Orientador: Iracema de Mattos Paranhos Calderon / Coorientador: Adenilda Cristina Honório França / Banca: Yuri Karen Sinzato / Banca: Debora Cristina Damasceno Meirelles dos Santos / Banca: Mahmi Fujimori / Banca: Mara Sandra Hoshida / Resumo: Durante a gravidez, a hiperglicemia materna altera a expressão e a transferência de células imunorregulatórias e de imunoglobulinas e o perfil de citocinas na interface materno-fetal. Avaliar, em gestações complicadas por hiperglicemia, a expressão de células NK e o perfil de citocinas no sangue, materno e do cordão umbilical, e na placenta (artigo 1); quantificar a produção de anticorpos e a passagem de IgG total, e respectivas subclasses, via receptor FcRn (artigo 2). MÉTODO - Foram avaliadas 120 gestantes, distribuídas nos grupos: não-diabético (ND; N = 30), hiperglicemia gestacional leve (HGL; N = 30), diabetes mellitus gestacional (DMG; N = 30) e diabetes mellitus tipo 2 (DM2; N = 30). Técnicas de citometria de fluxo foram utilizadas para análise de células e citocinas e, de ELISA, para avaliação das concentrações de IgG total e subclasses. A transferência placentária de anticorpos totais, e respectivas subclasses, foi definida pela relação [(concentrações no sangue de cordão umbilical/sangue materno) x 100]. Na análise estatística foram realizadas análises de variância (ANOVA), seguida pelo teste de Tukey, e de correlação de Pearson, com p < 0,05. No sangue materno dos grupos hiperglicêmicos, as células NK CD16+CD56- aumentaram, enquanto que CD16+CD56+ foi menor no grupo DMG. No sangue do cordão do grupo DM2 mostrou uma maior proporção de células CD16+CD56- e CD16-CD56+. As camadas extravilosas da placenta dos grupos DMG e DM2 most... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: - During pregnancy, the immune response associated with diabetes alters the expression and the transfer of immune cells, including regulatory, immunoglobulins and the profile of cytokines in the maternal-fetal interface. To evaluate the expression of NK cells, and the profile of cytokines in maternal blood, umbilical cord and placenta, quantify the production of antibodies, as well as, the passage of IgG and subclasses, via receptors FcRn in pregnancies complicated by diabetes or hyperglycemia.Wereessed 120 pregnant women, distributed as non-diabetic (ND; n=30), Mild Gestational Hyperglycemia (MGH; n=30), Gestational Diabetes Mellitus (GDM; n=30) and type 2 Diabetes Mellitus (DM2; N=30). The cells and cytokines were evaluated by flow cytometry. The concentrations of total IgG and subclasses were analyzed by ELISA. Placental transfer of the total and subclasses antibodies were defined in each assay by the ratio [(cord concentrations/maternal concentrations) x 100]. In the statistical analysis we used analysis of variance (ANOVA), followed by Tukey test, and Pearson's linear correlation, with p < 0.05. RESULTS - In the maternal blood from the hyperglycemic groups, the CD16+CD56− NK cells increased, whereas that of CD16+CD56+ decreased in GDM group. Cord blood from DM2 showed a higher proportion of CD16+CD56− and CD16−CD56+. The placental extravillous layer of GDM and DM2 showed an increase of CD16+CD56− cells and, irrespective of region, the proportion of CD16−CD56+ cells was higher in MGH and GDM and lower in DM2. IL-2 was lower in maternal blood and IFN-���� higher in maternal and cord blood from the GDM group. IL-17 was higher in maternal and cord blood from the DM-2 group. The placental extravillous layer of the MGH showed high levels of IL-4, IL-6, IL-10, IL- 17, and IFN-���� and low levels of IL-1���� and IL-8, whereas the placental villous layer... (Complete abstract click electronic access below) / Doutor

京都大学 / 0048 / 新制・論文博士 / 博士(人間健康科学) / 乙第13006号 / 論人健博第1号 / 新制||人健||3(附属図書館) / 32934 / (主査)教授 藤井 康友, 教授 澤本 伸克, 教授 一山 智 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

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O sistema imune de mucosas representa uma importante ferramenta de defesa contra a colonização da cavidade oral, especialmente pela presença das imunoglobulinas salivares no início da vida. Além das imunoglobulinas, as citocinas salivares também possuem funções importantes, pois utilizam células linfoides para amplificar ou deprimir a resposta inflamatória e podem ser consideradas biomarcadores proteicos. No entanto, pouco se sabe sobre a ontogenia dos componentes do sistema imune de mucosas em recém-nascidos a termo (AT) e especialmente em pré-termo (PT, com gestação inferior a 37 semanas) que reconhecidamente apresentam imaturidade imunológica. O objetivo do presente estudo foi o de analisar os níveis de imunoglobulinas (Ig) A (IgA) e M (IgM), citocinas: interleucinas (IL) IL-6, IL-10, IL-12 e interferon (IFN) (IFN-?) e 20 tipos distintos de bactérias orais em amostras de salivas de crianças ao nascer (T0) e após 3 meses de vida (T3) em PT e AT. Para isto, 123 amostras de saliva (70 AT e 53 PT) foram coletadas e os níveis de IgA e IgM foram quantificadas por ensaios ELISA. Em um subgrupo de 50 amostras (25 AT e 25 PT) foram analisadas a presença de 20 tipos diferentes de espécies bacterianas através de Checkerboard e os níveis de citocinas através de ensaio Luminex®. Dados socioeconômicos e do nascimento foram avaliados através de questionários. Os resultados do Checkerboard mostraram que nenhuma criança apresentou níveis detectáveis das bactérias testadas. Houve diferenças estatisticamente significantes (p<0.05) nos níveis de IgA em T0, sendo que PT apresentaram níveis inferiores aos AT, o que não aconteceu em T3. Diferentemente, não houve diferenças nos níveis de IgM entre os grupos e visitas (p>0.05). A análise prospectiva mostrou que houve um aumento dos níveis de IgA de T0 para T3 em ambos os grupos. Quanto à citocinas, houve diferenças somente em T0 entre PT e AT, nos níveis de IL-6 e IL10 que foram significantemente superiores em PT (p<0.05). Desta maneira, os dados sugerem que a prematuridade pode levar a algumas diferenças no status da resposta imunológica de mucosas no nascimento o que não acontece após 3 meses. / The mucosal immune system through the secretion of salivary immunoglobulins represent an important line of defense against initial acquisition and bacteria colonization of the oral cavity especially early in life. Cytokines are protein signaling molecules that lymphoid cells use to amplify or down regulate the inflammatory response. Little is known about the ontogeny of the mucosal immune system and bacterial colonization in saliva from neonates at fullterm (FT) and especially preterm (PT, with less than 37 weeks gestation), known to have an immunological immaturity. The aim of this study was to analyze the levels of immunoglobulin A (IgA) and M (IgM), four cytokines: interleukin (IL) IL-6, IL-10, IL-12 and interferon gamma (IFN-?) and 20 types of bacteria in saliva samples at birth (T0) and after 3 months (T3) in PT and FT. For this, 123 saliva samples (70 FT and 53 PT) were collected and levels of IgA and IgM were quantified by ELISA assays. In a subgroup of 50 samples (25 FT and 25 PT) was analyzed for the presence of 20 different types of bacterial species using checkerboard and the levels of cytokines through Luminex® assay. Economic data and birth partners were assessed through questionnaires. The results the Checkerboard showed that no child had detectable levels of bacteria. There were statistically significant (p <0.05) in IgA levels at T0, and PT showed lower levels than FT, which has not happened in T3. In contrast no differences in levels of IgM and visits between the groups (p> 0.05). The prospective analysis showed that there was an increase of IgA levels from T0 to T3 in both groups. As for cytokines, there were only differences at T0 between PT and AT, in IL-6 and IL-10 that were significantly higher in PT (p <0.05). Thus, the data suggest that prematurity may lead to differences in the status of the mucosal immune response at birth but not at 3 months of age.

by Kwok Fung, Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 89-97). / Acknowledgement --- p.i / Table of Content --- p.ii / Abstract --- p.vi / List of Abbreviation --- p.viii / List of Tables and figures --- p.ix / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The immunoglobulin --- p.1 / Chapter 1.1.1 --- Immunoglobulin structure --- p.1 / Chapter 1.1.2 --- Immunogloblulin genes --- p.1 / Chapter 1.1.3 --- Immunogloblulin gene recombination --- p.3 / Chapter 1.1.4 --- Antibody diversity --- p.4 / Chapter 1.1.4.1 --- Imprecise joining --- p.5 / Chapter 1.1.4.2 --- N region addition --- p.5 / Chapter 1.1.4.3 --- Somatic mutation --- p.6 / Chapter 1.2 --- Hypermutation --- p.6 / Chapter 1.2.1 --- Features of hypermutation --- p.6 / Chapter 1.2.2 --- Germinal centre & Affinity maturation --- p.8 / Chapter 1.2.3 --- Mutational Hotspots --- p.9 / Chapter 1.2.4 --- Intrinsic characteristics of hypermutation --- p.10 / Chapter 1.2.5 --- Models for the mechanism of hypermutation --- p.11 / Chapter 1.2.5.1 --- DNA replication --- p.11 / Chapter 1.2.5.2 --- DNA repair --- p.12 / Chapter 1.2.5.3 --- Gene conversion --- p.13 / Chapter 1.2.5.4 --- Transcription --- p.15 / Chapter 1.2.5.5 --- Homologous recombination of reverse transcribed mRNA --- p.16 / Chapter 1.2.5.6 --- Transcription-coupled repair --- p.17 / Chapter 1.3 --- Scope of investigation --- p.17 / Chapter Chapter 2 --- Material and Method --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Methods (first generation of LMPCR) --- p.21 / Chapter 2.2.1 --- Animal and cell lines --- p.21 / Chapter 2.2.2 --- Oxazolone antigen immunization --- p.21 / Chapter 2.2.2.1 --- Preparation of Bordetella pertussis --- p.21 / Chapter 2.2.2.2 --- Coupling of phenyloxazolone with CSA or BSA --- p.22 / Chapter 2.2.2.3 --- Preparation of aluminium hydroxide adjuvant --- p.23 / Chapter 2.2.2.4 --- Mice immunization --- p.23 / Chapter 2.2.3 --- Detection of anti-phOx antibody by enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.3.1 --- Reagents --- p.24 / Chapter 2.2.3.2 --- Assay procedure --- p.24 / Chapter 2.2.4 --- Extraction of genomic DNA (mice/cell line) --- p.25 / Chapter 2.2.4.1 --- Reagents --- p.25 / Chapter 2.2.4.2 --- Isolation of DNA from cell line (NQ2.12.4 & NQ5.4.3) --- p.25 / Chapter 2.2.4.3. --- DNA extraction from mice --- p.26 / Chapter 2.2.5 --- Ligation-mediated polymerase chain reaction (LMPCR) --- p.26 / Chapter 2.2.5.1 --- Procedure --- p.26 / Chapter 2.2.5.1.1 --- First primer extension --- p.29 / Chapter 2.2.5.1.2 --- Ligation --- p.29 / Chapter 2.2.5.1.3 --- PCR amplification --- p.30 / Chapter 2.2.5.1.4 --- Labelling of LMPCR product --- p.30 / Chapter 2.2.6 --- Marker preparation --- p.31 / Chapter 2.2.7 --- Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.7.1 --- Reagents --- p.32 / Chapter 2.2.7.2 --- Procedure --- p.32 / Chapter 2.2.8 --- Southern blot hybridization --- p.33 / Chapter 2.2.8.1 --- Reagents --- p.33 / Chapter 2.2.8.2 --- DNA blotting --- p.34 / Chapter 2.2.8.3 --- Preparation of 32P labelling DNA probe --- p.34 / Chapter 2.2.8.4 --- Prehybridization and Hybridization --- p.35 / Chapter 2.2.9 --- Simplified protocol for the first generation of LMPCR --- p.36 / Chapter 2.3 --- Method (second generation of LMPCR) --- p.37 / Chapter 2.3.1 --- Excess linker removal --- p.37 / Chapter 2.3.1.1 --- Exonuclease III Treatment --- p.37 / Chapter 2.3.1.2 --- Mung bean nuclease Treatment --- p.37 / Chapter 2.3.1.3 --- Chroma Spin Treatment --- p.37 / Chapter 2.3.2 --- HindIII digestion after LMPCR --- p.38 / Chapter 2.3.3 --- DNA sequencing --- p.38 / Chapter 2.3.3.1 --- Cloning of amplified sequences to M13mpl9 plasmid --- p.38 / Chapter 2.3.3.2 --- Plaque hybridization --- p.39 / Chapter 2.3.3.3 --- Preparation of single-stranded templates --- p.39 / Chapter 2.3.3.4 --- Sanger dideoxy sequencing of single-stranded DNA --- p.40 / Chapter 2.3.4 --- Simplified protocol for second generation of LMPCR --- p.41 / Chapter Chapter 3 --- First generation of LMPCR --- p.42 / Chapter 3.1 --- General design --- p.42 / Chapter 3.1.1 --- LMPCR protocol and its modification --- p.42 / Chapter 3.1.2 --- Oligonucleotide design --- p.44 / Chapter 3.1.3 --- Experimental design --- p.48 / Chapter 3.2 --- Result --- p.50 / Chapter 3.2.1 --- Anti-phOx Ig level in normal and immunized mice --- p.50 / Chapter 3.2.2 --- LMPCR analysis of the sense strand of VkOxl --- p.50 / Chapter 3.2.2.1 --- Overall patterns of the LMPCR signals --- p.50 / Chapter 3.2.2.2 --- Southern hybridization --- p.50 / Chapter 3.2.2.3 --- Distribution of signals --- p.57 / Chapter 3.2.2.4 --- LMPCR analysis of the VkOxl-Jk5 anti-phOx transgene --- p.61 / Chapter 3.2.2.5 --- Effect of the number of cells carrying the VkOxl-Jk5 gene on LMPCR --- p.61 / Chapter 3.2.3 --- LMPCR analysis of the antisense strand of VkOxl --- p.64 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Second generation of LMPCR --- p.72 / Chapter 4.1 --- Introduction(experi mental modification) --- p.72 / Chapter 4.1.1 --- Tagging the specific LMPCR products by addition of a Hin dIII site in the linker --- p.72 / Chapter 4.1.2 --- "Removal of excess linker, OXUH" --- p.72 / Chapter 4.1.2.1 --- Exonuclease III treatment --- p.73 / Chapter 4.1.2.2 --- Chroma spin treatment --- p.73 / Chapter 4.1.2.3 --- Mung Bean Nuclease treatment --- p.75 / Chapter 4.1.3 --- Other modifications in LMPCR --- p.75 / Chapter 4.2 --- Results --- p.75 / Chapter 4.2.1 --- Effect of including Exonuclease III treatment --- p.75 / Chapter 4.2.2 --- Effect of including Mung Bean Nuclease treatment --- p.76 / Chapter 4.2.3 --- Effect of including Chroma spin treatment --- p.76 / Chapter 4.2.4 --- Strand break positions detected at the sense strand --- p.76 / Chapter 4.2.5 --- DNA sequence analysis of the antisense strand LMPCR products --- p.82 / Chapter 4.3 --- Discussion --- p.84 / References --- p.89 / Appendix I --- p.98

O sistema imune de mucosas representa uma importante ferramenta de defesa contra a colonização da cavidade oral, especialmente pela presença das imunoglobulinas salivares no início da vida. Além das imunoglobulinas, as citocinas salivares também possuem funções importantes, pois utilizam células linfoides para amplificar ou deprimir a resposta inflamatória e podem ser consideradas biomarcadores proteicos. No entanto, pouco se sabe sobre a ontogenia dos componentes do sistema imune de mucosas em recém-nascidos a termo (AT) e especialmente em pré-termo (PT, com gestação inferior a 37 semanas) que reconhecidamente apresentam imaturidade imunológica. O objetivo do presente estudo foi o de analisar os níveis de imunoglobulinas (Ig) A (IgA) e M (IgM), citocinas: interleucinas (IL) IL-6, IL-10, IL-12 e interferon (IFN) (IFN-?) e 20 tipos distintos de bactérias orais em amostras de salivas de crianças ao nascer (T0) e após 3 meses de vida (T3) em PT e AT. Para isto, 123 amostras de saliva (70 AT e 53 PT) foram coletadas e os níveis de IgA e IgM foram quantificadas por ensaios ELISA. Em um subgrupo de 50 amostras (25 AT e 25 PT) foram analisadas a presença de 20 tipos diferentes de espécies bacterianas através de Checkerboard e os níveis de citocinas através de ensaio Luminex®. Dados socioeconômicos e do nascimento foram avaliados através de questionários. Os resultados do Checkerboard mostraram que nenhuma criança apresentou níveis detectáveis das bactérias testadas. Houve diferenças estatisticamente significantes (p<0.05) nos níveis de IgA em T0, sendo que PT apresentaram níveis inferiores aos AT, o que não aconteceu em T3. Diferentemente, não houve diferenças nos níveis de IgM entre os grupos e visitas (p>0.05). A análise prospectiva mostrou que houve um aumento dos níveis de IgA de T0 para T3 em ambos os grupos. Quanto à citocinas, houve diferenças somente em T0 entre PT e AT, nos níveis de IL-6 e IL10 que foram significantemente superiores em PT (p<0.05). Desta maneira, os dados sugerem que a prematuridade pode levar a algumas diferenças no status da resposta imunológica de mucosas no nascimento o que não acontece após 3 meses. / The mucosal immune system through the secretion of salivary immunoglobulins represent an important line of defense against initial acquisition and bacteria colonization of the oral cavity especially early in life. Cytokines are protein signaling molecules that lymphoid cells use to amplify or down regulate the inflammatory response. Little is known about the ontogeny of the mucosal immune system and bacterial colonization in saliva from neonates at fullterm (FT) and especially preterm (PT, with less than 37 weeks gestation), known to have an immunological immaturity. The aim of this study was to analyze the levels of immunoglobulin A (IgA) and M (IgM), four cytokines: interleukin (IL) IL-6, IL-10, IL-12 and interferon gamma (IFN-?) and 20 types of bacteria in saliva samples at birth (T0) and after 3 months (T3) in PT and FT. For this, 123 saliva samples (70 FT and 53 PT) were collected and levels of IgA and IgM were quantified by ELISA assays. In a subgroup of 50 samples (25 FT and 25 PT) was analyzed for the presence of 20 different types of bacterial species using checkerboard and the levels of cytokines through Luminex® assay. Economic data and birth partners were assessed through questionnaires. The results the Checkerboard showed that no child had detectable levels of bacteria. There were statistically significant (p <0.05) in IgA levels at T0, and PT showed lower levels than FT, which has not happened in T3. In contrast no differences in levels of IgM and visits between the groups (p> 0.05). The prospective analysis showed that there was an increase of IgA levels from T0 to T3 in both groups. As for cytokines, there were only differences at T0 between PT and AT, in IL-6 and IL-10 that were significantly higher in PT (p <0.05). Thus, the data suggest that prematurity may lead to differences in the status of the mucosal immune response at birth but not at 3 months of age.

x, 156 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1988

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

Diss. (sammanfattning) Linköping : Univ., 2006. / Härtill 4 uppsatser.