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Diploma thesis deal with the non-compliance questions by allergic patients. The thesis describe in detail the alergic disease with focusing on the forms, levels, risk factors and impact of non-compliance phenomenon. The practical part use the obtained informations from the quantitative research. These data were processed and used for reach the goals of the thesis. The data analysis evaluates and confirms eventually deny the theoretical supposition.

Diisocyanates (dNCO) such as methylene diphenyl diisocyanate (MDI) are used primarily as cross-linking agents in the production of polyurethane products such as paints, elastomers, coatings and adhesives, and are the most frequently reported cause of chemically induced immunologic sensitization and occupational asthma (OA). Immune mediated hypersensitivity reactions to dNCOs include allergic rhinitis, asthma, hypersensitivity pneumonitis and allergic contact dermatitis. There is currently no simple diagnosis for the identification of dNCO asthma due to the variability of symptoms and uncertainty regarding the underlying mechanisms. Immunological sensitization due to dNCO exposure is traditionally thought to require initial conjugation of the dNCO to endogenous proteins to generate neoantigens, which trigger production of dNCO specific T lymphocytes and ultimately dNCO specific IgE. Testing for dNCO-specific IgE, for diagnosis of dNCO asthma is however, only specific (96-98%) but not sensitive (18-27%). The low prevalence of detectable dNCO specific IgE has been attributed to both assay limitations and a potential IgE-independent dNCO asthma mechanism(s). The identity of the conjugated proteins responsible for the sensitization also remains unknown. It is also not clear whether dNCOs bind to extracellular, cell membrane, or intracellular proteins as a way of triggering non-IgE asthma. Standardization and optimization of immunoassays used to screen for dNCO specific antibodies in sera is important if its utility as a dNCO asthma diagnostic tool is to be achieved. This will potentially improve sensitivity and allow comparison of results across studies. Current studies on assays of dNCO-specific IgE and IgG lack or have limited characterization of the conjugates used. Diisocyanates bound to hemoglobin (Hb), human serum albumin (HSA), and THP-1 proteins were quantified by HPLC with fluorescence detection. Proteomic tandem mass spectrometry (MS) was used to delineate TDI and MDI specific amino acid binding sites on Hb as well as identification of proteins from MDI exposed THP-1 cells. The trinitrobenzene sulfonic acid assay (TNBS) and SDS gel electrophoresis were used to evaluate extent of intra and intermolecular cross-linking in dNCO-HSA conjugates. Binding of monoclonal antibodies (mAbs) to dNCO bound proteins in enzyme-linked immunosorbent assay (ELISA) was used to evaluate antigenicity of dNCO-protein conjugates. The amount of dNCO binding to HSA and Hb increased with the concentration of the dNCO used for conjugation. All the dNCOs reacted with HSA more than with Hb. Eight binding sites were observed with both MDI and TDI on Hb. The N-terminal valines of both the alpha and beta subunits on Hb, lysine 40 of the alpha subunit and lysine 61 of the beta subunit were common binding sites for both TDI and MDI. Lysine 7 of the alpha subunit and lysines 8, 65 and 66 of the beta subunit were unique to MDI. On the other hand, lysines 11, and 16 of the alpha subunit and lysines 17 and 144 of the beta subunit were unique to TDI. Protein bound MDI was detected in a dose-dependent manner in membrane and cytoplasm fractions of MDI exposed THP-1 cells. MDI was also detected in 11 of the 13 cytoplasmic protein bands. The extent of MDI intracellular protein binding was not affected by cytochalasin D, a chemical that binds actin filaments and inhibits active uptake into cells. The extent of cross-linking shown using the TNBS assay was found to increase with amount of dNCO used. Clear bands from both intra and intermolecular cross-linking were observed on all dNCO-Hb/HSA SDS gels. Using ELISA, both TDI-Hb and TDI-HSA conjugates were reactive to monoclonal antibodies produced against TDI conjugated HSA indicating that dNCO-Hb is also antigenic. The best characterization of dNCO-protein conjugates is achieved by the quantitative determination of conjugated dNCO per mole of protein as well as determining the extent of dNCO cross-linking. Although HSA is more reactive to dNCOs than other serum proteins such as Hb, contribution from other serum proteins to development of OA should not be overlooked as dNCO-Hb was found to be reactive to dNCO specific mAbs. dNCO-conjugated proteins identified in the soluble fraction of MDI exposed THP-1 cells were all of intracellular origin suggesting that MDI can cross the cell membrane and react with intracellular proteins. The entry of MDI into live cells is a passive process, as the extent of intracellular binding was not affected by cytochalasin D. The present study support the potential involvement of dNCO-haptenated membrane and intracellular proteins in development of non-IgE dNCO asthma.

Introduction: The incidence of food allergy has increased substantially in developed countries, with limited treatment and/or prevention options. Milk oligosaccharides have shown to modulate immune responses by serving as prebiotic substrates for the intestinal microbiota. However, some studies suggest that oligosaccharides may exert direct immunomodulatory effects, suggesting their therapeutic potential in preventing allergic diseases. We hypothesized that specific milk oligosaccharides including 6’sialyllactose, 2’fucosylactose, 3’sialyllactose and lacto-N-neotetraose may directly exert immunomodulatory effects on dendritic cells (DCs) and epithelial cells (ECs) by altering their phenotype and/or function in vitro. Methods: The effects of milk oligosaccharides (MOs) on bone-marrow derived DCs and the T84 and MODE-K epithelial cell lines were studied via direct treatment, in vitro. The expression of immunomodulatory cytokines and maturation markers were assessed to measure the effect of MOs on DC phenotype. Pro- and anti-inflammatory cytokines as well as NFκB p65 activity were measured to assess the effect of MOs on DC and EC function. In addition, in vitro stimulation of CD23 with IgE-Antigen complexes were used to study the effects of MOs on ECs in relation to allergy. Lastly, inhibitory antibodies for Siglec-F and PPARγ were used to elucidate the mechanism used by specific MOs to exert their effects. Results: Of the oligosaccharides studied, 6’siallylactose has direct immunomodulatory effects on DC phenotype and on DC and EC function at high concentrations. 6’sialyllactose increased DC expression of IL-10 and HO-1; it also increased CpG- and LPS- induced IL-10 release and decreased IL-12p70 release. Blocking the PPARγ receptor with GW9662 resulted in attenuation of this latter effect on IL-12p70 release. 6’sialyllactose reduced TNF-α induced IL-8 to a small but statistically significant extent and mKC to a great extent in T84 and MODE-K cells, respectively. In addition, 6’sialyllactose reduced IgE-Antigen stimulated release of IL-8 and CCL20, as well as NFκB p65 activity. Pre-treatment of cells with GW9662 resulted in attenuation of the effect of 6’SL on IL-8 release and p65 activation. In addition, 2’fucosylactose reduced CCL20 release and NFκB activity substantially, but these effects were not exerted via PPARγ. Conclusion: Some oligosaccharides are able to directly modulate the inflammatory response in DCs and ECs, via pathways involving PPARγ activation and/or NFκB inhibition. / Thesis / Master of Science (MSc)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

<h4> </h4> / <p>Asthma is an inflammatory disorder of the airways, and there has been growing insight into the cellular and molecular mechanisms underlying the inflammatory basis of this disease. Research into the inflammatory mechanisms of asthma has progressively shifted focus from downstream effectors, such as mast cells and eosinophils, up to Th2 lymphocytes and their proallergic cytokines. Even more upstream in the allergic cascade are dendritic cells (DCs), potent APCs that orchestrate immune responses. Evidence supporting a role of DCs in regulating airway allergic inflammation is derived mainly from animal studies. In animal models of asthma, myeloid DCs (mDCs) induce and maintain airway inflammation, while plasmacytoid DCs (pDCs) mediate tolerance and lung homeostasis. It remains uncertain, however, whether this concept of pro-allergic mDCs and anti-allergic pDCs translates from animal to human models. The overall objective of this thesis was to investigate the biology of DC subsets in allergen-induced asthma in asthmatic subjects. Initially, we demonstrate that both mDCs and pDCs increase in the airways of subjects with mild asthma after allergen inhalation. Next, we describe a distinct subpopulation of mDCs, called mDC2s, and demonstrate their association with allergy and asthma severity. Expanding on these findings, we show that mDC2s increase in the airways of mild asthmatics after allergen challenge. Lastly, we explore the potential of pharmacological therapies, anti-OX40L MAb and anti-TSLP MAb, to affect DCs in subjects with mild asthma, and demonstrate no effect of either drug on circulating DC subsets. The studies presented here provide evidence for multiple DC subtypes being involved in the regulation of allergen-induced inflammatory responses, and support continued investigations into the biology of different DC subsets in allergen-induced asthma.</p> / Doctor of Philosophy (Medical Science)

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Dependency is defined as authentic or pathological and is seen as a component important to the treatment of patients with chronic illness. It is hypothesized that a significant portion of ecology patients will meet the criteria for pathological dependence and differ on psychological and physiological parameters from those who do not. This study strongly supports the first two hypotheses but does not find that the two groups differ physiologically. One hundred eleven variables are surveyed. Fifty-two show significant differences between the groups and 29 are significant at greater than the .0001 level. A discriminant analysis was used to determine the least number of orthogonal variables that best discriminate between the groups. These are MMPI Scales 8, 3, subscale Ma2, employment status, and early childhood illness.

Background: Food allergy, an abnormal immunologic response to food protein, has an estimated prevalence of 6% in young children and 3.7% in adults in the U.S. The only proven therapy for food allergy is strict elimination of the offending allergens. As a result, caregivers and patients could experience constant anxiety and stress that affects their quality of life. Additionally, food allergy can lead to significant economic impact on the health care system, since severe reactions often lead to ED visits and hospitalizations. Objectives: The first major objective was to determine the economic burden of Food Allergy and Anaphylaxis (FAA) patients in the U.S. by estimating the direct medical and indirect costs. The second principal objective involved assessing the Health Related Quality of Life (HRQL) of food allergic patients by measuring their health utilities and disease specific quality of life. Methods: Economic burden was estimated by measuring certain direct medical and indirect costs from a societal perspective. Costs were estimated using a bottom-up approach -- calculating the average cost of illness per patient and multiplying it by reported prevalence estimates. FAA patients with an emergency department (ED) visit, office based physician visit, outpatient department visit, and hospital admission were identified from a list of federally administered databases using ICD-9 codes. Sensitivity analyses were conducted to measure the robustness of the estimates. The cross-sectional HRQL study measured health utilities in food allergic adults and children, and quality of life in allergic adults using EQ-5D and FAQL-AF questionnaires respectively. These questionnaires were administered in an online survey format. Regression models were specified to explore the deviations in HRQL scores between patients with different disease related characteristics. Results: The findings reveal that for a given year (2007), direct medical costs worth $225 million and indirect costs worth $115 million were incurred. Owing to the irregularities in the reporting and diagnosis of food allergy, these values might be an underestimation. Simulations from probabilistic sensitivity analysis generated mean direct medical costs of $307 million and indirect costs of $203 million. Survey responses were collected online for eight months, during which 45 adults and 94 parents (acting as proxy for their food allergic child) responded. Adults reported a mean utility of 0.874 compared to 0.918 for children. Gender, number of food allergies and frequency of carrying epinephrine device had significant impacts on HRQL scores. An effect size of 0.003 was estimated comparing health utilities of food allergic adults with the general U.S. population. Conclusions: This was the first research to examine economic burden of FAA, and elucidate health utilities for food allergic patients. A large proportion of costs were incurred due to ambulatory visits. Effect size calculation revealed that health utilities of food allergic patients were very similar to the general U.S. population.

Mast cells respond to a variety of signals, are associated with both increased inflammation and regulation of the immune response, and are able to interact with a variety of hematopoietic and non-hematopoietic cells. The majority of the work that highlights mast cell pleiotropic abilities has been completed in murine models. Though these models have significantly advanced our understanding of what mast cells can do, they cannot inform us as to what mast cells actually do in human beings. The goal of this dissertation is to assess fully mature, primary human mast cell function beyond the well-defined type 1 hypersensitivity function and place mature human mast cells in the context of interactions with other immune cells. The first project addresses the ability of IFNγ, a historically Th1 associated cytokine, to dramatically alter mast cell phenotype. In particular, IFNγ stimulation allows mast cells to act as antigen presenting cells to CD4+ T cells. The second project describes and addresses the T cell suppressive function of myeloid derived suppressor cells in Mastocytosis, a disease of clonal mast cells.