Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:34:20Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-04-13T19:42:38Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) / Made available in DSpace on 2017-04-13T19:42:38Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tiago Gallina Corrêa.pdf: 1514650 bytes, checksum: d88af72f3dfc4c6107504dc1ec16ea8a (MD5) Previous issue date: 2005-10-14 / Sem bolsa / Dermatite alérgica estacional foi estudada em um rebanho de ovinos Hampshire Down em um estabelecimento no município de Capão do Leão, Rio Grande do Sul. A epidemiologia, sinais clínicos e lesões macroscópicas foram observadas no estabelecimento verificando-se a época de ocorrência da enfermidade, a idade e sexo dos animais afetados e localização das lesões. A patologia microscópica foi estudada em biopsias de pele de ovinos afetados. Para a verificação da etiologia, insetos foram capturados com aspiradores entomológicos, semanalmente, das 16h00minh às 09h00minh, entre os meses de janeiro a abril de 2005. Os dados relativos à temperatura média, umidade relativa do ar e velocidade média dos ventos na região de ocorrência da doença foram obtidos na Estação Agro-climatológica da Universidade Federal de Pelotas. A prevalência da doença no período estudado foi de 40% e animais de qualquer idade e ambos os sexos foram afetados. A enfermidade ocorreu principalmente entre dezembro e março e alguns animais permaneceram com lesões durante todo o ano. Os sinais clínicos eram de inquietação e prurido e as lesões ocorriam principalmente nas orelhas, ao redor dos olhos e na região ventral do abdômen, caracterizando-se pela presença de eritema e pápulas, nos casos agudos, e espessamento da pele com rachaduras, exsudação, formação de crostas, e alopecia, nos casos crônicos; esses freqüentemente apresentavam infecções secundárias. As lesões histológicas eram principalmente de dermatite eosinofílica perivascular, observando-se, também, hiperqueratose, acantose e presença de pústulas sub corneais ou intra-epidérmicas nos casos crônicos. Os insetos capturados no período de estudo da doença foram identificados como Anopheles albitarsis e Culicoides insignis. Durante a captura observou-se que os animais apresentavam prurido intenso quando picados por Culicoides, mas não por Anopheles. As temperaturas média mínima e máxima no período, foram de 17ºC e 28ºC, respectivamente, e a velocidade média dos ventos foi de 12,84km/h. Ambas as espécies de insetos capturados têm hábitos noturnos aproximando-se dos animais para hematofagia em torno de 30min após o ocaso do sol.Testes intradérmicos realizados com antígenos obtidos a partir desses dípteros revelaram reação de hipersensibilidade aos antígenos de C. insignis. Os resultados obtidos indicam que a enfermidade estudada ocorre em conseqüência de hipersensibilidade imediata causada pela picada desse díptero. / A seasonal dermatitis was studied in a Hampshire Down flock in a farm at the minicipality of Capão do Leão, state of Rio Grande do Sul, southern Brazil. Frequency and seasonal distribution of the disease, breed, sex and age of affected sheep, and clinical signs, macroscopic pathology and distribution of the lesions were obtained by visiting the farm. Histological lesions were studied in skin biopsies of affected sheep. Biting insects were collected weekly between 4:00 p.m. to 9:00 a.m., from January to April of 2005 to identify the etiological agent of disease. Data about temperature, humidity and wind speed in the region where the disease occurs were obtained in the agro climatologic Station of Pelotas Federal University. The prevalence of disease during the study period was 40% and the age of affected animals was variable. The disease occurs between December and March, but some animals stayed with lesions during the whole year. Clinical signs were characterized by pruritus and skin lesions in the ears, around the eyes, and ventral abdomen. Erythema and papules were observed in acute cases. In chronic cases, the skin was thicken, exsudative, crusty and alopecic, frequently with secondary infections. Histological lesions of affected sheep were characterized by perivascular eosinofilic dermatitis. Hyperkeratosis, acanthosis and intraepidermal pustules were observed in chronic lesions. Anopheles albitarsis and Culicoides insignis were captured during the observation period. Sheep showed pruritus mainly during de Culicoides bites. The mean minimum and maximum temperature in the period were 17ºC e 28ºC, respectively, and the mean speed wind was 12,84km/h. Both insects were caught when they approached the sheep bait 30minutes after sunset. Intradermal tests performed with de antigens of the insect extracts showed hypersensitivity reaction to C.insignis antigens. Based on the results it was suggested that the disease occurs as a consequence of an immediate hypersensitivity caused by C. insignis bite.

Objetivos: Determinar a reatividade dos soros e determinação dos isotipos IgG e IgE a proteínas de sementes da dieta de crianças com alergia ao leite de vaca IgE mediada. Métodos: Foram avaliados soros de três grupos de crianças: alérgicas ao leite de vaca IgE mediada, crianças tolerantes ao leite e um grupo controle com crianças não atópicas. Foram usados extratos protéicos de diferentes tipos de sementes utilizando o teste de ELISA para análise da reatividade dos isotipos IgG e IgE. Resultados: Comparando as concentrações séricas de IgG dos diferentes grupos, observou-se concentrações mais elevadas e estatisticamente significante no grupo alérgico em relação aos grupos tolerante e controle, exceto para as sementes de soja e feijão roxinho. Em relação ao isotipo IgE observou-se os mesmos padrões de reatividade mostradas nas analises para IgG, com diferença significante do grupo alérgico em relação ao controle, exceto para milho. Observou-se que para a soja houve grande dispersão das concentrações séricas tanto no grupo alérgico quanto no tolerante, em valores superiores ao do grupo controle. Conclusão: A comparação entre os diversos grupos avaliados mostra que pacientes alérgicos ao leite e os tolerantes apresentam concentrações mais elevadas de IgG e IgE a outros alimentos que as crianças do grupo controle, o que pode sugerir possível alteração de permeabilidade da mucosa intestinal nestes grupos, mesmo na ausência de sintomatologia gastrintestinal / Objective: To determine the reactivity of serum and determination of IgG and IgE isotypes to seed proteins included in the diet of children with cow\'s milk allergy IgE mediated. Methods: We evaluated sera from three groups of children: cow\'s milk allergic patients, tolerant children and a control group with non-atopic children. It was used protein extracts from different types of seeds using an ELISA assay to analyze the reactivity of IgG and IgE isotypes. Results: Comparing the IgG serum from different groups, it was observed higher concentrations and statistically significant in the allergic group compared to the tolerant and control groups, except for soybeans and kidney beans. To the IgE isotype it was observed the same patterns of reactivity shown in the analysis for IgG, with significant difference in the allergic group compared to control, except for corn. It was observed that for soybeans there were values of serum, both in the allergic and tolerant group higher than in the control group. Conclusion: Our study showed that the allergic and tolerant groups of CMA patients presented higher IgG and IgE concentrations to many seeds than the control group. These findings may suggest possible changes in permeability intestinal mucosa in these groups

As proteínas do leite de vaca são os principais alérgenos relacionados à alergia alimentar em crianças e o diagnóstico inclui a realização do teste de provocação oral duplo cego placebo controlado (TPODCPC). Apesar da acurácia, este teste envolve riscos, necessita condições especiais para sua realização e apresenta limitações em crianças de baixa idade e em pacientes anafiláticos. Assim, há necessidade de métodos diagnósticos alternativos, entre eles o estabelecimento de pontos de corte de concentrações de IgE sérica específica para leite de vaca, que permitam o diagnóstico mesmo sem a realização do TPODCPC. O objetivo deste estudo foi estabelecer uma concentração discriminante de IgE sérica específica para leite de vaca e suas frações protéicas para o diagnóstico de alergia à proteína do leite de vaca (APLV) e avaliar se há diferentes concentrações discriminantes de IgE específica para este alérgeno no grupo de pacientes com anafilaxia. Realizou-se um estudo de coorte histórica incluindo pacientes com APLV e grupo controle composto por pacientes com suspeita não confirmada de APLV. Para estes objetivos, foram construídas curvas ROC para os seguintes alérgenos: leite de vaca, caseína, a- lactoalbumina e b-lactoglobulina. Os níveis de IgE específica foram avaliados posteriormente, em separado, nos pacientes com anafilaxia. Foram incluídos 123 pacientes (1,3M:1F mediana = 1,91 anos, com idade de 3,5 meses a 13,21 anos) com diagnóstico confirmado de APLV através de TPODCPC (n=26), presença de anafilaxia à proteína do leite de vaca (n=46) ou história clínica fortemente sugestiva de APLV associada à pesquisa positiva de IgE específica através de teste cutâneo (n=51). Entre os 65 pacientes com anafilaxia, 19 confirmaram o diagnóstico posteriormente, através de testes de provocação. O grupo controle foi composto por 61 pacientes (1M:1,1F) com idade variando entre 0,66 e 16,7 anos (mediana= 6,83 anos). A metodologia adotada para estabelecimento dos pontos de corte foi a construção de curva ROC e posterior cálculo dos valores preditivos positivos e negativos. Os pontos de corte obtidos considerando-se uma especificidade de 98% e um valor preditivo positivo acima de 95% foram: 3,06 kU/L para o leite de vaca, 2,06 kU/L para a- lactoalbumina, 1,85 kU/L para b-lactoglobulina e 1,47 kU/L para caseína. O valor de IgE específica para leite de vaca revelou-se com maior capacidade discriminante que os encontrados para as frações, tornando estes últimos dispensáveis. Com relação à anafilaxia, a curva ROC construída a partir de uma análise comparativa entre pacientes com e sem esta manifestação, indicou um ponto de corte elevado de 39kU/L, tornando impossível sua utilização na prática clínica. O presente estudo mostrou que um ponto de corte de IgE específica para leite de vaca é suficiente para o diagnóstico de APLV, não sendo necessária a avaliação das frações protéicas. Na presença de anafilaxia desencadeada pelo leite de vaca não há necessidade da utilização de pontos de corte de IgE específica, sendo os dados clínicos e a sensibilização ao leite suficientes para o diagnóstico de APLV. / Cow\'s milk proteins are the main allergens related to food allergy in children and the diagnosis include the double blind placebo controlled food challenge (DBPCFC). Although this test presents accuracy, it involves risks, it is necessary special conditions and it presents limitations in infants and anaphylactic patients. It is necessary other diagnostic methods, among them the cut off values for IgE specific for milk to allow the cow\'s milk allergy (CMA) diagnosis without the need to perform the DBPCFC. The objective of this study was to establish a discriminating concentration of specific IgE to cow\'s milk and its proteins fractions for the diagnosis of allergy to CMA and to assess if there are different discriminating concentrations of specific IgE to this allergen in the group of patients with anaphylaxis. It was carried out a historical cohort study including patients with CMA and the control group was composed by patients with excluded CMA suspicion. To obtain the cutoff points for diagnosis of CMA , ROC curves were constructed for the following allergens: cow\'s milk, casein, a-lactalbumin and a b-lactoglobulin. The levels of specific IgE were later evaluated, separately, in patients with anaphylaxis. The study included 123 patients (1.3 M: 1F median = 1.91 years, ranging from 3.5 months to 13.21 years) with confirmed diagnosis of CMPA through DBPCFC (n = 26), the presence of anaphylaxis triggered by cow\'s milk protein (n = 46) or strongly suggestive clinical history associated with positive specific IgE through skin test (n = 51). Among the 65 patients with anaphylaxis, 19 confirmed the diagnosis through challenge tests . The control group was consisted of 61 patients (1M: 1.1 F) with ages ranging between 0.66 and 16.7 years (mean 6.86, median = 6.83 years). The methodology to stablish the cutoff was the construction of a ROC curve and subsequent calculation of positive and negative predictive value. The cutoff points obtained considering a 98% specificity and positive predictive value above 95% were: 3.06 kU / L for cow\'s milk, 2.06 kU/ L for/ a-lactalbumin, 1.85 kU/ L for/ b-lactoglobulin and 1.47 kU / L for casein. The value of specific IgE to cow\'s milk showed better discriminant capacity than those found for the fractions, being these last values dispensable. In relation to anaphylaxis, the ROC curve constructed from a comparative analysis between patients with and without this manifestation indicated a very high cutoff of 39kU/L, being without value for the clinical practice. This study showed that the cut off point detected for hole cows milk was enough for CMA diagnosis, without necessity of the milk fractions evaluation. In the presence of cows milk anaphylaxis, it is not necessary the IgE specific cut off value, being the clinical data and the sensitization to cows milk enough for the CMA diagnosis.

Kung, Wing Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 92-115). / Abstracts in English and Chinese. / Abstract --- p.ii / Acknowledgements --- p.vii / Table of contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1. --- General introduction --- p.1 / Chapter Chapter 2. --- Literature review --- p.4 / Chapter 2.1 --- Food allergy and its prevalence --- p.4 / Chapter 2.2 --- Mechanism and clinical symptoms of food allergy --- p.6 / Chapter 2.3 --- Tropomyosin as the major allergen in shellfish --- p.15 / Chapter 2.4 --- Cross reactivity and epitope mapping of tropomyosin --- p.21 / Chapter 2.5 --- Novel approaches for the treatment of food allergy --- p.29 / Chapter Chapter 3. --- Expression of shrimp recombinant tropomyosin and sensitization of mice --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Materials and Methods --- p.40 / Chapter 3.2.1 --- "Recovery of E, coli with tropomyosin-carrying plasmid" --- p.40 / Chapter 3.2.2 --- Preparation of tropomyosin-carrying plasmid --- p.41 / Chapter 3.2.3 --- Confirmation of DNA sequence of the tropomyosin --- p.41 / Chapter 3.2.4 --- Identification of the recombinant protein --- p.43 / Chapter 3.2.5 --- Purification of the recombinant protein --- p.43 / Chapter 3.2.6 --- Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 3.2.7 --- Concentration measurement of the recombinant tropomyosin --- p.45 / Chapter 3.2.8 --- Mice --- p.46 / Chapter 3.2.9 --- Mice sensitization and challenging --- p.46 / Chapter 3.2.10 --- Tropomyosin-specific IgE level in blood --- p.47 / Chapter 3.2.11 --- Statistical analysis --- p.49 / Chapter 3.3 --- Results --- p.52 / Chapter 3.3.1 --- DNA sequence of the cloned tropomyosin --- p.52 / Chapter 3.3.2 --- Expression and purification of tropomyosin --- p.52 / Chapter 3.3.3 --- Hypersensitivity symptoms after challenge --- p.53 / Chapter 3.3.4 --- Blood tropomyosin-specific IgE level --- p.53 / Chapter 3.4 --- Discussion --- p.62 / Chapter Chapter 4. --- Identification of T cell epitopes --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Materials and methods --- p.67 / Chapter 4.2.1 --- Soluble epitope peptide synthesis --- p.68 / Chapter 4.2.2 --- Isolation of spleen cells from mice --- p.69 / Chapter 4.2.3 --- T cell proliferation assay --- p.70 / Chapter 4.3 --- Results --- p.71 / Chapter 4.3.1 --- Splenocyte proliferation to synthetic peptide --- p.72 / Chapter 4.3.2 --- Splenocyte proliferation to synthetic peptides pool --- p.72 / Chapter 4.4 --- Discussion --- p.77 / Chapter Chapter5 --- General conclusion --- p.89 / References --- p.92

ABSTRACT Whiplash associated disorders (WAD) is one of the most debated musculoskeletal conditions. Sensory disturbances including hypersensitive responses to mechanical, thermal and electrical stimulation have been consistently shown to be a feature of both the acute and chronic stages of the whiplash condition. More importantly, such dysfunctions have also been found to be associated with higher risk of poor functional recovery. It is apparent that better understanding of the sensory disturbances in WAD is needed in order to elucidate mechanisms underlying the pain and disability of this recalcitrant condition and to facilitate the development of more effective management strategies. Comprehensive Quantitative Sensory Testing (QST) combining sensory detection and pain threshold measures is proving to be a valuable tool to advance the classification and illuminating the underlying mechanisms of an array of musculoskeletal pain disorders but such protocol has never been undertaken in a WAD cohort. In order to fill this gap in knowledge, the series of studies in the thesis aimed to utilize comprehensive QST to investigate the presence of somatosensory dysfunction in chronic WAD and to compare the somatosensory profile of WAD to cervical radiculopathy and idiopathic (non-traumatic) neck pain. Once a better understanding of the potential underlying mechanisms in chronic WAD was established, the research then focused on documenting the presence of such somatosensory disturbances soon after whiplash injury and its temporal development over a 6 months period. The results have provided a number of significant insights into some of the potential underlying mechanisms of the somatosensory dysfunction in WAD as well as other neck pain conditions of different aetiology. It is clear that generalised sensory hypoaesthesia coexisted with sensory hypersensitivity in chronic WAD and a combination of pain and detection measures best discriminated patients with WAD and controls. Similar sensory presentation was also found in patients with cervical radiculopathy but not in idiopathic neck pain patients. This finding indicates that different mechanisms underlie various musculoskeletal conditions with disordered central processes contributing to a greater degree in some conditions. Patients with whiplash and those with cervical radiculopthy may share similar underlying pain mechanisms involving the central nervous system and the discrepant findings in the idiopathic neck pain group may be due to the magnitude of nociceptive input required to induce/maintain altered central adaptive changes. Another important observation from the studies was that sensory hypoaesthesia was present in the majority of patients with whiplash injury in the acute stage. However, it persisted only in individuals who initially reported higher levels of pain and disability levels and sign of hypersensitivity. It was this group of individuals who predominantly developed persistent symptoms at six months post injury. The longitudinal findings indicate that such sensory impairments can be identified very early on and treatment interventions directed at these sensory disturbances (both sensory hypersensitivity and hypoaesthesia) should aim to reduce the nociceptive input and this may improve recovery post whiplash injury. The findings in this thesis demonstrated the clear association between sensory hypersensitivity and other sensory disturbances and their potential influence on recovery. Furthermore, the heterogeneity of the whiplash condition highlighted the importance of the early identification of “low-risk and “high-risk” patients in order to assist the clinicians to make clinical decisions on the best management approach. It cannot be overemphasised that the early assessment of whiplash injured patients should aim to identify features associated with poor recovery and a better classification system will be an important step. Implications for assessment and management of whiplash are vital in the acute stage of injury and may well go some way toward preventing the transition to chronicity, particularly in those with a more complex clinical presentation involving somatosensory disturbances. Further research directions have also been identified in order to improvement management of this complex clinical condition.

Diseases that affect dairy cattle have serious economic and animal welfare implications. The inclusion of immune response (IR) traits in breeding indices has been suggested to improve inherent animal health, and decrease the use of antimicrobials. The objectives of this research were to (1) evaluate cell-mediated (CMIR) and antibody-mediated immune responses (AMIR) on 680 Holstein cows from 58 herds across Canada, (2) estimate genetic parameters of these traits (3) examine the associations with routinely evaluated traits as well as the incidence of mastitis, (4) evaluate the correlation of natural and specific antibody and (5) perform a genome-wide association study (GWAS) to determine genetic markers associated with high or low IR. In collaboration with the Canadian Bovine Mastitis Research Network cows were immunized with both a type 1 and type 2 test antigen to stimulate CMIR and AMIR, respectively. A cutaneous delayed-type hypersensitivity test to the type 1 test antigen was used as an indicator of CMIR, and serum antibody (IgG1 and IgG2) to the type 2 test antigen as an indicator of AMIR. IR phenotypes varied significantly by cow, herd and region in Canada. Heritability estimates were moderate, 0.19 for CMIR and 0.16-0.43 for AMIR depending on time in the immunization protocol and antibody isotype. Beneficial associations between AMIR and some reproductive traits were found. Using estimated breeding values, cows were classified as high, average or low responders. High AMIR cows had significantly lower incidence rates of clinical mastitis compared to average and low cows. No difference was found when cows were classified based on CMIR. Natural antibody was not genetically correlated with specific antibody nor was it associated with mastitis. The GWAS found 198 genetic markers significantly associated with AMIR, with the majority on chromosome 23 where the major histocompatability complex is located. Other significant genes involved in IR include those associated with the complement system, interleukin 17 and tumor necrosis factor. This research confirms the benefit of identifying high IR cows and gives a glimpse of current IR profiles in Canadian Holsteins. This was the first GWAS for IR traits in dairy cattle and suggests it may be possible to include IR traits in genomic selection indices. / This research was financed by grants to B.A. Mallard from National Sciences and Engineering Research Council of Canada, Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, DairyGen council of Canadian Dairy Network, Agriculture and Agri-Food Canada, Public Health Agency of Canada, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. Kathleen Adele Thompson Crispi was funded by the Dairy Farmers of Ontario Doctoral Research Assistantship.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.