Dissertations / Theses on the topic 'Mucosal immune system'

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.

Chlamydia is responsible for a wide range of diseases with enormous global economic and health burden. As the majority of chlamydial infections are asymptomatic, a vaccine has greatest potential to reduce infection and disease prevalence. Protective immunity against Chlamydia requires the induction of a mucosal immune response, ideally, at the multiple sites in the body where an infection can be established. Mucosal immunity is most effectively stimulated by targeting vaccination to the epithelium, which is best accomplished by direct vaccine application to mucosal surfaces rather than by injection. The efficacy of needle-free vaccines however is reliant on a powerful adjuvant to overcome mucosal tolerance. As very few adjuvants have proven able to elicit mucosal immunity without harmful side effects, there is a need to develop non-toxic adjuvants or safer ways to administered pre-existing toxic adjuvants. In the present study we investigated the novel non-toxic mucosal adjuvant CTA1-DD. The immunogenicity of CTA1-DD was compared to our "gold-standard" mucosal adjuvant combination of cholera toxin (CT) and cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN). We also utilised different needle-free immunisation routes, intranasal (IN), sublingual (SL) and transcutaneous (TC), to stimulate the induction of immunity at multiple mucosal surfaces in the body where Chlamydia are known to infect. Moreover, administering each adjuvant by different routes may also limit the toxicity of the CT/CpG adjuvant, currently restricted from use in humans. Mice were immunised with either adjuvant together with the chlamydial major outer membrane protein (MOMP) to evaluate vaccine safety and quantify the induction of antigen-specific mucosal immune responses. The level of protection against infection and disease was also assessed in vaccinated animals following a live genital or respiratory tract infectious challenge. The non-toxic CTA1-DD was found to be safe and immunogenic when delivered via the IN route in mice, inducing a comparable mucosal response and level of protective immunity against chlamydial challenge to its toxic CT/CpG counterpart administered by the same route. The utilisation of different routes of immunisation strongly influenced the distribution of antigen-specific responses to distant mucosal surfaces and also abrogated the toxicity of CT/CpG. The CT/CpG-adjuvanted vaccine was safe when administered by the SL and TC routes and conferred partial immunity against infection and pathology in both challenge models. This protection was attributed to the induction of antigen-specific pro-inflammatory cellular responses in the lymph nodes regional to the site of infection and rather than in the spleen. Development of non-toxic adjuvants and effective ways to reduce the side effects of toxic adjuvants has profound implications for vaccine development, particularly against mucosal pathogens like Chlamydia. Interestingly, we also identified two contrasting vaccines in both infection models capable of preventing infection or pathology exclusively. This indicated that the development of pathology following an infection of vaccinated animals was independent of bacterial load and was instead the result of immunopathology, potentially driven by the adaptive immune response generated following immunisation. While both vaccines expressed high levels of interleukin (IL)-17 cytokines, the pathology protected group displayed significantly reduced expression of corresponding IL-17 receptors and hence an inhibition of signalling. This indicated that the balance of IL-17-mediated responses defines the degree of protection against infection and tissue damage generated following vaccination. This study has enabled us to better understand the immune basis of pathology and protection, necessary to design more effective vaccines.

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.

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.

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.

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.

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.

Each individual experiences many sequential infections throughout the lifetime. An increasing body of work indicates that prior exposure to unrelated pathogens can greatly alter the disease course during a later infection. This can be a consequence of a phenomenon known as heterologous immunity. Most viruses invade the host through the mucosa of a variety of organs and tissues. Using the intranasal mucosal route of infection, the thesis focused on studying modulation of lymphocytic choriomeningitis virus (LCMV)-specific memory CD8 T cells upon respiratory vaccinia virus (VV) infection and the role of these memory CD8 T cells in heterologous immunity against VV and altered immunopathology in the lung. The VV infection had a profound impact on memory T cells specific for LCMV. The impact included the up-regulation of CD69 expression on LCMV-specific CD8 memory T cells and the activation of their in vivoIFN-γ production and cytotoxic function. Some of these antigen-specific memory T cells selectively expanded in number, resulting in modulation of the original LCMV-specific T cell repertoire. In addition, there was a selective organ-dependent redistribution of these LCMV-specific memory T cell populations in secondary lymphoid tissue (the mediastinal lymph node and spleen) and the non-lymphoid peripheral (the lung) organs. The presence of these LCMV-specific memory T cells correlated with IFN-γ-dependent enhanced VV clearance, decreased mortality and marked changes in lung immunopathology. Thus, the participation of pre-existing memory T cells specific for unrelated agents can alter the dynamics of mucosal immunity. This is associated with an altered disease course in response to a pathogen. The roles for T cell cross-reactivity and cytokines in the modulation of memory CD8 T cells during heterologous memory CD8 T cell-mediated immunity and immunopathology were investigated. Upon VV challenge, there were preferential expansions of several LCMV-specific memory CD8 T cell populations. This selectivity suggested that cross-reactive responses played a role in this expansion. Moreover, a VV peptide, partially homologous to LCMV NP 205, stimulated LCMV-NP205 specific CD8 T cells, suggesting that NP205 may be a cross-reactive epitope. Poly I:C treatment of LCMV-immune mice resulted in a transient increase but no repertoire alteration of LCMV-epitope-specific CD8 T cells. These T cells did not produce IFN-γ in vivo. These results imply that poly I:C, presumably through its induced cytokines, was assisting in initial recruitment of LCMV-specific memory CD8 T cells in a nonspecific manner. VV challenge of LCMV-immune IL-12KO mice resulted in activation and slightly decreased accumulation of LCMV-specific CD8 T cells. Moreover, there was a dramatic reduction of in vivoIFN-γ production by LCMV-specific IL-12KO CD8 T cells in the lung. I interpreted this to mean that IL-12 was important to augment IFN-γ production by memory CD8 T cells upon TCR engagement by antigens and to induce further accumulation of activated memory CD8 T cells during the heterologous viral infection. This thesis also systematically examined what effect the sequence of two heterologous virus challenges had on viral clearance, early cytokine profiles and immunopathology in the lung after infecting mice immune to one virus with another unrelated viruses. Four unrelated viruses, [LCMV, VV, influenza A virus or murine cytomegalovirus (MCMV)], were used. There were many common changes observed in the acute response to VV as a consequence of prior immunity to any of three viruses, LCMV, MCMV or influenza A virus. These included the enhanced clearance of VV in the lung, associated with enhanced TH1 type responses with increased IFN-γ and suppressed pro-inflammatory responses. However, immunity to the three different viruses resulted in unique pathologies in the VV-infected lungs, but with one common feature, the substitution of lymphocytic and chronic mononuclear infiltrates for the usual acute polymorphonuclear response seen in non-immune mice. Immunity to influenza A virus appeared to influence the outcome of subsequent acute infections with any of the three viruses, VV, LCMV and MCMV. Most notably, influenza A virus-immunity protected against VV but it actually enhanced LCMV and MCMV titers. This enhanced MCMV replication was associated with enhanced TH1 type response and pro-inflammatory cytokine responses. Immunity to influenza A virus appeared to dramatically enhance the mild lymphocytic and chronic mononuclear response usually observed during acute infection with either LCMV or MCMV in non-immune mice, but LCMV infection and MCM infection of influenza A virus-immune mice each had its own unique features. Thus, the specific sequence of virus infections controls the outcome of disease.

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.

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.

Research Doctorate - Doctor of Philosophy (PhD)
The aim of the work presented in this thesis was to examine the role of T helper cells (Th) and other factors affecting the expression of antibody responses in the intestinal niucosa. The experiments described in this thesis demonstrate that in rats, a population of antigen specific Th cells appears in the gut mucosal tissues and PP are an enriched source of these cells if appropriately immunized. These cells have the capacity to migrate via the TDL and in addition to their role in induction sites may also have a role in effector sites such as the gut LP and possibly other mucosal sites such as the lung. This work provides further evidence for the hypothesis that IgA responses occurring after immunization of the intestine are as much a reflection of the activities and location of regulatory T cells as of the B-cell precursors of ACC. Immunization of PP by routes other than the natural route through the gut epithelium stimulates a vigorous response to antigens which elicit tolerance if given by the oral route.

Contains errata sheet in back pocket.
Bibliography: leaves 167-211.
xviii, 211, [8] leaves, [4] leaves of plates : ill. (some col.) ; 30 cm.
Explores the hypothesis that growth of the small intestine at weaning is promoted by an activated mucosal immune system in the gut. Tests by observing rats, guinea pigs and human infants.
Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1995?

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.

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...

碩士
國立陽明大學
生物藥學研究所
95
The gut has a complex system to maintain the homeostasis in the battle of limiting inflammatory responses to commensal bacteria while retaining the ability to initiate protective adaptive immune responses to pathogens. Recently, some researches show that intestinal epithelial cells (IECs) may play key role in maintaining the homeostasis, for example, thymic stromal lymphopeitin (TSLP) produced by the IEC is capable of directing dendritic cells towards a Th2 response, and influencing the differentiation properties of monocytes into tissue macrophages. We’d like to develop a system to elucidate the bioactivity of polysaccharides from Chinese herbs (Ganoderma Lucidum, Astragalus mongholicus, Dendrobium huoshanense, Dioscorea opposita) using IEC-6 as a platform, and how they regulate intestinal immune system. With the treatment of GaLuPS and AsMoPS，IEC-6 can up-regulate IL-6、TNF-α、TLR7、TLR9 gene expression, whileas DeCaPS can up-regulate TGF-刍 gene expression, which is known as immunosuppressive cytokine, leading to the proliferation of regulatory T cell. In our in vivo OVA-induced asthma animal model, oral treatment with 30 and 90 mg/kg/day DeCaPS was shown to suppress asthma induced by active immunization and challenged with OVA. Here we show that DeCaPS can reduce pulmonary eosinophil recruitment and OVA-specific IgE in serum and bronchoalveolar lavage fluids (BALF), attenuate inflammation in lung, and decrease bronchial hyper-reactivity stimulated by methacholine. We also found that IL-13 and eosinophil-chemokine eotaxin（CCL11） mRNA from lung homogenates are lower in DeCaPS group. Furthermore, the population of CD4+CD25+ increases in intestinal lamina propira with the treatment of DeCaPS, whereas there is no difference in mediastinal lymph node in lung and spleen. According to these results, we demonstrate that DeCaPS has potential to attenuate asthma by suppressing the recruitment of eosinophil and Th2 cell in lung. Our lab had previously demonstrated that DisPoPS has potential to be used as mucosal adjuvant, so we use BALB/c mice challenged with influenza vaccine mixed with DisPoPS intranasally to confirm this result. After sacrificing the mice at day 30, we found that DisPoPS was capable of inducing a significant increase in HA-specific total IgG and IgA from lung wash、nasal、vaginal wash and serum. Our results showed that DisPoPS is a good mucosal adjuvant.

Allergic rhinitis is a highly common disease prevalent worldwide. The symptoms include rhinorrhea, itchy eyes and swelling. It is driven by Immunoglobulin E and a type 2 T cell response resulting in inflammation in the upper airways. Allergic rhinitis is often under diagnosed and has no cure, only treatments with varying efficiency. The role of the peripheral nervous system has classically not been thought of as part of the immune system. Recent studies have however, been successful in demonstrating its involvement. One of its most studied mediators, substance P (SP), seems to have a role in priming the innate immune system for incoming dangers. SP and Toll-like Receptors (TLRs) have been shown to regulate one another’s expression in healthy individuals. This project aimed to compare this cross-talk in healthy and allergic subjects. Analysis was made for additional receptors CD10, NK1R and VIPR2 and will also involve the cytokine interleukin 6 (IL-6) which is an important pro-inflammatory cytokine expressed following TLR ligand binding. Healthy and allergic individuals volunteered to leave nasal epithelial cells for culturing. These samples were stimulated with either SP or with R837, the synthetic ligand for TLR7. The project included three major methods; Enzyme-linked immunosorbent assay (ELISA), Realtime quantitative PCR and fluorescent activated cell sorting (FACS). Stimulation with R837 did not give any significant differences in the transcription of genes coding for SP, tac1, and its receptor of choice, tacr1, between 7 healthy and 9 allergic subjects. Real-time qPCR for genes tlr4 and tlr9 showed that transcription of tlr4 was significantly elevated in allergic subjects and that the same was true for transcription of tlr9 but in healthy individuals, following SP exposure. Competitive-ELISA for SP proved that allergic individuals have a slower response to R837 stimulation, secreting significantly less of SP after 15 minutes compared to healthy individuals. The FACS results were hard to interpret and are unusable due to heavily split cells and persistent infections that resulted in a too small patient group for the allergic subjects (n=1). Future projects could include more patients for FACS, more extensive analysis for CD10, incorporating it in the qPCR analysis and to perform an ELISA measuring the antiinflammatory neuropeptide Vasoactive Intestinal Peptide (VIP) as it has antagonistic properties towards SP.