Multiple checkpoints control the survival and activation of auto-reactive B cells. The discovery of the TNF family cytokine BAFF has been crucial to understanding peripheral B cell tolerance mechanisms. Homeostatic levels of BAFF are tightly regulated to maintain tolerance in the periphery. Chronically increased levels of BAFF lead to the survival of autoreactive B cells. Autoimmune patients display elevated serum BAFF levels. BAFF Tg mice model this situation with systemically high levels of BAFF and the subsequent development of two separate but related autoimmune syndromes; systemic lupus erythematosus (SLE) and Sj??gren???s syndrome (SS). The work conducted in this thesis further investigates the defects in tolerance down-stream of self-reactive B cell survival, which may contribute to autoimmune disease development in BAFF Tg mice. Expansion of the Marginal zone (MZ) B cell population correlates with the pathogenesis of several models of autoimmune disease. BAFF Tg mice are unique in that they not only display an increased splenic MZ B cell population, but also MZ B cells are found in the salivary glands of mice developing SS. The examination of genes differentially regulated between MZ and Follicular (Fo) B cells led to the investigation of sphingosine-1-phosphate receptor biology. The expression of S1P receptors was shown to be required for the positioning of MZ B cells in the spleen. Chronic BAFF stimulation alters the retention of MZ B cells through the alteration of S1P receptors and decreased integrin activation. The alteration of S1P receptors and increased ligand sensitivity leads to the accumulation of MZ B cells in the inflamed salivary glands of BAFF Tg mice. This works provides a potential mechanism for the tissue specificity seen in systemic autoimmune disease. The provision of T cell help to auto-reactive B cells is thought to underlie the development of SLE. BAFF Tg mice deficient in T cells surprisingly developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did however require signals through the toll-like receptor (TLR)-associated signalling adaptor, MyD88, which controlled the production of pathogenic autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, which requires TLR signalling and is independent of T cell help. It is likely that autoimmune patients with elevated levels of BAFF show a similar basis for disease.

Multiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler’s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which the insulin producing β-cells in the pancreatic islets of Langerhans are selectively attacked by the immune system. The β-cells are destroyed resulting in a reduced or eliminated insulin production, which in turn lead to a high blood glucose level. The non-obese diabetic (NOD) mouse is the most commonly used animal model for human T1D. NOD mice develop diabetes spontaneously through a process that closely resembles the human pathogenesis. In both humans and the NOD mouse, disease is caused by a combination of genetic and environmental factors. In the NOD mouse, more than 30 insulin-dependent diabetes (Idd) loci on 15 chromosomes have been linked to disease susceptibility, however, most of the Idd-regions lack identification of a disease associated gene. B cells are required for T1D development, although the underlying mechanisms are not fully revealed. The aim of this thesis was to dissect B cell-related immune deviations in the NOD mouse, including the underlying genetics of these traits. The TACI receptor binds two ligands, i.e. the cytokines BAFF and APRIL.TACI ligation by APRIL mediates class switch, drives plasma cell differentiation and increases immunoglobulin production. In Paper I, a novel NOD-specific B cell-related trait was identified, i.e. the increased percentage of TACIhigh-expressing splenic B cells, by comparing NOD mice with non-autoimmune disease prone C57BL/6 mice. To investigate if the described TACI trait was controlled by genes linked to any Idd-region, an Idd-focused linkage analysis was performed. The TACI-trait mapped to regions on chromosome 1 and 8, more specifically to the vicinity of the Idd5.4 and Idd22. Interestingly, the linkage to Idd22 was explained by mice ≥61 days of age, suggesting a temporal genetic regulation of TACI expression possibly influenced by the ongoing autoimmune process. In Paper II, the linkage of the TACI trait to chromosome 1 and 8 was confirmed by analyzing the percentage of TACIhigh-expressing B cells in congenic NOD.C1/Idd22 mice. Moreover, the functional consequence of TACI upregulation was investigated, with the focus on plasma cell development and immunoglobulin production. NOD splenic B cells stimulated with APRIL displayed increased numbers of plasma cells and produced higher amounts of IgG and IgA compared to B cells from C57BL/6 mice. Thus, the TACI upregulation on NOD B cells possibly contribute to a B cell compartment which is more disposed to plasma cell differentiation and isotype switch. NOD mice display enhanced and prolonged immune response towards several antigens, including non-self immunoglobulins. In Paper III, the genetic factor(s) controlling the altered immune response against a BALB/c derived monoclonal antibody were dissected. Significant linkage to the Idd1/Idd24, Idd12, and Idd18.1 regions as well as to a proximal region on chromosome 2 (33.5 Mb) was detected. The linkage to Idd1/24 was verified by analyzing a set of H2-congenic NOD and C57BL/6 mice, and the linked region was narrowed down to ~8 Mb. Candidate gene analysis revealed a significant difference in the transcription of the H2-O/DO molecule. This suggests that multiple mechanisms contribute to the loss of immune response control, including an altered MHC class II peptide loading on NOD B cells. In Paper IV, a novel B cell intrinsic receptor for IgM and IgG was revealed. The receptor appeared to be more abundant in NOD mice compared to C57BL/6 mice, as the level of extramembranous IgG monomers and IgM pentamers on peripheral blood B cells from NOD mice was significantly higher compared to C57BL/6 mice. In addition, analysis of immune complex binding using IgG- or IgM-opsonized bacterial particles revealed a higher degree of binding in NOD mice compared with C57BL/6 mice. The enhanced capture of immunoglobulins and immune complexes could thus contribute to the development of T1D by altering normal B cell functions such as activation and immune complex transportation.

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. There is strong evidence that the rash is due to 12-hydroxynevirapine (12-OH-NVP), which is further metabolized to a reactive benzylic sulfate in the skin. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver appears to involve oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the hypothesis that the 12-hydroxylation pathway is involved in causing the rash. Some genes up-regulated by 12-OH-NVP were Trim63, S100a7a, and IL22ra2, etc. Up-regulation of genes such as S100a7a, which is considered a danger signal, supports the danger hypothesis. Up-regulation of genes such as the ubiquitin ligase and Trim63 are consistent with protein-adduct formation. Up-regulation of IL-22ra2 gene suggests an immune response. These results provide important clues to how NVP causes induction of an immune response, in some cases leading to an idiosyncratic drug reaction.

Nevirapine (NVP) can cause serious skin rashes and hepatotoxicity. It also causes an immune-mediated skin rash in rats but not hepatotoxicity. There is strong evidence that the rash is due to 12-hydroxynevirapine (12-OH-NVP), which is further metabolized to a reactive benzylic sulfate in the skin. This could both act as a hapten and induce a danger signal. In contrast, most of the covalent binding in the liver appears to involve oxidation of the methyl group leading to a reactive quinone methide. In this study we examined the effects of NVP and 12-OH-NVP on gene expression in the liver and skin. Both NVP and 12-OH-NVP induced changes in the liver, but the list of genes was different, presumably reflecting different bioactivation pathways. In contrast, many more genes were up-regulated in the skin by 12-OH-NVP than by NVP, which is consistent with the hypothesis that the 12-hydroxylation pathway is involved in causing the rash. Some genes up-regulated by 12-OH-NVP were Trim63, S100a7a, and IL22ra2, etc. Up-regulation of genes such as S100a7a, which is considered a danger signal, supports the danger hypothesis. Up-regulation of genes such as the ubiquitin ligase and Trim63 are consistent with protein-adduct formation. Up-regulation of IL-22ra2 gene suggests an immune response. These results provide important clues to how NVP causes induction of an immune response, in some cases leading to an idiosyncratic drug reaction.

Hantaviruses can cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). Hantaviruses are spread to humans mainly through inhalation of infectious virions, secreted from infected rodents. The human diseases are characterized by an increased capillary leakage syndrome. Hantaviruses are known to infect endothelial cells, but they are non-cytopathogenic. The mechanism behind human disease is not well understood, but an overactive immune response is implicated in the pathogenesis. The aim of my thesis has been to investigate parts of innate and adaptive immune responses in Puumala virus-infected patients. In paper I we found a sex difference in the cytokine profile during acute infection. Females had significantly higher plasma levels of IL-9, FGF-2, GM-CSF and lower levels of IL-8 and IP-10 compared to males. These differences may affect the activation and function of the immune response. In paper II we studied the phenotype and kinetics of NK cells. We observed that CD56dim NK cells were elevated during acute infection and that these, predominantly NKG2C+ NK cells, remained elevated for at least two months after symptom debut. Our novel finding of a prolonged NK cell response, implicates that NK cells may possess adaptive immunity features.  In paper III we observed a vigorous cytotoxic T cell (CTL) response during acute infection, which contracted in parallel with decrease in viral load. The CTL response was not balanced by an increase in regulatory T cells. The T cells expressed inhibitory immunoregulatory receptors, known to dampen intrinsic T cell activity.  In paper IV, we found that a low IgG response in patients was significantly associated with more severe disease, while the viral load did not affect the outcome. Our findings support the use of passive immunization as a treatment alternative for hantavirus-infected patients. In conclusion, my thesis contributes to an increased knowledge about the immune response in hantavirus-infected patients. The findings, combined with future studies, will hopefully lead to a better understanding of the pathogenesis and possible treatment alternatives.

The biomaterial component of a tissue engineered device has been shown to enhance the immune response to a co-delivered model shed antigen. The purpose of this research was to investigate in vivo the differential level of the immune response toward different forms of the biomaterial. A model shed antigen, ovalbumin (OVA), was incorporated into polymeric biomaterial carriers made of 50:50 poly(lactic-co-glycolic acid) (PLGA) in the form of microparticles (MP) or scaffolds (SC). These MP and SC biomaterial carrier vehicles with incorporated antigen were then injected or implanted, respectively, into C57BL6 mice to investigate the differential level of the immune response towards OVA controlled release from PLGA MP and PLGA SC. For each polymeric carrier, the resulting time-dependent systemic humoral immune response towards the incorporated OVA, the OVA-specific IgG concentration and isotypes (IgG2a or IgG1, indicating a predominant Th1 or Th2 response, respectively) were determined using ELISA. To assess the differential level of the immune response depending on the form of PLGA, the total amounts of polymer and OVA delivered were kept constant as well as the release rate of OVA. The in vitro protein release kinetics were studied for both PLGA MPs and PLGA scaffolds to examine the release rate of OVA from the polymeric carriers. The level of the humoral immune response was higher and sustained for OVA released from PLGA SC which were implanted with associated tissue damage, and lower and transient when the same amount of polymer and OVA were delivered from PLGA MP, which were minimally invasively delivered by injection. This immune response was primarily Th2 helper T cell-dependent as exemplified by the predominance of IgG1 isotype, although for the strong adjuvant, Complete Freunds adjuvant (CFA), and PLGA SC carriers the anti-OVA IgG2a isotype levels were also significant, potentially indicating both a Th2 and Th1 response. The PLGA SC and PLGA MP exhibited similar protein release kinetics, releasing similar amounts of OVA at each time point. Each carrier incubated contained the same ratio of OVA to polymer. In vitro protein release kinetics experiments suggest that the rate of release of OVA from PLGA SC and PLGA MP was similar, and therefore the enhanced immune response induced by PLGA SC is most likely due to danger signals from implantation which primed the system for an enhanced immune response and not from a difference in concentration of OVA released from the carriers.

Multiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler’s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS.

Dietary effects of a nucleotide mixture that contained AMP¡BGMP¡BCMP¡BUMP and IMP in equivalent amount were evaluated in 4 supplemental levels of 0, 0.05, 0.2, and 0.4 g/100g diet. Weight gain (519-616%) of juvenile groupers fed for 14 weeks with the test diets was not significantly affected by the supplements. Serum concentrations of total protein, albumin, and cholesterol, A/G ratio and activities of AST were also not significantly affected. Serum total bilirubin concentration in the 0.4 g/100g group was significantly higher than the 0 or 0.05 g/100g group. Head-kidney macrophage phagocytosis and intracellular superoxide anion production as well as serum lysozyme activity were also not different among treatments. Histological sections of fish gut indicated that villous heights of intestinal fold were significantly greater in the 0.2g/100g group than the 0.4 g/100g group, muscle layer thickness, in contrast, was not affected. When challenged with Photobacterium damsela subsp. damselae after fed the test for 3 weeks, relative percent survivals of the groupers were 87.8, 91.1, 90.0 and 94.4% (0, 0.05, 0.2, and 0.4 g/100g diet, respectively). The survivals were 74.5, 82.4, 86.3, and 85.3%, respectively when the feeding trial was extended to 18 weeks. The results show that a diet containg 38% protein and asupplemented with nucleotides at the tested levels was not effective in promoting growth, but was moderately effective in enhancing immune responses and pathogen resistance of juvenile grouper.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM&reg / -T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p&lt / 0.01). On the other hand, immunization with 160 &micro / g recombinant putative PPIase provided a significant increase in IgG level (p&lt / 0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 &micro / g and 80 &micro / g recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-&gamma / level. No significant difference was observed between control and vaccinated mice in terms of IFN-&gamma / production.