<p><strong>Background</strong><strong></strong></p> <p>IgE antibodies have high antigen specificity and are the hallmark biomarkers of allergy. IgE binds to high-affinity IgE receptors, known as FcεRI, which are expressed especially on mast cells and basophils. In allergic individuals, antigen binding to IgE that is associated with FcεRI leads to crosslinking of adjacent receptors and subsequently to cell activation, degranulation and/or secretion of bioactive molecules. These molecules together cause minor local tissue reactions such as oedema or itch, but also can cause major systemic reactions such as hypotension, cardiac and respiratory distress or even laryngeal swelling and death. The role of the nervous system in these reactions is usually thought of as secondary. However, in recent years there have been a number of studies suggesting the expression of FcεRI on neurons, opening the possibility that nerves are directly involved in antigen-specific responses and making a previously unrecognized contribution to allergic disease. <strong></strong></p> <p>Based on these previous observations regarding neuronal FcεRI, the current study employed both <em>in vivo</em> and <em>in vitro</em> approaches with the following objectives:<strong></strong> <ol> <li>To confirm the presence of FcεRI on peripheral nerves and demonstrate that they are functionally active under different conditions of IgE sensitization.</li> <li>To examine the pathways involved in neuronal activation by IgE bound to FcεRI and compare and contrast these to those already established for mast cells and basophils.</li> </ol></p> <p><strong>Methods and Results</strong></p> <p>A potential role of neuronal FcεRI in the IgE-dependent allergen avoidance behaviour of sensitized mice presented with antigen in sucrose solution was assessed based on published evidence for the involvement of peripheral nerves in this response.</p> <p>Chimeric mice with a wild-type nervous system but lacking FcεRI on hematopoietic cells including mast cells and basophils, failed to exhibit aversive behaviour, whereas mice with FcεRI-bearing hematopoietic cells demonstrated the normal aversive response confirming that FcεRI expression on mast cells is necessary for development of allergen avoidance. While immunohistochemical staining could detect IgE bound to mast cells in tissue samples, no IgE was detected on nerves where the nerves were identified by using a pan-neuronal marker, PGP 9.5, in the intestine of either normal or passively sensitized C57BL/6 and BALB/c mice. Similarly, traditional FACS analysis clearly identified FcεRI on cultured mast cells, but these methods provided no evidence for expression of FcεRI or IgE binding on superior cervical ganglion (SCG) and dorsal root ganglion (DRG) neurons in culture.</p> <p>To determine evidence for functional FcεRI on neurons <em>in vitro</em>, intracellular calcium increase was assessed as a measure of cell activation following sensitization and antigen challenge. Using both microscopy and FACS analysis, calcium fluorophore (Fluo-3, AM) increase could be detected in SCG or DRG that were activated with the calcium ionophore A23187 but not following antigen challenge.</p> <p><strong>Summary: </strong>The current study found no evidence for the presence of the FcεRI on neurons <em>in situ</em> or their sensitization by IgE actively or passively using several different approaches both in tissues and cultured SCG or DRG neurons. Possible explanations for the resultant discrepancy with previously published works are discussed.</p> / Master of Science (MSc)

The non-obese diabetic (NOD) mouse is widely used model Type 1 diabetes (T1D), a chronic inflammatory disease characterized by destruction of the insulin producing β cells in the islets of Langerhans by immune cells. The classical symptoms include increased glucose levels in urine and blood, frequent urination and enhanced thirst. The disease has a strong genetic component and is also influenced by the environment. NOD mice develop T1D spontaneously. The disease occurs in two phases; insulitis - the infiltration of immune cells in the islets of Langerhans and overt diabetes caused by the destruction of insulin producing β cells. Several disease associated gene regions or loci [termed insulin dependent diabetes (Idd) loci] have been associated with T1D development. Although, T1D is recognized as a T cell mediated disease in both mouse and man, many studies have shown the importance of B cells in the pathogenesis of the disease. Autoantibodies appear prior to islet infiltration and several molecular and cellular events precede this beta-cell autoimmunity. Although the pathogenesis of T1D is well characterized, less is known about the environmental and immunological factors that trigger the disease. In this thesis, we studied the contribution of B cell anomalies to the skewed immune response observed in the NOD mouse. In our studies covered in the thesis we observed that NOD mice display enhanced IgE in the serum already at one week of age. In addition, upon treatment of pre-diabetic NOD mice with anti-IgE antibodies, diabetes incidence was delayed. We hypothesize that the presence of IgE in the system may be explained due to enhanced class switching. Antibody feedback however, is an essential component of the immune response and can lead to either enhanced or dampened responses. Thus, increased IgE may provide positive feedback that might sustain an immune response. We also aimed to analyze the biological consequence of this feature. In vitro stimulation of B cells by the TACI ligand APRIL resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. In addition, TACI+ cells were observed in NOD germinal centers facilitating increased BAFF uptake and subsequent escape of low affinity antibody producing clones. NOD mice elicited an enhanced and prolonged immune response towards T-dependent antigens such as hen-egg lysozyme (HEL). Serum HEL-specific IgG level was significantly increased and was predominantly of the IgG1 isotype. Immunofluorescence analysis of NOD spleen revealed the presence of spontaneous germinal centers which others have perceived to provide a ready niche for the entry of naïve B cells that encountered novel antigen. Adoptive transfer experiments of purified B and T cells from NOD into NOD.Rag2-/- (NOD-RAG) mice illustrated the importance of B cell intrinsic defects in the reproduction of the original phenotype as observed in NOD.

Kainate receptor knockout mice have unique differences within their immune system. They exhibit an attenuated TH2 branch, while maintaining a robust TH1 response. Specifically, blocking the formation of functional kainate receptors affects mast cells and their related pathologies. While they seem to develop and activate normally in vivo and in vitro, KAR KO mast cells release more inflammatory mediators upon degranulation. These mice experience severe anaphylactic shock due to two compounding abnormalities. First, KAR KO mast cells release significantly more histamine in vivo upon IgE-mediated activation. Second, the animals over-respond to exogenous histamine with drastic temperature drops compared to WT. This report shows that the kainate receptor plays an important role in mast cell-mediated immune responses.

In this study we show, to our knowledge, the first description of the role ADAM10 plays on mast cells. ADAM10 is abundantly expressed on mast cells both in vitro and in vivo. Its expression is inhibited by IL-10, a suppressive cytokine. siRNA depletion of ADAM10 on bone marrow-derived mast cells (BMMC) caused decreased IL-6 production following IgE cross-linking and also impaired BMMC stem cell factor (SCF)-induced migration through collagen IV. Mast cells and T helper cells (Th cells) in the peritoneum were reduced in ADAM10 KO mice. In addition, ADAM10 KO BMMC produced significantly less of all cytokines measured following IgE cross-linking, including IL-6, TNF-α, IL-13, and MCP-1, compared to wild type BMMC. Collectively these data show that mast cell ADAM10 can be regulated by a T regulatory cell cytokine, IL-10, and describes key ways in which ADAM10 loss affects prototypical mast cell functions and distribution.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Polymorphonuclear neutrophils (PMNs) are important effector cells in host defense and the inflammatory response to antigen. The involvement of PMNs in inflammation is mainly mediated by the Fc receptor family, including IgE receptors. Recently, we have shown that human PMNs from allergic asthmatic subjects express the high affinity receptor, FceRI. In this study, we have examined the regulation of FceRI by human PMNs in vitro and in vivo during the allergic pollen season. First we studied the pattern of expression of FceRI in PMNs during the pollen allergic and outside the pollen season. Peripheral blood neutrophils were isolated from adult atopic asthmatics (AA) (n=17), allergic non asthmatics (ANA) (n=15) and healthy donors (n=16) by dextran, ficoll gradient centrifugation and magnetic cell sorting (MACS). Surface, total protein and mRNA expression of FceRI were investigated in the three groups by FACS, immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) respectively. Secondly, we investigated the effect of Th-2 cytokines which are known to regulate IgE receptor expression. PMNs from atopic asthmatic subjects were stimulated in vitro with Th-2 cytokines (IL-4, IL-9, GM-CSF) and Th-1 cytokine IFN-gamma. Finally we determined whether the expression of FceRIbeta chain correlated with the surface expression of FceRIalpha chain in PMNs. Irrespective of the season, PMNs from atopic asthmatic subjects showed increased expression of FceRIalpha chain in surface, total protein and mRNA compared to atopic non asthmatics and healthy donors (n=20). Interestingly, FceRIalpha chain surface and mRNA expression increased significantly during pollen season compared to non pollen season (P=0.001) in PMNs isolated from AA (n=9) in contrast to healthy donors and ANA (n=8). Furthermore similar pattern of FceRI expression were observed in vitro when PMNs were stimulated with Th2 cytokines. IL-4, IL-9 and GM-CSF showed increased protein and mRNA expression of FceRIalpha chain at 6 and 18hrs (n=6) whereas IFN-gamma down regulated the mRNA expression of FceRIalpha chain at 6hrs. Also, irrespective of season AA (n=11) subjects showed increased expression of FceRI beta chain when compared to ANA (n=10) and healthy donors (n=9). Western blot analysis showed increased FceRI beta protein in atopic asthmatic subjects (n=4). Interestingly irrespective of the groups, there was a positive correlation r = 0.8054 between total protein expression of beta chain with surface expression of alpha chain of FceRI in neutrophils. Our data suggest that the expression of FceRI in neutrophils of atopic asthmatic patients is highly regulated. Our in vitro studies provide evidence that Th-2 cytokines such as IL-9, IL-4 and GM-CSF up-regulate the expression of FceRI. Furthermore we show evidence of increased expression of FceRIbeta chain in neutrophils of atopic asthmatic subjects. Collectively these results suggest that FceRI mediated neutrophil dependent activation may play a key role in allergic diseases. / May 2005

This thesis consists of two topics. A frequency-shift readout circuit is integrated for the rapid IgE measurement biomedical system in the first half. Secondly, we present a ROM-less DDFS (direct digital frequency synthesis) using a non-equal division parabolic polynomial interpolation method, which is used as the frequency generator in the measurement system. The first topic investigates the IgE concentration measurement system and realizes the readout circuit using TSMC 1P6M 0.18 £gm CMOS technology. We integrate the flexural plate wave (FPW) sensor chips and an ASIC comprising control block, digital to analog convertor (DAC), OTA-C oscillators, amplifiers, peak detectors, registers, and a subtractor. By taking advantages of the characteristics that the central frequencies of the loaded FPW sensors will be shifted, sine waves with various frequencies are generated and swept through one pair of FPW sensors. The frequency difference of these sensors is then readout to get concentration by look-up table. The second topic investigates the division method of a quarter sine wave to improve the spurious free dynamic range (SFDR) and realizes a ROM-less DDFS which is used as the frequency generator in the mentioned IgE measurement system. The proposed non-equal division parabolic polynomial interpolation method will generate a complete sine wave by a quarter of a sine digital signal owing to the symmetry. We combine the quasi-linear interpolation and an offset adjustment to derive the quarter sine wave digital signals. The proposed method not only reduces the absolute error between ideal sine wave and generated sine wave, it also improves SFDR.

No description available.

No description available.

No description available.