<p>Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of <i>Salmonella enterica</i> serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic <i>Escherichia coli</i>.</p><p>The immune response elicited using secreted proteins from <i>S. enterica</i> serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant <i>Salmonella</i> atypical fimbriae (SafB/D) complexes, a conserved structure within <i>Salmonella</i> species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. </p><p>Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. </p><p>Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.</p>

<p>Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. </p><p>This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. </p><p>The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed.</p><p>The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.</p>

<p>Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response.</p><p>When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. </p><p>Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. </p><p>Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.</p>

In the present studies we have explored the link between food hypersensitivity and IgA nephropathy (IgAN) and evaluated treatment options in primary and recurrent disease. Approximately one third of our IgAN patients had a rectal mucosal sensitivity to gluten, as demonstrated by increased local mucosal nitric oxide production and/or myeloperoxidase release after gluten challenge. The gluten sensitivity seemed to be an innate immune reaction unrelated to the pathogenesis of celiac disease. Approximately half of the patients had a rectal mucosal sensitivity to soy or cow’s milk (CM). The levels of IgG antibodies to alfa-lactalbumin, beta-lactoglobulin and casein were significantly higher in CM sensitive as compared with non-sensitive IgAN patients, indicating that an adaptive immune response might be involved in addition to the innate immune reaction observed. With the knowledge of gastrointestinal reactivity enteric treatment was considered as a potential new treatment approach of IgAN. A 6-month prospective trial demonstrated proof-of-concept for the use of enteric budesonide targeted to the ileocaecal region of IgAN patients. We observed a modest, but significant reduction in urine albumin, a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation. eGFR calculated from the Cockcroft-Gault formula and cystatin C was not changed. In a retrospective study recurrence of IgAN and graft loss was evaluated in Norwegian and Swedish patients having received a primary renal transplant due to IgAN. Adjusting for relevant covariates, a multiple Cox-regression analysis on time to IgAN recurrence showed that use of statins was associated with reduced risk of recurrence and reduced risk of graft loss. The time lag from diagnosis to first transplantation and female gender were also associated with lower risk of recurrence. Improved graft survival was associated with related donor, low donor age and no or low number of acute rejection episodes.

The gastric mucosa is frequently exposed to endogenously secreted hydrochloric acid of high acidity. Gastric mucosal defense mechanisms are arranged at different levels of the gastric mucosa and must work in unison to maintain its integrity. In this thesis, several mechanisms underlying gastric mucosal resistance to strong acid were investigated in anesthetized rats and mice. The main findings were as follows: Only when acid secretion occurred did the pH gradient in the mucus gel withstand back-diffusion of luminal acid (100 mM or 155 mM HCl), and keep the juxtamucosal pH (pHjm) neutral. Thus, when no acid secretion occurred and the luminal pH was 0.8-1, the pH gradient was destroyed. Bicarbonate ions, produced concomitant with hydrogen ions in the parietal cells during acid secretion and blood-borne to the surface epithelium, were carried transepithelially through a DIDS-sensitive transport. Prostaglandin-dependent bicarbonate secretion seemed to be less important in maintaining a neutral pHjm. Removal of the loosely adherent mucus layer did not influence the maintenance of the pHjm. Hence, only the firmly adherent mucus gel layer, approximately 80µm thick, seemed to be important for the pHjm. Staining of the mucus gel with a pH-sensitive dye revealed that secreted acid penetrated the mucus gel from the crypt openings toward the gastric lumen only in restricted paths (channels). One crypt opening was attached to one channel, and the channel was irreversibly formed during acid secretion. Gastric mucosal blood flow increased on application of strong luminal acid (155 mM HCl). This acid-induced hyperemia involved the inducible but not the neural isoform of nitric oxide synthase. These results suggest a novel role for iNOS in gastric mucosal protection and indicate that iNOS is constitutively expressed in the gastric mucosa. It is concluded that a pH gradient in the gastric mucus gel can be maintained during ongoing acid secretion, since the acid penetrates the mucus only in restricted channels and bicarbonate is carried from the blood to the lumen via a DIDS-sensitive transporter.

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.

Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed. The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

Background: Tobacco smoking is the main cause of chronic obstructive pulmonary disease, COPD, but the mechanisms by which cigarette smoke induces COPD are still elusive. T lymphocytes have been implicated in the pathogenesis of the disease, but their role in the airway inflammation in COPD is not fully understood. The aim of this thesis was therefore to address T lymphocyte subsets and their activation in the airways of subjects with COPD, in comparison to smokers with normal lung function (S) and never smokers (NS). Methods: Subjects with moderate to severe COPD were recruited along with controls. They were all non-atopic and clinically stable, without any exacerbation during at least three months prior to inclusion. Only medication with short-acting β2-agonists and/or anti-cholinergic drugs was permitted. All subjects underwent bronchoscopy with endobronchial mucosal biopsy sampling as well as bronchial wash, BW, and bronchoalveolar lavage, BAL, collection. Biopsies were immunohistochemically stained for inflammatory cells and markers. BW and BAL fluids were prepared for differential cell counts. Soluble markers were measured in BW and lymphocyte subsets were determined in BAL using flow cytometry. Results: In biopsies, an increase in epithelial CD3+ and CD8+ cells was found in COPD, compared to NS. In BAL fluid, CD8+ cells were enhanced, whereas CD4+ cells were reduced in subjects with COPD and S, compared to NS. Furthermore, CD4+ and CD8+ cells were more activated both in COPD and S, in terms of increased expression of CD25, CD69 and HLA-DR. NKG2D-expressing CD8+ T cells in BAL fluid were enhanced in both COPD and S. CD4+CD25bright cells were upregulated in COPD and S, suggesting the presence of regulatory T cells. Further analyses of T cell subsets with the more specific markers for regulatory T cells, FoxP3 and CD127, indicated a smoking-induced expansion of non-regulatory T cells, which tended to normalize after smoking cessation in COPD. Currently smoking subjects with COPD still expressed high proportions of activated non-regulatory CD4+ T cells. The data on FoxP3 expression further indicated that the increase in CD25 expression in COPD and S was not only associated with the expansion of regulatory T cells. As CD127 expression is reported to be inversely associated with FoxP3, the data indicate the expansion of a non-regulatory CD25+ population in smokers and patients with stable COPD. The immunohistochemical staining for the NKG2D ligands MICA and MICB on epithelial cells was unchanged. Conclusion: The results of this thesis suggest a role for CD4+ and CD8+ T-cells in clinically stable COPD, indicating that T-cells are of importance in the long-term inflammatory response in COPD. Regardless of current smoking habits, activated CD8+ T lymphocytes were found to be increased in BAL fluid from subjects with COPD, suggesting that changes in CD8+ T cells are associated with a persistent immune response and, thus, of importance in COPD pathogenesis. In contrast, the expansion of non-regulatory CD25+CD4+ cells in BAL fluid seemed to be preferentially smoke-related. In summary, the data indicate that, among airway T cells, changes in CD8+ cells seem to be highly associated with COPD pathogenesis, whereas changes in CD4+ cells appear to be related to cigarette smoke-induced responses. Further, a non regulatory population of helper T cells was identified in BAL fluid of COPD patients, which may contribute to the persistent cytotoxic T cell responses.

No description available.