The increasing prevalence of allergic diseases in affluent societies has been associated with changes in microbial exposure early in life and a less diverse gut flora. The objective of this thesis was to assess the development of allergic sensitisation and symptoms during the first four years of life in a non-selected birth cohort in relation to environmental factors, family history, gut microbiota and salivary IgA antibodies. The cohort comprised all 1,228 infants living in a Swedish county who were born over a one-year period. The parents replied to questionnaires, and 817 children (67 %) were skin prick tested both at 1 and 4 years of age. Saliva (n=279), faecal (n=139) and blood (n=253) samples were collected at 1 year of age from children with a positive skin prick test at 1 year and from a sample of children with a negative skin prick test. Faecal samples were also obtained from 53 children at 4 years of age. Dog keeping during infancy was associated with a decreased risk of sensitisation to pollen and late-onset wheezing at age 4, and the reduced odds ratios persisted after adjustment for heredity and avoidance measures, OR 0.3, 95% CI 0.1-0.9 and OR 0.5, 95% CI 0.2-1.0, respectively. In contrast, early dog keeping was associated with an increased risk of earlyonset transient wheezing but only in children with parental asthma (OR 2,8, 95% CI 1.3-6.4). Levels of short chain fatty acids (SCFAs) in faeces were assessed both at 1 and 4 years of age and related to the development of sensitisation and symptoms. The levels of acetic (p<.01) and propionic (p<.01) acids decreased from one to four years of age, whereas valeric acid (p<.001) increased which is in line with a more complex gut microbiota with age. Allergic children, compared with non-allergic children, had lower levels of i-butyric, i-valeric and valeric acid in faeces both at 1 and 4 years of age. Low levels of secretory IgA (SIgA) in saliva were associated with wheezing but only in sensitised children. In children with positive SPT to at least one allergen both at 1 and 4 years of age and in children with circulating IgE antibodies to egg or cat at one year of age, those who developed late-onset wheezing had lower levels of SIgA than those who did not, p=.04 and p=.02 respectively. Of 9 children with levels of SIgA in the upper quartile and persistent sensitisation, none developed wheezing, compared with 10/20 children with lower levels, (p=. 01). Having older siblings, more than three infections during infancy, at least one smoking parent and male gender were all associated with high levels (in the upper quartile) of total IgA and SIgA. The findings in this thesis indicate that the microbial load early in life could affect the development of allergy. A functional assessment of the gut flora demonstrated differences between allergic and non-allergic children both at 1 and 4 years of age. Salivary IgA was associated with infections during infancy, and high levels of secretory IgA protected from symptoms in sensitised children. Finally, dog keeping in infancy may offer protection from allergy, but the mechanism is uncertain.

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Streptococcus mutans is the main causative agent of dental caries, which is a widespread infectious disease. A number of surface molecules are involved in the pathogenicity of this organism, including adherence and aggregation factors. The wall-associated protein A (WapA) of Streptococcus mutans GS-5 was previously demonstrated to be a sucrose-dependent adherence and aggregation factor, and is a larger precursor to extracellular antigen A (AgA), a candidate antigen for a dental caries vaccine.The full-length wapA gene and a C-terminal truncated version agA encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The above constructs were mixed with a cationic lipid and used to transfect Chinese hamster ovary (CHO) cells. Transient expression of the wapA and agA genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis. In CHO, cells WapA containing the membrane and wall-spanning region was found in apoptotic bodies, whereas the soluble AgA, which lacked the hydrophobic region, was found in extracellular medium. A higher salivary IgA level was observed in mice immunized with the pcDNA-wapA vaccine as compared to those immunized with the pcDNA-agA vaccine. Furthermore, the anti-WapA antibody inhibited S. mutans sucrose-dependent adherence, suggesting potential protection of the tooth against S. mutans colonization, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs that are absent from AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not.

Submitted by Pós Imunologia (ppgimicsufba@gmail.com) on 2017-06-05T19:24:17Z No. of bitstreams: 1 Dissertação Taiana final corrigida 16.02.2017 (1).pdf: 1211793 bytes, checksum: a20533e20682931dfb2ee45359c2edcf (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-07T14:02:20Z (GMT) No. of bitstreams: 1 Dissertação Taiana final corrigida 16.02.2017 (1).pdf: 1211793 bytes, checksum: a20533e20682931dfb2ee45359c2edcf (MD5) / Made available in DSpace on 2017-06-07T14:02:20Z (GMT). No. of bitstreams: 1 Dissertação Taiana final corrigida 16.02.2017 (1).pdf: 1211793 bytes, checksum: a20533e20682931dfb2ee45359c2edcf (MD5) / Capes / INTRODUÇÃO: O lúpus eritematoso sistêmico é uma doença reumática autoimune caracterizada por múltiplas manifestações clínicas e laboratoriais, entre estas, a dislipidemia. Contudo, a imunopatogenia desta alteração metabólica no LES ainda não é bem conhecida. OBJETIVO: O presente estudo investigou o envolvimento de anticorpos na patogênese da doença obstrutiva coronária crônica de pacientes portadores de LES de um serviço de reumatologia de Salvador-Bahia. Especificamente, investigou a presença de anticorpos IgA anti-β2GPI e IgG anti-Saccharomyces cerevisiae em pacientes lúpicos e buscou associar a soropositividade para estes anticorpos com achados clínicos e laboratoriais indicadores de doença obstrutiva coronária crônica e história de acidente cardiovascular. MATERIAL E MÉTODOS: Cento e cinquenta mulheres portadoras de LES foram incluídas no estudo e classificadas para dislipidemia a partir dos seus lipidogramas. A atividade do LES foi avaliada com o SLEDAI-2K. Foram determinados os seguintes índices aterogênicos de risco cardiovascular: razão apoB/apoA, índices I e II de Castelli (CT/HDL-C e LDL-C/HDL-C, respectivamente) e a razão TG/HDL-C. Anticorpos antinucleares foram detectados por IFI, enquanto anticorpos contra autoantígenos, anti-S. cerevisiae (ASCA) e IgA anti-β2GPI foram detectados por testes de ELISA indiretos. RESULTADOS: Cento e dezesseis de 151 pacientes (76,8%) eram dislipidêmicas. Destas, 13 tinham relato de evento cardiovascular (AVC = 12 e IAM = 01). Uma importante proporção dessas pacientes tinha atividade lúpica moderada a alta (69/116, 59,5%), observando-se nas mesmas, níveis baixos de HDL-C e índices aterogênicos de risco cardiovascular mais elevados. Os títulos de ANA foram mais altos nos pacientes dislipidêmicos, enquanto a prevalência e os níveis dos autoanticorpos contra autoantígenos, de C3, C4 e PCR foram semelhantes entre pacientes sem dislipidemia e dislipidêmicas. Existiu uma maior prevalência de ASCA nas pacientes lúpicas em relação às mulheres sem LES, mas não foram observadas diferenças na prevalência e níveis destes anticorpos nos dois grupos de lúpus. Anticorpos IgA anti-β2GPI foram também detectados de forma similar nestes grupos. Não existiram correlações entre ASCA e anticorpos IgA anti-β2GPI e SLEDAI, índices aterogênicos e níveis de PCR. CONCLUSÕES: A maioria das pacientes lúpicas dislipidêmicas apresenta índices aterogênicos de risco cardiometabólico elevados, sugerindo alta predisposição aos eventos cardiovasculares. Existe uma importante produção de anticorpos IgG anti-S. cerevisiae no LES, mas sem relação com dislipidemia ou atividade da doença, sugerindo uma homologia estrutural entre a manana da levedura e autoantígenos. / INTRODUCTION: Systemic lupus erythematosus is an autoimmune rheumatic disease characterized by multiple clinical manifestations, among them, dyslipidemia. However, the immunopathogenesis of this metabolic alteration in SLE is still not well known. OBJECTIVE: The present study investigated the involvement of antibodies in the pathogenesis of chronic obstructive coronary disease in female patients with SLE from a rheumatology service in Salvador-Bahia. Specifically, it examined the presence of anti-β2GPI IgA and anti-Saccharomyces cerevisiae IgG antibodies (ASCA) in lupus patients and sought to associate seropositivity to these antibodies with clinical and laboratory findings indicative of chronic obstructive coronary disease and history of a cardiovascular event. MATERIAL AND METHODS: One hundred and fifty-one women with SLE were included in the study and classified for dyslipidemia from their blood lipid profile. The SLEDAI-2K protocol measured the SLE activity. The following atherogenic indexes of cardiovascular risk were determined: apoB / apoA ratio, Castelli indexes I and II (CT / HDL-C and LDL-C / HDL-C, respectively) and TG / HDL-C ratio. Antinuclear antibodies were detected by an indirect fluorescent antibody test. Indirect ELISAs detected autoantigen antibodies, ASCA and anti-β2GPI IgA antibodies. RESULTS: One hundred and sixteen patients (76.8%) were dyslipidemic. Of these, 13 had a previous cardiovascular event (stroke = 12 and AMI = 01). A significant proportion of these patients had moderate to high lupus activity (69/116, 59.5%), with high levels of non-HDL-C and higher atherogenic cardiovascular risk rates. ANA titers were higher in dyslipidemic patients, while the prevalence and levels of autoantibodies against autoantigens and of C3, C4, and C-reactive protein were similar among patients with and without dyslipidemia. There was a higher prevalence of ASCA in lupus patients compared to women without SLE, but both lupus patients had similar prevalence and levels of these antibodies. Anti-β2GPI IgA antibodies were also detected similarly in these SLE groups. There was no correlation between SLEDAI and either ASCA and IgA anti-β2GPI antibodies, atherogenic indexes and CRP levels. CONCLUSIONS: The majority of dyslipidemic lupus patients present elevated atherogenic cardiometabolic risk, suggesting a high predisposition to cardiovascular events. There is a significant production of ASCA in SLE, but it is unrelated to dyslipidemia or disease activity, suggesting a structural homology between S. cerevisiae mannan and autoantigens.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-09-17T17:06:21Z No. of bitstreams: 1 Dissertação_ICS_Márcia Fernanda dos Santos Melo Carneiro.pdf: 3252453 bytes, checksum: 8e1c2cfe0185223bd295094f77aa813a (MD5) / Approved for entry into archive by Patricia Barroso(pbarroso@ufba.br) on 2013-09-17T17:43:10Z (GMT) No. of bitstreams: 1 Dissertação_ICS_Márcia Fernanda dos Santos Melo Carneiro.pdf: 3252453 bytes, checksum: 8e1c2cfe0185223bd295094f77aa813a (MD5) / Made available in DSpace on 2013-09-17T17:43:10Z (GMT). No. of bitstreams: 1 Dissertação_ICS_Márcia Fernanda dos Santos Melo Carneiro.pdf: 3252453 bytes, checksum: 8e1c2cfe0185223bd295094f77aa813a (MD5) / FABAMED, que apóia a pesquisa e fornece ajuda com os custos de material de escritório e tem convênio com a APAE e FIOCRUZ; O projeto conta também com a presença de estudantes bolsistas do CNPq e FAPESB e de outros estudantes de Medicina da UFBA que são voluntários; / Introdução. As glomerulopatias são a 3ª causa de doença renal crônica dialítica no Brasil. Tem-se observado variações regionais na prevalência dos diferentes tipos de glomerulopatias, possivelmente em decorrência de variações na condição social, raça, idade e no acesso e indicação à biópsia renal. Na Bahia temos uma peculiaridade, pois a população é formada predominantemente por pretos e pardos, podendo o padrão histológico ser diferente do restante do país. Os estudos epidemiológicos de prevalência são escassos no Brasil e em nosso estado, onde os dados iniciais mostravam maior prevalência de glomerulonefrites associadas à causas infecciosas. Objetivos. Identificar as glomerulopatias mais prevalentes em nosso meio e fazer correlações anátomoclínicas. Material e Métodos. Estudo de coorte prospectivo observacional, realizado no Hospital Geral Roberto Santos (HGRS), que incluiu dados clínicos, laboratoriais e histológicos de 165 pacientes adultos internados com glomerulopatia, confirmada por biópsia renal, registrados prospectivamente de outubro 2007 a outubro de 2012. Resultados. A maioria dos pacientes tinha entre 18-40 anos (62,4%) e eram não brancos (70,3%). Houve ligeira predominância de mulheres (54,5%). Chamou atenção que 52,7% dos pacientes vinham do interior do estado. A maioria estava sintomática (90,9%), e o período de início de sintomas foi ≤ 3 meses em apenas 38,8%. Havia diagnóstico de hipertensão arterial sistêmica há ≤ 5 anos em 39,4 %. A mediana da creatinina foi 1,6mg/dL e 46,7% dos pacientes tinham proteinúria nefrótica, com mediana da proteinúria de 3,35 g/24h. Necessitaram de diálise 25,5 % dos pacientes, sendo que 20,6 % permaneciam em diálise na alta hospitalar. No geral, nefrite lúpica foi a doença mais frequente (29%), a glomeruloesclerose segmentar e focal (GESF) foi diagnosticada em 17,6% dos casos, seguida por complexo lesão mínima/GESF (CLM/GESF) (10,3%), nefropatia membranosa (NM) (8,5%), lesão mínima (LM) (6,7%), membranoproliferativa (GNMP) (4,8%), proliferativa difusa (2,4%) e nefropatia por IgA (4,2%). Glomerulonefrite esclerosante ocorreu em 6,7 % dos casos. Comparamos os grupos com glomerulonefrite primária e secundária, e observamos que o último estava mais sintomático (p=0,037), necessitou mais de diálise (p=0,001), usou mais terapia imunossupressora (p=0,000) e teve mais complicações (p=0,006), porém recebeu mais alta da diálise antes da alta hospitalar (p=0,002). Conclusões. Trata-se de uma coorte de pacientes jovens, não brancos, com predomínio de glomerulopatias primárias, sendo GESF a mais frequente dentre as primárias e nefropatia por IgA incomum. No geral, nefrite lúpica foi a doença mais comum, achado compatível com o restante do país e que sugere mudanças no perfil epidemiológico dessas doenças em nosso meio. Observamos que o grupo das glomerulonefrites secundárias era mais grave, foi tratado mais agressivamente, mas em compensação recebeu mais alta da diálise. / Salvador

The aim of the thesis was to develop an immunoassay for the detection of dangerous bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA). MRSA infections cause global problems in health facilities which provide acute and follow-up care in inpatient and outpatient parts. That is why early and rapid diagnosis and targeted thereapies are very important for the subjects. E. coli was purified using the QIAquick PCR kit Purificatin isolated bacterial constructs, which were subsequently purified and dialyzed. The recombinant protein PBP2a in different concentrations was applied to a nitrocellulose membrane in the form of lines. Furthermore was performed optimized blot-line method for the detection of specific antibodies against the recombinant antigen PBP2a in the classes IgG, IgA and IgM. Several different concentrations of the conjugate Goat Anti-human IgG-AP, Goat Anti-human IgA-AP or Goat anti-Human IgM-AP were used for the detection. The color intensity of each line of the strip was evaluated with Immunoblot software. The measured values were used to determine diagnostic sensitivity, specificity and overall diagnostic match. Further testing of precision was carried out under repeatability conditions (intra-assay) and reproducibility (inter-assay). Precision of the method was expressed by coefficient of variation. As the most suitable for the manufacture of a IgG kit was determined the concentration of the antigen PBP2a from 0.30 mg/ml to 0.45 mg/ml and the concentration of IgG conjugate from 1:1500 to 1:1800. For class IgA as the most appropriate antigen was determined concentration PBP2a from 0.40 mg/ml to 0.52 mg/ml and conjugate concentrations of IgA from 1:500 to 1:1000. The coefficient of variation under repeatability conditions for the entire range of the IgG class is 10.09 %, and for IgA is 8.91 %. Variation coefficient reproducibility conditions for the entire range of the IgG class is 9.23 % and for IgA is 9.60 %. Precision of the method under conditions of repeatability and reproducibility for classes IgG and IgA meets the criteria for the manufacture of a diagnostic kit. Titration results showed that particular batch of cards made of nitrocellulose membrane coated with antigen PBP2a must always be verified on the panel of reference samples and the values of concentrations (for both antigen and conjugate) should be set according to the needs, but differently for the class of immunoglobulins IgG and IgA.

京都大学 / 新制・課程博士 / 博士(社会健康医学) / 甲第24536号 / 社医博第128号 / 新制||社医||12(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 近藤 尚己, 教授 西浦 博, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

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