Embora faltem evidências diretas da relação causal entre a vacinação recente e o desenvolvimento da anemia hemolítica imunomediada (AHIM) em cães, pode ser identificada uma associação temporal entre elas. Constituem-se em objetivos deste trabalho avaliar: a presença de imunoglobulinas (IgG e IgM) e complemento (C3>) na superfície eritrocitária e o potencial do estímulo mitogênico de membranas eritrocitárias autólogas sobre os linfócitos periféricos de cães nos momentos pré-vacinal (imediatamente antes da vacinação com vacinas polivalente e antirrábica) e pós-vacinal (28 a 38 dias após a imunização). Vinte e um cães adultos e hígidos, machos e fêmeas, foram submetidos à anamnese, exame físico e avaliações laboratoriais nos dois momentos do estudo. O teste da antiglobulina direta (n=15) foi realizado com o reagente de Coombs polivalente, nas diluições de 1:2 a 1:8. A detecção de imunoglobulinas (IgG, IgM) e complemento (C3) na superfície de eritrócitos por citometria de fluxo (n=21) foi realizada utilizando anticorpos anti-IgG de cão produzido em ovelha cadeia pesada, anti-IgM produzido em cabra e anti-C3 de cão produzido em cabra, todos conjugados com fluoresceína de isotiocianato (FITC). As células mononucleares do sangue periférico foram isoladas por gradiende, marcadas com CFSE e estimuladas com Concanavalina A (ConA) e com membranas eritrocitárias autólogas em duas concentrações (ME1 e ME2). Foi utilizado o Índice de Proliferação (IP) como indicador da proliferação celular, obtido pela divisão das intensidades de fluorescência obtidas por citometria de fluxo das amostras basal e estimulada. As comparações das variáveis \"hemácias marcadas com anti-Ig/C3\" e \"IP de linfócitos\" foram realizadas utilizando-se o t-Student para amostras pareadas. As comparações dos IP de linfócitos frente aos diferentes antígenos (Con A, ME1 e ME2) foram realizadas por meio da ANOVA com medidas repetidas. Quando houve diferença significante entre os índices, foram realizadas comparações múltiplas (teste de Bonferroni). Foi considerado um nível de significância de 5%. Observou-se que os cães se encontravam em boas condições de saúde, nos dois momentos do estudo, com as variáveis hematológicas e bioquímicas mantidas próximas entre si e resultado do teste da antiglobulina direta negativo (n=21). A porcentagem de hemácias marcadas com IgG e IgM nos momentos pré-vacinal (1,06±0,49% e 1,42±1,59%) e pós-vacinal (0,83±0,56% e 1,35±1,71%) não foi alterada, com p=0,261 e p=0,699, respectivamente. A porcentagem de hemácias com C3 na superfície no momento pós-vacinação (0,40±0,38%) foi, em média, menor do que no momento pré-vacinação (0,71±0,33%), com p=0,019. Os índices de proliferação obtidos com a ConA, ME1 e ME2 no momento pré-vacinal (2,15±0,83; 1,03±0,07; 1,05±0,11) e pós- vacinal (2,13±0,58; 1,02±0,05; 1,02±0,05) não se modificaram, com p=0,935; p=0,845 e p=0,222, respectivamente. Em ambos os momentos, os índices de proliferação celular observados com o uso de ConA foram, em media, maiores do que os índices com ME1 e ME2 (p<0,001). A baixa porcentagem de hemácias com IgG, IgM ou C3 na superfície e a ausência de resposta proliferativa dos linfócitos quando estimulados com membranas eritrocitárias, indicam que, neste experimento, não houve nenhuma evidência de que o estímulo vacinal pudesse estar relacionado ao desenvolvimento da AHIM. / Despite the lack of evidence regarding a causal link between recent vaccination and development of immune mediated hemolytic anemia (IMHA) in dogs, a temporal association between them has been identified in some cases. The aim of this study was to evaluate: the presence of immunoglobulins (IgG and IgM) and complement (C3) on the surface of red blood cells and the potential for mitogenic stimulation of peripheral lymphocytes against autologous red blood cell membranes on dogs at pre-vaccination (immediately prior to vaccination with polyvalent and anti-rabies vaccines) and post-vaccination (after 28 and 38 days after vaccination). Twenty-one healthy adult dogs (both males and females) were subjected to physical examination and complementary laboratory exams in the aforementioned two instances of the study (i.e. pre-vaccination and post-vaccination). Direct antiglobulin test (n=15) was performed using the polyvalent Coombs reagent in 1:2 to 1:8 dilutions. Immunoglobulins (IgG, IgM) detection and identification of complement (C3) on the surface of red blood cells were done by flow cytometry (n=21) using antibodies anti-dog IgG heavy chain produced in sheep, anti-dog IgM produced in goat and anti-dog C3 produced in goat - all in conjunction with fluorescein isothiocyanate (FITC). The peripheral blood mononuclear cells were isolated by gradient, labeled with CFSE and stimulated with concanavalin A (Con A) and autologous erythrocyte membranes in two concentrations (EM1 and EM2). The Proliferation Index (PI), used as an indicator of cell proliferation, was obtained by dividing the fluorescence intensities of basal and stimulated samples, both obtained by flow cytometry. A comparison was made between the variables \"labeled red blood cells with anti-Ig/C3\" and \"PI lymphocyte\" using the paired Student\'s t-test. Comparisons of PI lymphocytes to different antigens (Con A, EM1 and EM2) were performed using ANOVA of repeated measures. Whenever significant differences between the indices were found, multiple comparisons (Bonferroni test) were then performed. A 5% significance level was considered. At the two instances of the study, dogs were presented in good health status with both hematological and biochemical variables kept close together and negative results for direct antiglobulin test (n=21). The percentage of red blood cells labeled with IgG and IgM in the pre-vaccination (1.06±0.49% and 1.42±1.59%) and post-vaccination (0.83±0.56% and 1.35±1.71%) were kept similar, with p=0.261 and p=0.699, respectively. The percentage of erythrocytes with C3 on the surface at the time post-vaccination (0.40±0.38%) was on average lower than in the pre-vaccination (0.71±0.33%), p=0.019. The proliferation index obtained with ConA, ME1 and ME2 in the pre-vaccination (2.15±0.83, 1.03±0.07, 1.05±0.11) and post-vaccination (2.13±0.58, 1.02±0.05, 1.02±0.05) did not differ significantly, with p=0.935, p=0.845 and p=0.222, respectively. In both instances, rates of cell proliferation observed with the use of ConA were, on average, higher than the rates with ME1 and ME2 (p<0.001). The low percentage of erythrocytes with IgG, IgM or C3 on the surface and the absence of lymphocyte proliferative response when stimulated with erythrocyte membranes, indicate that, at least in this experiment, there is no evidence of any association between vaccine stimulation and development of IMHA in dogs.

Embora faltem evidências diretas da relação causal entre a vacinação recente e o desenvolvimento da anemia hemolítica imunomediada (AHIM) em cães, pode ser identificada uma associação temporal entre elas. Constituem-se em objetivos deste trabalho avaliar: a presença de imunoglobulinas (IgG e IgM) e complemento (C3>) na superfície eritrocitária e o potencial do estímulo mitogênico de membranas eritrocitárias autólogas sobre os linfócitos periféricos de cães nos momentos pré-vacinal (imediatamente antes da vacinação com vacinas polivalente e antirrábica) e pós-vacinal (28 a 38 dias após a imunização). Vinte e um cães adultos e hígidos, machos e fêmeas, foram submetidos à anamnese, exame físico e avaliações laboratoriais nos dois momentos do estudo. O teste da antiglobulina direta (n=15) foi realizado com o reagente de Coombs polivalente, nas diluições de 1:2 a 1:8. A detecção de imunoglobulinas (IgG, IgM) e complemento (C3) na superfície de eritrócitos por citometria de fluxo (n=21) foi realizada utilizando anticorpos anti-IgG de cão produzido em ovelha cadeia pesada, anti-IgM produzido em cabra e anti-C3 de cão produzido em cabra, todos conjugados com fluoresceína de isotiocianato (FITC). As células mononucleares do sangue periférico foram isoladas por gradiende, marcadas com CFSE e estimuladas com Concanavalina A (ConA) e com membranas eritrocitárias autólogas em duas concentrações (ME1 e ME2). Foi utilizado o Índice de Proliferação (IP) como indicador da proliferação celular, obtido pela divisão das intensidades de fluorescência obtidas por citometria de fluxo das amostras basal e estimulada. As comparações das variáveis \"hemácias marcadas com anti-Ig/C3\" e \"IP de linfócitos\" foram realizadas utilizando-se o t-Student para amostras pareadas. As comparações dos IP de linfócitos frente aos diferentes antígenos (Con A, ME1 e ME2) foram realizadas por meio da ANOVA com medidas repetidas. Quando houve diferença significante entre os índices, foram realizadas comparações múltiplas (teste de Bonferroni). Foi considerado um nível de significância de 5%. Observou-se que os cães se encontravam em boas condições de saúde, nos dois momentos do estudo, com as variáveis hematológicas e bioquímicas mantidas próximas entre si e resultado do teste da antiglobulina direta negativo (n=21). A porcentagem de hemácias marcadas com IgG e IgM nos momentos pré-vacinal (1,06±0,49% e 1,42±1,59%) e pós-vacinal (0,83±0,56% e 1,35±1,71%) não foi alterada, com p=0,261 e p=0,699, respectivamente. A porcentagem de hemácias com C3 na superfície no momento pós-vacinação (0,40±0,38%) foi, em média, menor do que no momento pré-vacinação (0,71±0,33%), com p=0,019. Os índices de proliferação obtidos com a ConA, ME1 e ME2 no momento pré-vacinal (2,15±0,83; 1,03±0,07; 1,05±0,11) e pós- vacinal (2,13±0,58; 1,02±0,05; 1,02±0,05) não se modificaram, com p=0,935; p=0,845 e p=0,222, respectivamente. Em ambos os momentos, os índices de proliferação celular observados com o uso de ConA foram, em media, maiores do que os índices com ME1 e ME2 (p<0,001). A baixa porcentagem de hemácias com IgG, IgM ou C3 na superfície e a ausência de resposta proliferativa dos linfócitos quando estimulados com membranas eritrocitárias, indicam que, neste experimento, não houve nenhuma evidência de que o estímulo vacinal pudesse estar relacionado ao desenvolvimento da AHIM. / Despite the lack of evidence regarding a causal link between recent vaccination and development of immune mediated hemolytic anemia (IMHA) in dogs, a temporal association between them has been identified in some cases. The aim of this study was to evaluate: the presence of immunoglobulins (IgG and IgM) and complement (C3) on the surface of red blood cells and the potential for mitogenic stimulation of peripheral lymphocytes against autologous red blood cell membranes on dogs at pre-vaccination (immediately prior to vaccination with polyvalent and anti-rabies vaccines) and post-vaccination (after 28 and 38 days after vaccination). Twenty-one healthy adult dogs (both males and females) were subjected to physical examination and complementary laboratory exams in the aforementioned two instances of the study (i.e. pre-vaccination and post-vaccination). Direct antiglobulin test (n=15) was performed using the polyvalent Coombs reagent in 1:2 to 1:8 dilutions. Immunoglobulins (IgG, IgM) detection and identification of complement (C3) on the surface of red blood cells were done by flow cytometry (n=21) using antibodies anti-dog IgG heavy chain produced in sheep, anti-dog IgM produced in goat and anti-dog C3 produced in goat - all in conjunction with fluorescein isothiocyanate (FITC). The peripheral blood mononuclear cells were isolated by gradient, labeled with CFSE and stimulated with concanavalin A (Con A) and autologous erythrocyte membranes in two concentrations (EM1 and EM2). The Proliferation Index (PI), used as an indicator of cell proliferation, was obtained by dividing the fluorescence intensities of basal and stimulated samples, both obtained by flow cytometry. A comparison was made between the variables \"labeled red blood cells with anti-Ig/C3\" and \"PI lymphocyte\" using the paired Student\'s t-test. Comparisons of PI lymphocytes to different antigens (Con A, EM1 and EM2) were performed using ANOVA of repeated measures. Whenever significant differences between the indices were found, multiple comparisons (Bonferroni test) were then performed. A 5% significance level was considered. At the two instances of the study, dogs were presented in good health status with both hematological and biochemical variables kept close together and negative results for direct antiglobulin test (n=21). The percentage of red blood cells labeled with IgG and IgM in the pre-vaccination (1.06±0.49% and 1.42±1.59%) and post-vaccination (0.83±0.56% and 1.35±1.71%) were kept similar, with p=0.261 and p=0.699, respectively. The percentage of erythrocytes with C3 on the surface at the time post-vaccination (0.40±0.38%) was on average lower than in the pre-vaccination (0.71±0.33%), p=0.019. The proliferation index obtained with ConA, ME1 and ME2 in the pre-vaccination (2.15±0.83, 1.03±0.07, 1.05±0.11) and post-vaccination (2.13±0.58, 1.02±0.05, 1.02±0.05) did not differ significantly, with p=0.935, p=0.845 and p=0.222, respectively. In both instances, rates of cell proliferation observed with the use of ConA were, on average, higher than the rates with ME1 and ME2 (p<0.001). The low percentage of erythrocytes with IgG, IgM or C3 on the surface and the absence of lymphocyte proliferative response when stimulated with erythrocyte membranes, indicate that, at least in this experiment, there is no evidence of any association between vaccine stimulation and development of IMHA in dogs.

Rates of immune-mediated diseases (IMDs) have rapidly increased. Although the exact etiology has not yet been fully elucidated, disruptions to the microbiome has been proposed as a potential mechanism. We conducted a retrospective, longitudinal, birth cohort study utilizing electronic health records (EHR) to investigate the association between early life antibiotic exposure and the risk of developing juvenile idiopathic arthritis (JIA), pediatric psoriasis, or type 1 diabetes. Incident rate ratios (IRR) were estimated using modified Poisson regression models and adjusted for significant confounders. Children exposed to two or more antibiotics prior to 12 months of age had a 69% increased risk of developing JIA (1.69 IRR, 95% CI [1.04-2.73]), which rose to 97% when exposed prior to 6 months (1.97 IRR, 95% CI [1.11-3.49]). Children exposed to a penicillin antibiotic had a 62% increase in risk for psoriasis (1.62 IRR, 95% CI [1.06-2.49]), which rose slightly to 64% when exposure occurred between 6 and 12 months of age [(1.64 IRR, 95% CI [1.04-2.59]). We found a moderate to strong association between early antibiotic exposure and risk for JIA and psoriasis when exposure was examined by age, frequency, and type of antibiotic, but not for type 1 diabetes. Potential interactions effects between infection and antibiotics with an increased susceptibility to early life infections among children with an IMD was also observed. Overall, children exposed to antibiotics at an early age have an increased probability of developing an IMD after 12 months of age. However, alternative explanations for this association should be considered. / Public Health

Twenty publications are submitted. All deal with the issues of control of two ends of the spectrum of immune-mediated respiratory disorders in children, namely atopic (asthma and allergic rhinitis) and HIV-related lung disease. This submission summarises the research by the author into this spectrum of lung diseases of children in South Africa, highlighting the diversity of conditions that are not only clinically important, but also common. Understanding of all conditions is required to improve the health of children in this region. Management of chronic conditions requires two major end points - adequate and timely diagnosis and - management to control the condition. The author has a passion for improving the quality of life of children and firmly believes that the research findings will, and have, led to transformation in management of both these common disorders. This document follows the progression of the authors research work and highlights how interesting and important is the scope of two disorders which could be thought to have a central origin, namely in the T-cell. T-cells form the basis of cellular immunity and an excess of T-helper 2 cell activity promotes atopy, whilst the human immunodeficiency (HI) virus infects T-helper cells and promotes cellular immune deficiency and its attendant clinical disorders. The author’s research work is not based on the immunological basis of these conditions but does deal with the clinical implications and especially aspects relating to control of these two extremes of a clinical spectrum of disorders. To take the clarity of two diseases at the end of a spectrum to its natural conclusion these extremes are defined in aetiology or pathophysiological differences (excess versus suppression of the immune system), occurring in the affluent and poor alike versus just the poor, control being required to improve quality of life versus to save lives and finally that management requires anti-inflammatory therapy versus antibiotic and anti-infective therapy. For the eight publications based on atopic respiratory disease in children the themes are firstly that children with asthma and chronic rhinitis are diagnosed late, that most individuals with these conditions are not well controlled and finally that the reasons for lack of control are becoming obvious. For the first time, the significant lack of asthma and allergic rhinitis control in South Africa is documented. These studies suggest that, like surveys from the rest of the world, asthma control is seriously under-estimated and neglected in all asthmatics in South Africa, in both the privileged and the under-privileged. The research also defines reasons for poor asthma and allergic rhinitis control in this region. As in many studies published from around the world it is now evident that poor asthma and allergic rhinitis control cannot be blamed on any one source. A multitude of reasons underlie this phenomenon and each of the subsequent papers in this section illustrates attempts at defining these principles. The three most important reasons for poor control are probably that most asthmatics are managed in the wrong hands (by doctors who don’t understand adequate control and who aren’t empowered to use the correct therapy), that control may actually be a pipe dream and practically difficult to do or even impossible to achieve and lastly that the allergic basis of asthma is over emphasised and may not in fact determine all asthma. The subsequent papers summarise research work in the field of HV infection in children and exposes the opposite end of a spectrum of Paediatric respiratory disease and highlight research into the conditions common in HIV-infected children. Eleven papers are presented. For the diseases associated with the HI virus the major complications of inadequate diagnosis and prevention in children are acute pneumonia (especially severe pneumonia) and bronchiectasis. Bronchiolitis is not common in HIV infected children, despite epidemics of this condition in non-infected children. Passive smoking does not aggrevate or worsen disease progression in children. The complications of HIV related diseases in children require the same principles of adequate diagnosis and control as would apply to the chronic atopic conditions. Once the author delved into the disorders at the other end of the clinical spectrum, namely those associated with immune deficiency secondary to HIVinfection he faced the question of a possible relationship between the conditions. One submission explores that relationship. This research has a unique perspective, conferred by the fact that these two conditions do not occur to the same extent anywhere else in the world. Atopic respiratory conditions and HIV-related lung diseases occur side by side in abundance in this region. This perspective has created a clarity for research to address the two most important aims in clinical medicine, namely to diagnose correctly and then to manage the condition so that control is achieved. These must be universal principles of the successful practice of medicine. / Thesis (DSc)--University of Pretoria, 2013. / gm2013 / Paediatrics and Child Health / unrestricted