Bibliography: leaves 111-137.

Bibliography: leaves 129-145.

<p>IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages <i>in vitro</i>. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.</p>

Graduation date: 2006

IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages in vitro. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.

NF-κB transcription factors are regulators of a wide spectrum of genes involved in immune responses and inflammation as well as cellular proliferation and survival. Transcriptionally competent NF-κB dimers are retained in the cytoplasm of resting cells by binding to inhibitors of NF-κB (IκBs). Stimuli that activate NF-κB converge on the activation of the IκB kinase (IKK), resulting in phosphorylation and subsequent proteasomal degradation of IκB. This releases functional NF-κB dimers that rapidly move to the nucleus where they regulate transcription of NF-κB-dependent target genes. The study of signalling to NF-κB from T and B lymphocyte antigen receptors is a field of intense investigation, and much attention is focused on the complex of the molecular scaffolding proteins Carma1, Bcl10 and MALT1. Together, these are crucial for the organisation of a structure beneath the activated receptor, termed the immunological synapse. IKK is recruited to this structure and becomes activated, subsequently leading to activation of NF-κB. Calcium (Ca2+) is a ubiquitous intracellular messenger that is involved in the regulation of numerous aspects of cellular function, including transcription. NF-κB activity is known to be regulated by changes in intracellular Ca2+ levels, such as those created by antigen receptor activation, but the mechanisms are to a large extent undefined. Ca2+ signals in cells are transmitted predominantly by the ubiquitous Ca2+ sensor protein calmodulin (CaM). Signalling that increases the intracellular Ca2+ concentration leads to binding of Ca2+ to CaM, which changes its structure, thereby allowing it to interact with a new range of target proteins. The studies of NF-κB signalling in lymphocytes presented here reveal that CaM is involved, both directly and indirectly, in the regulation of NF-κB. CaM was found to interact directly and in a Ca2+-dependent manner with the NF-κB proteins RelA and c-Rel after their signal-induced release from IκB. The interaction of CaM with c-Rel, but not RelA, was found to be inhibitory for its nuclear accumulation and transcriptional activity on Ca2+-regulated IL-2 and GM-CSF promoters; thus, CaM binding was found to differentially regulate c-Rel and RelA in lymphocytes. CaM was also shown to interact directly and in a Ca2+-dependent manner with Bcl10. The interaction was mapped to the Carma1-interacting CARD domain of Bcl10 and was found to have a negative effect on the ability of Bcl10 to bind to Carma1. Binding of CaM to Bcl10 also had a negative effect on activation of NF-κB after T cell receptor stimulation, since a point mutant of Bcl10 with reduced binding to CaM showed increased activation of an NF-κB reporter in Jurkat T cells, which was further enhanced by TCR-activating stimuli. In addition, CaM was found to positively regulate NF-κB activation indirectly through CaM-dependent kinase II (CaMKII). Inhibitors of CaM and CaMKII were shown to inhibit IκBα degradation in lymphocytes induced by phorbol ester or T cell receptor stimulation. The actions of CaMKII were mapped to a point upstream of IKK activation and further studies revealed that CaMKII is recruited to the immunological synapse, where it inducibly interacts with and phosphorylates Bcl10 at multiple sites. Phosphorylation of Bcl10 by CaMKII was shown to be important for the ability of Bcl10 to activate NF-κB, since mutation of the phosphorylation sites of Bcl10 inhibited Bcl10-induced transcriptional activity of NF-κB, in part by preventing signalinduced ubiquitination and degradation of Bcl10.

CHARACTERIZATION OF CARBOHYDRATE BASED VACCINES Variables influencing the immunogenicity and physicochemical properties of glycoconjugate vaccinesMany aspects can influence the immunogenicity of conjugate vaccines and the main variables investigated so far are the size of the saccharide moiety, the saccharide:protein ratio in the purified conjugate, the conjugation strategy, the nature of the spacer and the protein carrier. The size of the saccharide moiety and saccharide/protein ratio were investigated in different works such as Seppälä and Mäkelä in one of the first studies on the effect of size and chemistry on the immunogenicity of dextrans-protein conjugates found that dextrans of low molecular weight conjugated to chicken serum albumin, induced strong anti-dextran responses in mice, while increasing the dextrans' size resulted in reduced immunogenicity.47 Peeters et al. showed that a synthetic tetramer of Hib capsular polysaccharide repeating unit, conjugated to a protein carrier, induced in adult mice and non-human primates antibody levels comparable to a commercial Hib conjugate and higher than those induced by a trimer, indicating that for Hib a minimum of eight sugars is needed for a proper immunological response.48 Laferriere et al. found little influence of the carbohydrate chain length on the immunogenicity of pneumococcal conjugate vaccines in mice.49 Pozsgay et al. studied the immunogenicity in mice of synthetic Shigella dysenteriae type 1 LPS oligosaccharides conjugated to human serum albumin (HSA). The authors found that octa-, dodeca-, and hexadecasaccharide fragments induced high levels of lipopolysaccharide binding IgG antibodies in mice after three injections and were superior to a tetrasaccharide conjugate. The influence of the carbohydrate/protein ratio was different for the three conjugates. The octasaccharide-HSA conjugate with the highest density evoked a good immune response, while in the case of dodeca- and hexadecasaccharide conjugates, the median density was optimal.50 These studies suggest that oligosaccharide chain length and hapten loading might be interconnected in determining the immunogenicity of glycoconjugate vaccines. The spacer is a short linear molecule that is generally linked to the polysaccharide chain or to the protein or to both moieties, depending on the chemistry, used to facilitate the coupling between the protein and sugar. There are evidences in the literature which suggest that rigid, constrained spacers like cyclohexyl maleimide, elicit a significant amount of undesirable antibodies, with the risk of driving the immune response away from the targeted epitope on the hapten.51 52 The use of a flexible alkyl type maleimido spacer has been reported as a way to overcome the previous observed immunogenicity of cyclic maleimide linkers.53 A number of protein carriers have been used so far in preclinical and clinical evaluation of conjugate vaccines. 54 55 56 57 58 59 60Proteins such as diphtheria and tetanus toxoids, which derive from the respective toxins after chemical detoxification with formaldehyde, were initially selected as carrier because of the safety track record accumulated with tetanus and diphtheria vaccination. CRM197, a non-toxic mutant of diphtheria toxin61 which instead does not need chemical detoxification, has been extensively used as carrier for licensed Hib, pneumococcal, meningococcal conjugate vaccines and for other vaccines being developed. An outer membrane protein complex of serogroup B meningococcus has been used by Merck as carrier for their Hib conjugate vaccine.62 GSK in their multivalent pneumococcal conjugate vaccine introduced the use of the Hib-related protein D as carrier for most of the polysaccharides included into the vaccine.63 64 The team of John Robbins made extensive use of the recombinant non toxic form of Pseudomonas aeruginosa exo-toxin as carrier for Staphylococcus aureus type 5 and 8 as well as for Salmonella

Prostaglandin E2 (PGE2) is an immunosuppressive factor secreted by the murine Lewis lung carcinoma (LLC). This factor can help to insure the survival of a tumor by suppressing the functions of lymphocytes and macrophages in their defense against tumors.Four weekly assays of macrophage tumoristatic activity from tumor-bearing mice were performed during the course of tumor development. Macrophages from the peritoneal cavity were cultured with LLC cells and after 48 hours the LLC growth was measured.The results of this study showed that macrophages from tumorbearing mice were suppressed in their ability to halt LLC growth in vitro. This suppression could be prevented by the use of indomethacin, a prostaglandin synthesis inhibitor. Indomethacin administration to tumor-free mice also enhanced their macrophage tumoristatic activities suggesting that the tumor was not the exclusive source of prostaglandin which suppressed anti-tumor immunity. Finally, indomethacin-fed tumor-bearing mice had smaller tumors than did mice not fed indomethacin.Ball State UniversityMuncie, IN 47306

The immune regulatory abilities of alveolar macrophages from C57B1/6 mice bearing a metastatic variant of Lewis lung carcinoma were determined. During early stages of tumor development, or before tumors metastasized to the lungs, alveolar macrophages did not affect or slightly enhanced T-lymphocyte proliferation; as tumor growth progressed, or following tumor metastasis, alveolar macrophages suppressed the T-cell response. Macrophage suppressor activity was probably not mediated by their production of PGE, since macrophages of tumor-bearing mice secreted less 2 PGE than did macrophages of normal mice. Normal alveolar 2 macrophages or macrophages preincubated in tumor cell supernatant for a short period stimulated T-cell blastogenesis and secreted PGE during in vitro culture. However, with 2 longer exposure to tumor cell supernatant, alveolar macrophages lost the capacity to augment T-cell proliferation and secreted less PGE 2.Ball State UniversityMuncie, IN 47306

T helper (Th) cell activation is necessary for the adaptive immune response. Formation of an immunological synapse (IS) between Th cells and antigen-presenting cells is the first step in Th cell activation. In vitro studies indicate that formation of the IS induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2 influx across the plasma membrane and Ca2 -dependent Th cell activation. Earlier studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFA suppress CD4 T cell mitochondrial translocation during the early stages of IS formation and down-modulate Ca2 dependent Th cell activation. CD4 cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were co-cultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. fat-1 mice exhibited a significantly (P< 0.05) reduced percentage of CD4 T cells with mitochondria which translocated to the IS; fat-1 (30 percent) versus wild type control (82 percent). With respect to an effect on the mitochondrial-to-cytosolic Ca2 ratio, wild type cells showed significant increases at the IS (71 percent) and total cell (60 percent) within 30 min of IS formation. In contrast, fat-1 CD4 T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2 ratio was observed in wild type cells co-incubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2 (CRAC) channels, BTP2. Together, these observations provide evidence that n-3 PUFA modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2 entry.