Studies in cystic fibrosis patients have suggested that the balance between pro- and anti-inflammatory cytokines may be important in controlling infection and tissue destruction during bronchopulmonary Pseudomonas aeruginosa (PA) infection. Here, the inflammatory response was characterized in a murine model of bronchopulmonary PA infection in resistant BALB/c and susceptible C57BL/6 mice following intratracheal inoculation with 10 5 CFU of mucoid PA entrapped in agar beads. On day 7 post infection, BALB/c mice had significantly less bacteria and significantly more IL-10 in the lungs as compared to C57BL/6 mice while the levels of TNF-alpha and IFN-gamma levels were comparable. Bronchoalveolar lavage revealed that the significantly lower lung inflammatory response in BALB/c mice was composed of 45% PMN, 30% lymphocytes and 25% macrophages while that of C57BL/6 mice was composed of 62% PMN, 8% lymphocytes, and 30% macrophages. Alveolar macrophages from C57BL/6 mice spontaneously produced significantly higher NO levels while cells from BALB/c mice produced significantly more TNF-alpha, either spontaneously or following stimulation with LPS or heat-killed PA. There was no difference in IL-10 production. Lung PMN from either strain produced undetectable levels of NO and high levels of TNF-alpha. PMN from BALB/c mice, however, produced significantly higher levels of IL-10. The severity of infection in both strains of mice was exacerbated by administration of the anti-IL-10 mAb JES5-2A5. IL-10 neutralization resulted in an increased inflammatory response, increased bacterial counts, and increased TNF-alpha levels in both mouse strains. The increased inflammatory response in resistant BALB/c mice was due to a significant influx of macrophages whereas in susceptible C57BL/6 mice a significant influx of PMN and decreased numbers of macrophages were observed. Taken together, these results suggest that an intricate network of cells and cytokines regulates the pulmonary infla

Cytomegalovirus (CMV) is an ubiquitous pathogen which can cause severe opportunistic infection in immune compromised or suppressed patients. Current vaccine strategies use recombinant glycoprotein gB subunits; however, little is known about the immunogenicity of this glycoprotein and the possible induction of autoimmunity. Using established ELISA assays and immunoblotting, anti-viral and autoantibody responses were detected following intraperitoneal injection of a recombinant Adenovirus-gB construct. The immune response to gB was studied in MRL/mpj (H-2k), BALB/c (H-2d), C3H (H-2 k) and BALB.k (H-2k) mice. A significant IgG anti-viral response was induced in all four strains. IgG autoantibodies, including Rheumatoid Factor (RF), anti-DNA, and anti-U1 70K were also induced. Antibodies to U1 70K are typical of patients with Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD). The most significant antibody responses were to gB and to U1 70K, with higher levels detected in the autoimmune prone mice. Since multiple genetic factors influence the immune response, immunization of this construct in genetically predisposed individuals could trigger the onset of an autoimmune disease state such as SLE.

Characterization of the phyenotypic, functional and proliferative properties of antigen-specific CD8 T cell subsets allowed us to delineate a differentiation pathway based on the expression of CD45 isotype and CC-chemokine receptor 7 (CCR7), and undergone by T cells following encounter with cognate antigen. Comparative assessment of HIV- and cytomegalovirus-specific responses exhibited by dually-infected patients evidenced a skewed maturation of HIV-specific memory CD8 T lymphocytes and their altered distribution in blood and lymph nodes. These findings contribute to our understanding of immunologic memory, may be relevant to HIV-1 pathology and are pertinent to the design, as well as evaluation of therapeutic regimens and vaccination strategies.

This work was aimed, firstly, at studying the effects or a form of immunotherapy utilizing the cytokine, interleukin-2 and the non-steroid, anti-inflammatory drug, indomethacin, on natural killer (NK) cells in young adult mice bearing a tumor of hemopoietic origin, i.e., lymphomas and leukemias. In the hands of others, recently, this form of immunotherapy has had strikingly ameliorative influences in mice and man bearing solid (melanoma) tumors. The second aim of this project was to test the universality, with respect to age, of this form of immunotherapy in mice bearing a tumor of hemopoietic origin. The third goal was to investigate the mechanism(s) responsible for the well known lack of NK cell functional activity in normal infant and aged mice. Under various combinations and applications of the cytokine and drug, in both normal and erythroleukemic mice, the following parameters were measured: (i) quantitation of NK cells in the bone marrow and spleen, and (ii) the lytic activity of those cells in all cases. In some cases, their (i) proliferative capacity, (ii) target binding capacity and (iii) microenvironmental influences were also assessed. The project has shown that rIL-2+indomethacin treatment of tumor-bearing young adult mice significantly elevated NK cell numbers and their function, reduced tumor cell numbers and increased the life span of such mice. However, in tumor-bearing infant mice, the drug and/or cytokine increased life span and stimulated NK cell numbers was/were unable to induce NK cell functional (lytic) activity from these numerically increased NK cells. Finally, tumor-bearing aged mice were totally unresponsive to such treatments. These results indicate that this form of immunotherapy is very effective in stimulating NK cells in mice bearing erythroleukemia. However, this form of immunotherapy is not universal with respect to age. The lack of NK cell functional activity in normal infant. and aged mice results from different mechanisms. Wher

About 40% of human pregnancies are unsuccessful and many of these are thought to be genetically normal. Our hypothesis is that decidual NK cells in abortion prone pregnancies produce IFN-gamma which primes macrophages. These primed macrophages are then triggered by a second signal to become the major effectors in early embryo resorption. Analysis of individual implantation sites was performed at day 9 of pregnancy to determine the cytokine profile of these NK cells and if NK cells selectively infiltrate the decidua of embryos that will undergo resorption. Use of a pan-NK cell marker (DX5) allowed labeling of decidual NK cells for flow cytometric analysis. Magnetic labeling and isolation of DX5+ cells from individual embryos was followed by RT-PCR and southern blot analysis. This thesis prevents evidence that a number of the embryos are infiltrated by higher numbers of DX5+ NK-cells with an incidence that is similar to the occurrence of early embryo loss in this experimental model. (Abstract shortened by UMI.)

Macrophages are considered as one of the early effector cells involved in the immunological response against infection. Following macrophages interaction with pathogens, macrophages become activated and attempt to eliminate the invader. Interferon gamma (IFN-gamma) is one of the most important activators of macrophage function. In macrophages IFN-gamma is promoting the expression of major histocompatibility complex class II (MHC-II) molecules and other proteins directly involved in the process of containing infection. / The effect of the Nramp1, on macrophage function is investigated in this thesis. Nramp1 has been shown to determine the resistance or susceptibility of mice to infections with several intracellular microorganisms, including Mycobacterium bovis BCG. Although the precise mechanism of Nramp1 action is unknown, there are several well-established effects associated with the Nramp1. In general, it has been shown that macrophages derived from mice susceptible to infections with M. bovis BCG are less efficient in the production of nitric oxide (NO), reactive oxygen intermediates (ROI), TNF-alpha, and MHC-II antigens in response to IFN-gamma. / Using macrophage cell lines derived from mice that are either resistant (B10R) or susceptible (B10S) to M. bovis BCG infection, we have demonstrated that lower levels of IFN-gamma-induced expression of MHC-II antigens is correlated with less efficient phosphorylation of the STAT1 protein in B10S macrophages compared to B10R macrophages. We have shown that low levels of MHC-II expression in B10S macrophages correlate with less efficient expression of CIITA (Class II Transactivator). We have observed that infection of macrophages with M. bovis BCG has an inhibitory effect on both CIITA and MHC-II expression in macrophages stimulated with IFN-gamma. / We have also studied the effect of lipopolysaccharide (LPS) on MHC-II expression in macrophages. We have found that the inhibitory effect of LPS on CIITA gene transcription does not involve changes in the binding of STAT1 to CIITA promoter IV. We have also demonstrated that unlike M. bovis BCG, the inhibitory effect of LPS on MHC-II expression is mediated by Toll-like receptor 4 (TLR4). In addition, we have shown that inhibitory effects of both LPS and M. bovis BCG depend on the adaptor protein MyD88. / We have also analyzed the regulation of IFN-gamma- or/and LPS-stimulated expression of TLR2 in macrophages. We have shown that regulation of TLR2 expression by IFN-gamma depends on TLR4 expression. We have also determined that the phenol extractable fraction present in the commercial preparations of endotoxins from Gram-negative bacteria is able to synergize with IFN-gamma and activate TLR4-deficient macrophages. / Overall, we believe that these studies significantly contribute to the understanding of the molecular mechanism of the process of macrophage activation.

Recent studies have determined that IgE-producing B lymphocytes are present in the respiratory mucosa of patients with asthma and rhinitis, but not in respiratory mucosa of normal individuals. Our laboratory has shown that B lymphocytes in human nasal mucosa stain strongly for interleukin 13 (IL-13), a crucial cytokine for airway hyper-responsiveness and remodelling, and that inhibiting IL-13 decreases IgE secretion significantly in B cells. We sought to determine the role of IL-13 in sustaining IgE-producing B lymphocytes in the absence of a lymph node-like environment. Human B lymphocytes were isolated from tonsils and purified by means of rosetting with sheep RBCs. Purified B cells were transfected by NucleofectionTM with IL-13- or IL-13Rα1-gene specific siRNA or anti-IL-13 antibodies. They were then stimulated with the LTK4A1 fibroblast line transfected with CD40-ligand or anti-CD40 antibodies, and recombinant IL-4. Total mRNA was extracted, and plasma cell related genes were measured by means of real-time PCR. Proliferation and colony formation were also observed to determine phenotypical differences. IL-13 siRNA and anti-IL-13 decreases IgE secretion in stimulated cultured B cells. Anti-IL-13 treatment also markedly increases prdm1 and april mRNA transcripts. Anti-IL-13 treatment caused stimulated B cells to produce more colonies that were smaller in size compared to control. Blocking production of IL-13 through anti-IL-13 may trigger B cells to mature into short-lived plasma cells by increasing prdm1 and april. IL-13 may be an important autocrine factor for long-lived IgE-producing B lymphocytes, and therefore be the reason for sustained IgE in the airways. / Des études récentes ont déterminé que les lymphocytes B produisant l’IgE sont présents dans la muqueuse respiratoire des patients avec l'asthme et le rhinite, mais pas dans la muqueuse respiratoire d'individus normaux. Notre laboratoire a démontré que les lymphocytes B dans la muqueuse nasale humaine sont marqués fortement pour l’interleukine 13 (IL-13), une cytokine cruciale pour l’hypersensibilité des voies respiratoires et le remodelage, et en inhibant l’IL-13, la sécrétion d’IgE est diminuée de façon significative dans les cellules B. Nous avons cherché à déterminer le rôle d'IL-13 dans la production prolongée d'IgE par les lymphocytes B dans l’absence d’un environnement ressemblant à un environnement d’un ganglion lymphatique. Des lymphocytes B humaines ont été isolés d’amygdales et purifiés avec une méthode qui utilise le sang de mouton. Les cellules B purifiées ont été transfecté avec la technologie NucleofectionTM avec du ARN interférent spécifique pour l’IL-13 ou son récepteur ou avec des anticorps anti-IL-13. Ils ont été ensuite stimulés avec une lignée cellulaire de fibroblast (LTK4A1) transfectée avec le ligand CD40 ou stimulés avec des anticorps anti-CD40, et du IL-4 recombinante. L’ARNm total a été extrait, et les gènes reliés avec des cellules plasmocytaires ont été mesurés par la PCR temps réel. La prolifération et la formation des colonies ont été aussi observées pour déterminer les différences phénotypiques. L’ARN interférent d’IL-13 et l’anti-IL-13 diminue la sécrétion d'IgE dans les cellules B stimulées. De plus, le traitement d’anti-IL-13 augmente nettement les transcriptions de prdm1 et d'april. Le traitement d’anti-IL-13 a causé les cellules B stimulées à produire plus de colonies qui étaient plus petites comparée aux cellules contrôles. En bloquant la production d'IL-13 avec des anticorps anti-IL-13, les cellules B peuvent préférentiellement m

An initial study comparing cytokine gene expression in the skin of control vs. anergic patients lacking delayed type hypersensitivity reactivity revealed no difference; but disclosed an apparent absence of detectable CD3+ T cells in the skin of anergic individuals. To assess its significance for anergy, an investigation of the role of skin T cells in DTH-reactive healthy individuals was undertaken. To do so, the phenotypic and functional characteristics of T cells isolated from skin and blood were compared. Analysis by flow cytometry has shown that 74% of skin T cells expressed cell surface HLADR, 66% were positive for the IL-2 receptor CD25, and less than 43% have displayed the VLA integrin $ alpha$4 chain as compared to 28%, 7%, 79% for peripheral blood mononuclear cells respectively. The expression of a cutaneous lymphocyte antigen (CLA) was 61% in the former and 14% in the latter. Functionally, skin T cells failed to proliferate in response to all ligands including IL-2, anti-CD3, lectins and phorbol esters with ionomycin, as well as showed a reduced Ca++ flux to phytohemagglutinin. Skin tissue co-cultured with autochtonous PBL could inhibit its proliferative reaction. Despite their ability to proliferate, lymphocytes from skin were shown to be able to produce IFN$ gamma$ in response to PHA+IL-12 as well as anti-CD3+IL-2. Inhibition by anti-cytokine mAbs revealed that in both instances IL-12 was obligatory for this production. In an additional study it was established that a hitherto uncharacterized subset of T cells in blood which could secrete IFN$ gamma$ consisted of CLA+ cells. This observation established a functional link between these CLA+ skin-seeking T cells and the CLA+ T cells in skin. / A major difference between IFN$ gamma$-producing cells from blood and skin was found to be the tempo of synthesis: whereas, PBMC was first detected to contain IFN$ gamma$ 42 hours following activation, lasting for days, skin cells were positive after 2.5 hrs of activation, (or 16x faster) for a duration of only 90 minutes. These kinetics were confirmed using intact skin in culture. Experiments designed to reveal the mechanism of this fast action have shown that mRNA for IFN$ gamma$ is present in unstimulated isolated skin T cells as well as in intact skin, but not in PBMC, and its presence may be attributed to ongoing constitutive transcription. Activation of skin T cells, which has been shown to elicit prompt translation in IFN$ gamma$ synthesis has also been shown, at the same time, to terminate IFN$ gamma$ gene transcription in an apparently selective manner. Accordingly, it can be seen that the amount of IFN$ gamma$ synthesized in skin and the duration of its synthesis is preprogrammed. This mode of regulation may be unique to the skin, and unique for IFN$ gamma.$ / The results presented are interpreted to indicate that r cells present in human skin may play an essential role in the DTH response, and provide evidence for "peripheral sensitization", or lymphocyte activation outside organized lymphoid tissue. Because of its speed, it may represent the antigen-specific component of a first line cutaneous host defence system. The absence of such T cells in the skin of anergic patients may indeed be responsible for a lack of DTH reactivity, and its clinical consequences.

The bursa of Fabricius is central to the establishment of the chicken B cell compartment. In juvenile chickens B cells emigrate from the bursa at a rate of about 1% of the peripheral blood and spleen B cell pools per hour. Three distinct B cell populations have been identified in the blood: population 1 is the largest, consisting of short lived cells that express the surface marker LT2 and emigrate directly from the bursal follicular cortex. Population 2, the next largest population, also consists of direct bursal emigrants that are longer lived cells, and lack surface LT2. Population 3 B cells are short lived, and are derived from a bursal-independent and rapidly dividing source, which is, however, ultimately dependant on the bursa for its establishment. Only 5% of the lymphocytes generated daily in the bursa emigrate to establish the peripheral B cell compartment. The remainder die in situ by apoptosis. Cell death in the bursa is preceded by loss of surface immunoglobulin, a threshold level of which appears essential for progression through the cell cycle and subsequent emigration.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by leukocytic infiltration of the central nervous system (CNS) and demyelination and remission/relapse. It is induced by CD4$ sp{+}$ T cells. We used reverse transcriptase/polymerase chain reaction to analyse T cell and cytokine gene expression in the CNS of SJL/J mice with myelin basic protein-induced EAE. Undetectable in normal CNS and cerebrospinal fluid, the expression of CD3, IL-2, IFN-$ gamma$, and TNF$ alpha$ increased in EAE, correlating with disease severity, then dropped to background levels during remission. IL-2 and IFN-$ gamma$ were produced by CD4$ sp{+}$ CD45RB$ sp{ rm low}$ T cells isolated from LN and CNS. In contrast, TNF$ alpha$ was predominately made by macrophages and microglia in the CNS. Purified microglia from normal CNS were induced to express TNF$ alpha$ by activated TH1 supernatant, suggesting that TNF$ alpha$ expression by cells in the CNS could be regulated by cytokines from infiltrating T cells. IL-4 was not detectable in total CNS or in isolated CD4$ sp{+}$ CD45RB$ sp{ rm low}$ cells from the CNS, but was readily amplified from CD4$ sp{+}$ CD45RB$ sp{ rm low}$ LN T cells. This suggests an enrichment of TH1 cells in autoimmune CNS. / To determine the effect of IFN-$ gamma$ expression in the CNS, we produced transgenic mice using an IFN-$ gamma$ cDNA downstream of an MBP promoter. Expression of the transgene was CNS-specific. MHC class I was induced in the CNS of transgenic mice. Transgenic animals that were backcrossed up to 5 generations with SJL/J did not develop spontaneous pathology. However, when they were immunized with MBP in adjuvant, the penetrance of EAE was greater, symptoms were more severe, and the duration of the first episode significantly longer than in non-transgenic littermates, suggesting a role for IFN-$ gamma$ in the amplification and perpetuation of EAE.