Academic literature on the topic 'Intestinal mucosal immune system'

ABSTRACT Immunization(s) fostering the induction of genital mucosa-targeted immune effectors is the goal of vaccines against sexually transmitted diseases. However, it is uncertain whether vaccine administration should be based on the current assumptions about the common mucosal immune system. We investigated the relationship between mucosal sites of infection, infection-induced inflammation, and immune-mediated bacterial clearance in mice using the epitheliotropic pathogenChlamydia trachomatis. Chlamydial infection of the conjunctival, pulmonary, or genital mucosae stimulated significant changes in tissue architecture with dramatic up-regulation of the vascular addressin, VCAM, a vigorous mixed-cell inflammatory response with an influx of α4β1+ T cells, and clearance of bacteria within 30 days. Conversely, intestinal mucosa infection was physiologically inapparent, with no change in expression of the local MAdCAM addressin, no VCAM induction, no histologically detectable inflammation, and no tissue pathology. Microbial clearance was complete within 60 days in the small intestine but bacterial titers remained at high levels for at least 8 months in the large intestine. These findings are compatible with the notion that VCAM plays a functional role in recruiting cells to inflammatory foci, and its absence from the intestinal mucosa contributes to immunologic homeostasis at that site. Also, expression of type 1 T cell-mediated immunity to intracellular Chlamydia may exhibit tissue-specific variation, with the rate and possibly the mechanism(s) of clearance differing between enteric and nonenteric mucosae. The implications of these data for the common mucosal immune system and the delivery of vaccines against mucosal pathogens are discussed.

The mammalian intestine harbors a remarkable number of microbes and their components and metabolites, which are fundamental for the instigation and development of the host immune system. The intestinal innate and adaptive immunity coordinate and interact with the symbionts contributing to the intestinal homeostasis through establishment of a mutually beneficial relationship by tolerating to symbiotic microbiota and retaining the ability to exert proinflammatory response towards invasive pathogens. Imbalance between the intestinal immune system and commensal organisms disrupts the intestinal microbiological homeostasis, leading to microbiota dysbiosis, compromised integrity of the intestinal barrier, and proinflammatory immune responses towards symbionts. This, in turn, exacerbates the degree of the imbalance. Intestinal adaptive immunity plays a critical role in maintaining immune tolerance towards symbionts and the integrity of intestinal barrier, while the innate immune system regulates the adaptive immune responses to intestinal commensal bacteria. In this review, we will summarize recent findings on the effects and mechanisms of gut microbiota on intestinal adaptive immunity and the plasticity of several immune cells under diverse microenvironmental settings.

The gastrointestinal tract represents the largest mucosal membrane surface and is the one of the most complex human organs. The intestinal barrier dysfunction contributes to systemic immune activation. The mucosal immune system has extremely arduous tasks to resist invaders and promote tolerance of food antigens and the microbiota. The intestinal mucosal immune system fulfills these tasks through complex interactions between immune cells and the local microenvironment in intestine. Intestinal Epithelial Cells (IECs) play important roles in these complex interactions. IECs not only constitute the first barrier of the intestine but also are crucial for integrating external and internal signals and for coordinating the ensuing immune response. Dendritic Cells (DCs) play key roles in shaping the intestinal immune response by their ability to coordinate protective immunity and immune tolerance in the host. DCs are pivotal actors in the connection between innate and adaptive immune responses. The IECs coordinate with the DCs in immune recognition, tolerance and host defense mechanisms. In this review, we will summarize how IECs orchestrate intestinal DCs in intestinal homeostasis and diseases.

Listeria monocytogenesis an opportunistic foodborne pathogen causing listeriosis, an often fatal infection leading to meningitis, sepsis, or infection of the fetus and abortion in susceptible individuals. It was recently found that the bacterium can also cause acute, self-limiting febrile gastroenteritis in healthy individuals. In the intestinal tract,L. monocytogenespenetrates the mucosa directly via enterocytes, or indirectly via invasion of Peyer’s patches. Animal models forL. monocytogenesinfection have provided many insights into the mechanisms of pathogenesis, and the development of new model systems has allowed the investigation of factors that influence adaptation to the gastrointestinal environment as well as adhesion to and invasion of the intestinal mucosa. The mucosal surfaces of the gastrointestinal tract are permanently exposed to an enormous antigenic load derived from the gastrointestinal microbiota present in the human bowel. The integrity of the important epithelial barrier is maintained by the mucosal immune system and its interaction with the commensal flora via pattern recognition receptors (PRRs). Here, we discuss recent advances in our understanding of the interaction ofL. monocytogeneswith the host immune system that triggers the antibacterial immune responses on the mucosal surfaces of the human gastrointestinal tract.

The evidence of the beneficial effects of dairy products on the intestinal microflora was given for the first time in 1908 by I.I. Mechnikov in the famous article «A few words about the sour milk». Since that time probiotics - the living microorganisms for regulation of intestinal microbiota are the case of interest. Interactions between the probiotics and macroorganism are very complex and include a network of genes receptors, signaling molecules and a variety of other factors that determine the natural course of the disease.

Dynamic interactions between gut microbiota and a host’s innate and adaptive immune systems are essential in maintaining intestinal homeostasis and inhibiting inflammation. Gut microbiota metabolizes proteins and complex carbohydrates, synthesizes vitamins, and produces an enormous number of metabolic products that can mediate cross-talk between gut epithelium and immune cells. As a defense mechanism, gut epithelial cells produce a mucosal barrier to segregate microbiota from host immune cells and reduce intestinal permeability. An impaired interaction between gut bacteria and the mucosal immune system can lead to an increased abundance of potentially pathogenic gram-negative bacteria and their associated metabolic changes, disrupting the epithelial barrier and increasing susceptibility to infections. Gut dysbiosis, or negative alterations in gut microbial composition, can also dysregulate immune responses, causing inflammation, oxidative stress, and insulin resistance. Over time, chronic dysbiosis and the leakage of microbiota and their metabolic products across the mucosal barrier may increase prevalence of type 2 diabetes, cardiovascular disease, autoimmune disease, inflammatory bowel disease, and a variety of cancers. In this paper, we highlight the pivotal role gut bacteria and their metabolic products (short-chain fatty acids (SCFAs)) which play in mucosal immunity.

Intestinal inflammation has traditionally been viewed as a process in which effector immune cells cause the destruction of other mucosal cells that behave as passive bystander targets. Progress in understanding the process of intestinal inflammation has led to a much broader and more integrated picture of the various mucosal components, a picture in which cytokines, growth factors, adhesion molecules, and the process of apoptosis act as functional mediators. Essentially all cellular and acellular components can exert immunelike activities, modifying the classical concept of selected immune cells acting on all other cells that has been the dogma of immunologically mediated tissue damage for decades. The existence of specialized communication pathways between epithelial cells and T cells is well documented, including abnormal epithelial cell-mediated T cell activation during inflammation. Mesenchymal cells contribute to fibrosis in the inflamed gut but are also responsible for retention and survival of leukocytes in the mucosa. In chronically inflamed intestine the local microvasculature displays leukocyte hyperadhesiveness, a phenomenon that probably contributes to persistence of inflammation. The extracellular matrix regulates the number, location, and activation of leukocytes, while metalloproteinases regulate the quantity and type of deposited matrix proteins. This evidence from the intestinal system, consolidated with the use of data from other organs and systems, reveals a rich network of reciprocal and finely orchestrated interactions among immune, epithelial, endothelial, mesenchymal, and nerve cells and the extracellular matrix. Although these interactions occur under normal conditions, the dysfunction of any component of this highly integrated mucosal system may lead to a disruption in communication and result in pathological inflammation.

Bile acids are a class of cholesterol derivatives that have been known for a long time for their critical roles in facilitating the digestion and absorption of lipid from the daily diet. The transformation of primary bile acids produced by the liver to secondary bile acids appears under the action of microbiota in the intestine, greatly expanding the molecular diversity of the intestinal environment. With the discovery of several new receptors of bile acids and signaling pathways, bile acids are considered as a family of important metabolites that play pleiotropic roles in regulating many aspects of human overall health, especially in the maintenance of the microbiota homeostasis and the balance of the mucosal immune system in the intestine. Accordingly, disruption of the process involved in the metabolism or circulation of bile acids is implicated in many disorders that mainly affect the intestine, such as inflammatory bowel disease and colon cancer. In this review, we discuss the different metabolism profiles in diseases associated with the intestinal mucosa and the diverse roles of bile acids in regulating the intestinal immune system. Furthermore, we also summarize recent advances in the field of new drugs that target bile acid signaling and highlight the importance of bile acids as a new target for disease intervention.

The intestinal epithelium is a selective barrier where incompletely-digested food antigens are transmitted to the immune system. Food antigens are often the starting point of intestinal diseases such as food allergy or coeliac disease. The intestinal epithelial cells (IEC) take up and process food antigens mainly by fluid-phase transcytosis involving two functional pathways, one minor direct pathway without degradation and another major lysosomal degradative pathway. Among the peptidic metabolites generated during transepithelial transport of luminal antigens, some have a molecular mass compatible with a binding to restriction (major histocompatibility complex; MHC) molecules; the latter can be up regulated on enterocytes, especially in inflammatory conditions. Indeed, interferon-γ not only increases the paracellular absorption of antigens, but also their transcytosis across epithelial cells. It has been reported that enterocytes may even directly present peptidic epitopes to underlying T-cells. As a new potential way of transmitting peptidic information to the local or systemic immune system, the secretion by IEC of antigen-presenting vesicles called exosomes and bearing MHC–peptide complexes has recently been proposed. Many other factors such as nutritional or environmental factors can also influence the properties of the epithelial barrier and the outcome of the immune response to lumen antigens.

Host health depends on the intestinal homeostasis between the innate/adaptive immune system and the microbiome. Numerous studies suggest that gut microbiota are constantly monitored by the host mucosal immune system, and any slight disturbance in the microbial communities may contribute to intestinal immune disruption and increased susceptibility to inflammatory bowel disease (IBD), a chronic relapsing inflammatory condition of the gastrointestinal tract. Therefore, maintaining intestinal immune homeostasis between microbiota composition and the mucosal immune system is an effective approach to prevent and control IBD. The overall theme of this review is to summarize the research concerning the pathogenesis of IBD, with particular focus on the factors of gut microbiota-mucosal immune interactions in IBD. This is a comprehensive and in-depth report of the crosstalk between gut microbiota and the mucosal immune system in IBD pathogenesis, which may provide insight into the further evaluation of the therapeutic strategies for IBD.

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.

The gastrointestinal (GI) system is responsible for digestion and absorption, but also has important endocrine, immune and barrier functions. Additionally, the GI system plays a major role in fluid, electrolyte and acid-base balance. The GI system is regulated by complex myogenic, neural and humoral mechanisms, and, in health, these are affected by the presence of luminal nutrient, thereby modulating function of the GI system. Accordingly, GI function varies depending on whether a person is fasted or in the postprandial state. Adequate fasting and postprandial perfusion, motility and exocrine secretion are required for ‘normal’ functioning. The protective mechanisms of the GI system consist of physical (intact gut mucosa), non-immune (gastric acid, intestinal mucin, bile and peristalsis) and immune (gut-associated lymphoid tissue, GALT) elements. Disruption of GI protection is a putative mechanism underlying the development of multiple-organ dysfunction syndrome. Maintenance of GI function is increasingly recognised as an important factor underlying survival in critical illness.

Mevalonate kinase deficiency (MKD) is an autosomal recessive inborn error of isoprenoid biosynthesis, a pathway yielding sterols and nonsterol isoprenoids.In patients, the enzyme activity of mevalonate kinase is severely reduced due to mutations in the encoding gene, MVK. The substrate, mevalonate, accumulates and is elevated in blood and urine. Shortage of certain downstream products of the pathway, nonsterol isoprenoids, leads to dysregulation of the innate immune system, activation of inflammasomes, and interleukin (IL)-1 mediated inflammation.Symptoms start in early childhood with recurrent attacks of fever, vomiting, diarrhea, headache, sore throat, abdominal pain, arthralgias, painful lymphadenopathy, hepatosplenomegaly, skin rash, and mucosal ulcers. Severely affected patients have additional symptoms, such as intellectual impairment, progressive cerebellar ataxia, and tapetoretinal degeneration. Complications include intestinal obstruction, AA-amyloidosis, hemophagocytosis, and severe infection.Management of MKD is directed at controlling inflammation.

The analysis of the variation in the number, intensity and direction of correlations between the immune system and the gut microbiota of rats revealed that the T-, B-system and humoral immunity changes as well as cellular and humoral factors of an organism nonspecific defense are accompanied by changes of the Intestinal microbiota of intact and experimental rats

Ingestible capsule endoscope technology has been a topic of research since the middle of the 20th century and has become a prominent area of study since the commercialization of capsule endoscopy in 2000. Ingestible telemetry capsules have been investigated by NASA in the last 20 years as a means for monitoring human body temperature during periods of physical exhaustion, but are limited in sensing time due to passage through the digestive system. In this work, we present a feasibility study on a sensor that attaches to the intestinal mucosa after being delivered to the bowel via ingestible capsule to be used on long distance space flights. This study included experiments conducted on NASA’s Weightless Wonder aircraft and replicated in a laboratory setting on the ground. During these experiments, a capsule was activated, manually inserted into excised porcine small intestine, and then automatically implanted a sham sensor onto the mucosal lining. The purpose of the experiment was to determine if the automated implantation sequence is affected by microgravity. Eight trials conducted in each setting yielded successful implantation of four sham sensors in microgravity and three in earth gravity. Results suggest that automated implantation is feasible in both 1G and microgravity environments though design changes are necessary to significantly improve repeatability in both environments. Though improvements in reliability of the device are needed, this experiment is a benchmark for transferring capsule technology currently used only for visual screening of the bowel to health monitoring systems for space flights.

Cervical cancer is the second most cancer in women worldwide with over 500000 new cases and 275000 deaths being registered every year. With nearly 73000 women dying every year, India now tops the world in cervical cancer deaths. India represents 26.4% of all women dying of cervical cancer globally. Cervical cancer estimated to be responsible for about 5% of human cancers worldwide. Currently available vaccines may not provide complete protection against all HPV types as the protection is primarily type specific. Furthermore, the available vaccines are delivered via intramuscular route and require three doses and require cold chain supply which increases the cost of vaccine. Therefore a single dose vaccine delivered via non-invasive route (nasal) that protects against multiple HPV types would be a cost effective and better alternative to the currently available HPV vaccines. The main objective of this study was to prepare HPV antigen loaded poly (lactic-co-glycolic acid) (PLGA) and Tri Methyl Chitosan (TMC) coated PLGA microparticles and compare their efficacy as nasal vaccine. The developed formulations were characterized for size, zeta potential, entrapment efficiency, mucin adsorption ability, in vitro and in vivo studies. PLGA microparticles demonstrated negative zeta potential whereas PLGA-TMC microparticles showed higher positive zeta potential. The protein loading efficiency was found as above 80%. Results indicated that PLGA-TMC microparticles demonstrated substantially higher mucin adsorption when compared to PLGA microparticles. HPV antigen encapsulated in PLGA-TMC particles elicited a significantly higher secretory (IgA) immune response compared to that encapsulated in PLGA particles. Present study demonstrates that PLGA-TMC microparticles with specific size range can be a better carrier adjuvant for nasal subunit vaccines. Surface modified PLGA microparticles proved great potential as a nasal delivery system for HPV infections where systemic and mucosal responses are necessary particularly in conditions after viral pathogens invade the host through the mucosal surface.

The lymphatic system has long been thought of as little more than a series of passive ducts as they serve to return fluid and proteins from interstitial spaces back to the blood, provide a route for immune cell trafficking, and transport dietary lipid from the intestine to the blood. Recent evidence has revealed that the lymphatics play an active role in lipid trafficking, and alterations in this function have been correlated with the presence of lymphatic diseases (Dixon, 2010). Here we describe the use of a two-cell, tissue engineered model to explore mechanisms of lipid transport across lymphatic endothelial cells (LEC). Previously this model was demonstrated to recapitulate essential features of the intestinal-lacteal interface with in the mammalian gut (Dixon et al., 2009). With our model we demonstrate, not only that lipid transport across the lymphatics is transcellular and ATP dependent, but also, this mechanism of transport utilizes the molecular motors dynein and kinesin.