. The DNA binding with CLPEI and HA at the mixing ratio N / P ratio 100 can be effectively protected from the digestion of restriction enzymes. Good transfection efficiency and low cytotoxicity were observed in cell culture system. The complex caused mild to no damage to the intestine in mice ingested orally the complex. Thus, the oral gene delivery system developed in this study had potentials to become a platform for oral vaccines used to fight intestinal pathogens.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
A doença de Crohn e a Colite Ulcerosa são duas doenças que, genericamente, se designam por doenças inflamatórias do intestino. Estas são doenças crónicas que se caracterizam por períodos de inflamação (com exacerbação dos sintomas), seguidos por períodos de remissão. As lesões podem ser limitadas à região do reto e do colon (colite ulcerosa) ou estenderem-se por todo o trato digestivo (doença de Crohn), podendo essas lesões serem relativamente simples ou mais complexas, levando à estenose ou à formação de fístulas. Estas patologias são características dos países desenvolvidos, especialmente da Europa do Norte e da América do Norte embora se tenha vindo a verificar um aumento de incidência em países em desenvolvimento. Até à data ainda não se conseguiu definir os fatores que desencadeiam o seu aparecimento, no entanto, supõe-se que resultem de uma resposta descontrolada do sistema imunitário a fatores ambientais que ocorre em indivíduos geneticamente predispostos. Uma vez que ainda se desconhece a causa para estas patologias, a terapêutica disponível não leva à cura, mas foca-se em manter os doentes em período de remissão durante o máximo de tempo possível. Esta deve ser adaptada às necessidades de cada doente, sendo que existem orientações para o estabelecimento do regime terapêutico. Os fármacos de primeira linha são os anti-inflamatórios aminossalicilados, seguidos pelos corticosteroides (tópicos ou orais), derivados da tioguanina e metotrexato. Para aqueles doentes refratarios às terapêuticas anteriores existe ainda a possibilidade de tratamento com fármacos biológicos como o Infliximab ou o Adalimumab, dois anticorpos anti-TNF.

博士
國立臺灣大學
生化科技學系
104
Chronic inflammation and immune disorder are associated with insulin resistance, allergy, inflammatory bowel disease, etc. Regulatory T lymphocytes (Treg) suppress a variety of inflammatory and pathological immune responses. Wild bitter melon (WBM) has been demonstrated to improve the insulin resistance, but its effect on immune is still unclear. Therefore, the aim of this study is to investigate the effect of WBM on the intestinal immune system and Treg development. First, to investigate the improvement in insulin resistance of wild bitter melon is involved with its immune modulatory effects. High-fat diet-induced obese C57BL/6J mice were fed the diet containing 5% WBM powder for 12 weeks. The results showed that WBM treatment reduced fasting glucose and triglyceride in the serum, and improved insulin resistance. WBM reduced the M1/M2 ratio and CD4+ T cells infiltration in stromal vascular fraction (SVF). Moreover, WBM increased the percentage of CD4+Foxp3+ Treg in mesenteric lymph nodes (MLN). To confirm the effects of WBM on intestinal immune system and Foxp3+ Treg cells population, Foxp3-EGFP mice co-expressing EGFP and Foxp3 in Treg were fed WBM experimental diet. The results showed that WBM also increased the percentage of CD4+ Foxp3+ Treg in MLN and interleukin (IL)-10 and TGF (transforming growth factor)-β secretion by ConA-stimulated Peyer’s patches and MLN cells. These results suggested that WBM could enhance the Foxp3+ Treg development in the intestinal immune system. The bioactive compounds of WBM and mechanisms of promoting the development of Treg were investigated by using the primary spleen cells, MLN cells, CD4+ T cells and bone marrow derived dendritic cells (BMDC) culture in vitro. The results showed that M-Res and BuE extract of WBM increased IL-10 and TGF-β secretion by splenocytes and promoted the development of Foxp3+ Treg through the aryl hydrocarbon receptor (AhR) pathway. WE extract increased IL-10 secretion by BMDC and promoted CD4+ T cells differentiate into IL-10+ T cells and Foxp3+ T cells in CD4+ T cells cocultured with WE pretreated BMDC. Moreover, tryptophan is the bioactive compound from BuE extract was found that increase Foxp3+ Treg development in vitro. The anti-inflammatory and immunomodulatory effects of WBM and tryptophan on inflammatory bowel disease were investigated by using dextran sodium sulphate (DSS)- induced colitis murine model. A diet containing 5% WBM or 0.5% tryptophan was administered to mice for 2 or 6 weeks and then gave 7 days DSS treatment. WBM intake significantly improved weight loss, disease activity index and colon shortening. Colonic cytokine IL-6 and IL-1β were significantly decreased, and the anti-inflammatory cytokine IL-10 was increased in WBM-fed mice. The mRNA expression of TGF-β and Foxp3 in colon, and Foxp3+ Treg cell population in the MLN were also significantly higher in the WBM group. Tryptophan supplement ameliorated disease activity index and reduced the colonic IL-6 levels. The significantly negative correlation between Foxp3 mRNA expression and pro-inflammatory cytokines in colon is in accordance with the critical role of Treg cell for regulation of intestinal immune homeostasis. These results suggested that WBM reduces the inflammatory response and induces higher level of Treg cells in DSS-induced colitic mice. In summary, WBM enhanced Treg development in intestinal immune system in both in vitro and in vivo. The mechanism of enhancing Treg development by WBM are not only through AhR pathway, but also increased the CD103+ dendritic cells and IL-10 secretion. Furthermore, WBM inhibited the inflammatory response caused by colitis and induces higher level of Treg cells in the intestinal immune system. These results suggested that WBM could exert the anti-inflammatory and immunoregulatory effects in maintaining intestinal immune balance and alleviating the severity of the symptoms on inflammatory bowel disease.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
No âmbito da Unidade Curricular "Estágio Curricular" do Mestrado Integrado em Ciências Farmacêuticas, foi redigido o seguinte documento, que engloba os relatórios dos estágios referentes a Farmácia Comunitária e a Indústria Farmacêutica. Em cada um deles, é feita e descrita uma análise SWOT, analisando pontos internos - Forças (S) e Fraquezas (W) - e pontos externos - Oportunidades (O) e Ameças (T). Atualmente, os probióticos são definidos pela Food and Agriculture Organization e pela World Health Organization, como sendo “microrganismos vivos que, quando administrados em quantidades adequadas, conferem um benefício para a saúde do hospedeiro”. Os benefícios aliados ao seu consumo estão directamente dependentes da espécie e estirpe em questão, mas também do indivíduo. Sendo o sistema imunitário um dos principais mecanismos de defesa contra agentes patogénicos, uma correta manutenção das respostas aliadas a este sistema traz vantagens no tratamento e prevenção de diversas doenças e na manutenção de uma boa qualidade de vida. Assim, as aplicações destes microrganismos são vastas, indo desde o controlo e tratamento de infecções associadas a Clostridium difficile, até à prevenção do desenvolvimento de reacções alérgicas. Esta monografia teve como objectivos, através de uma revisão bibliográfica, transmitir uma perspectiva global sobre o benefício da utilização de probióticos para a saúde, assim como analisar os possíveis mecanismos de acção, particularmente, no que respeita à modulação do sistema imunitário, através do reforço das respostas inata e adaptativa. Pretendeu-se ainda abordar os critérios de eficácia e segurança dos microrganismos utilizados como probióticos.
The following document was written as part of the curricular unit "Curricular Internship" of the Integrated Master in Pharmaceutical Science, which includes the reports of the internships referring to the Community Pharmacy and the Pharmaceutical Industry. In each of them, a SWOT analysis is made and described, analyzing internal points - Forces (S) and Weaknesses (W) - and external points - Opportunities (O) and Threats (T). Probiotics are currently defined by the Food and Agriculture Organization and the World Health Organization as "living microrganisms which, when administered in appropriate quantities, confer a benefit to the health of the host. The benefits allied to their consumption are directly dependent on the species and strains in question, but also on the individual. Since the immune system is one of the main mechanisms of defense against pathogens, a correct maintenance of the responses allied to this system brings advantages in the treatment and prevention of various diseases and in maintaining a good quality of life. Thus, the applications of these microorganisms are vast, ranging from the control and treatment of infections associated with Clostridium difficile, to the prevention of the development of allergic reactions.This monograph aimed, through a literature review, to provide a global perspective on the health benefit of probiotics, as well as to analyze the possible mechanisms of action, particularly regarding the modulation of the immune system, by strengthening the innate and adaptive responses. It was also intended to address the efficacy and safety criteria of microorganisms used as probiotics.

Mucosal immune system comprises not only the major compartment of the immune system but also important interface with the outer environment. It is responsible in maintaining an intricate balance with the danger and non-danger stimuli of the outer world by employing specific anatomical features and unique functional mechanisms. Mucosal immune system has been long understudied, perhaps due to the limited accessibility, and its biological importance is thus still underevaluated. However, it has become evident that it is important to study mucosal immune system not only in local mucosal affections but also when uncovering pathogenic mechanisms and novel prevention strategies of organ specific autoimmune diseases such as type 1 diabetes. Thus, the first, more clinically oriented part of this thesis is focused on mucosal immune system of the upper respiratory tract in disease conditions - in nasal polyposis (NP). Because there is a substantial accumulation of eosinophils and neutrophils in the most frequent type of NP, we investigated and described increased expression of chemokine receptors CCR1 and CCR3 in NP versus nasal mucosa. Both innate immune mechanisms as well as homeostasis of epithelial cells may participate in NP. We have documented increased numbers of iNOS-positive and insulin-like growth...

A hipótese da Higiene originalmente proposta por Strachan em 1989 é caraterizada pela associação do tamanho das famílias e o seu status social com a prevalência de doenças tais como a febre dos fenos e eczemas em crianças. Segundo a sua hipótese, o motivo pelo qual, atualmente, existem mais alergias e/ou doenças autoimunes deve-se a uma menor exposição aos patogénios enquanto criança. Mais tarde esta hipótese foi modificada por Rook, que afirmava que eram os microorganismos presentes na microflora comensal, se alterados, enquanto crianças, que podiam modular, intensificar ou inibir os mecanismos de resposta do sistema imunológico, podendo assim causar doenças autoimunes e/ou alergias. Para se perceber a hipótese da higiene, é necessária uma breve introdução ao sistema imunológico, desde o recém-nascido até à idade adulta, a sua maturação e diferenciação assim como este consegue proceder à sua regulação através, essencialmente, das células T reguladoras e Th17. Para chegar da hipótese da higiene à da microflora é necessário o estudo da microflora comensal, também desde o nascimento até à idade adulta e perceber os mecanismos de defesa do sistema imunológico contra os diversos tipos de patogénios, incluindo bactérias, parasitas e vírus e como estes podem influenciar a resposta imunitária à posteriori. Em ambas as hipóteses a intervenção de outros fatores, nomeadamente ambientais e genéticos, é importante e o estudo da associação de todos eles abrem portas para novas terapias imunológicas para alergias e/ou doenças auto-imunes.
The Hygiene Hypothesis originally proposed by Strachan in 1989 is characterized by the association of family size and social status with the prevalence of diseases such as hay fever and eczema in children. According to his hypothesis, the reason why there are currently more allergies and/or autoimmune diseases is due to less exposure to pathogens as a child. This hypothesis was later modified by Rook, who stated that it were the microorganisms present in the commensal microflora, if altered as children, that could modulate, intensify or inhibit the response mechanisms of the immune system, thus causing autoimmune diseases and/or allergies. To understand the hygiene hypothesis its necessary a brief introduction to the immune system, from the newborn to adulthood, its maturation and differentiation as well as its regulation essentially by regulatory T cells and Th17. To go from the hygiene hypothesis to microflora, it’s necessary the study of commensal microflora, also from birth to adulthood and understand the defence mechanisms of the immune system against various types of pathogens, such as bacteria, parasites and viruses and how these may influence the later immune response. In both hypotheses the intervention of other factors, namely environmental and genetics, are important, and the study of the association in between all open the door for new immunological therapies for allergies and/or autoimmune diseases.