Natália Helena Colombo FATORES MICROBIOLÓGICOS E IMUNOLÓGICOS ENVOLVIDOS NO DESENVOLVIMENTO DA CÁRIE PRECOCE DA INFÂNCIA Araçatuba-SP 2015 Natália Helena Colombo FATORES MICROBIOLÓGICOS E IMUNOLÓGICOS ENVOLVIDOS NO DESENVOLVIMENTO DA CÁRIE PRECOCE DA INFÂNCIA Araçatuba - SP 2015 Tese apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, para obtenção do título de Doutor em Ciência Odontológica – Área de Concentração: Saúde Bucal da Criança. Orientadora: Profa. Dra. Cristiane Duque Catalogação-na-Publicação Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Colombo, Natália Helena. C172d Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância / Natália Helena Colombo. – Araçatuba, 2015 101 f. : il. ; tab. + 1 CD-ROM Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientadora: Profa. Cristiane Duque 1. Cárie dentária 2. Sistema imunológico 3. Peptídeos catiônicos antimicrobianos 4. Bactérias 5. Scardovia wiggsiae I. Título Black D27 CDD 617.645 Dedicatória Dedico este trabalho, Aos meus pais Selma Maffei Colombo e Valdir Colombo, Pela vida maravilhosa e cheia de amor que me deram. Pelos exemplos que fizeram de mim quem eu sou. Obrigada por nunca terem medido esforços para ajudar os filhos. Agradecimentos A Deus, Por me dar saúde e força, guiar meus passos, iluminar meu caminho. Por fortalecer minha fé. Por permitir que eu recuperasse os movimentos da mão. À minha querida orientadora, Professora Cristiane Duque, Pelo carinho que acolhe seus alunos, pela paciência, amizade, ensinamentos transmitidos, grande incentivo e confiança. Obrigada por estar sempre amigavelmente disposta a ajudar. Aprendi muito com nosso convívio. Aos voluntários dessa pesquisa As queridas crianças que se esforçaram para me ajudar nessa pesquisa, e aos seus responsáveis por permitirem a participação dos filhos e pela paciência em responder os questionários. Ao meu namorado Fabiano Obrigada pela amizade, companheirismo, paciência e apoio. Ao meu amado irmão Gustavo Obrigada pela amizade e incentivo. Aos queridos amigos do laboratório Pela amizade, companhia, apoio e ajuda. Paula, obrigada pela grande ajuda com o qRT- PCR. Kelly, obrigada pela amizade e por ter sido meu braço direito na semana que eu tirei a tala. Karina, obrigada pela amizade e por estar sempre pronta para ajudar, obrigada por trazer materiais de Araraquara. Queridas Márjully e Dinah, obrigada por toda a ajuda com o projeto do ART, a ajuda de vocês foi essencial. Aos queridos alunos de iniciação cientifica que colaboraram com este projeto: Jesse e Laís. Marcelle e José Antônio, os primeiros a me acolherem no laboratório, me ajudando em pequenas coisas que faziam toda a diferença. Carlinha, amiga querida, companheira de clínica. Você é uma pessoa maravilhosa que está sempre pronta para ajudar sejam os amigos ou os pacientes. Obrigada por esses anos de amizade e convivência, aprendi muito com você. Conte sempre comigo. Naida, agradeço pelo companheirismo, pelas trocas de materiais e por dividirmos nossas experiências do qRT-PCR. À todos os amigos do laboratório, pelo agradável convívio e pelo companheirismo: Carla Favretto, Carla, Daniela Câmara, Daniela Oliveira, Dinah, Douglas, Gabriela, Jaime, Jaqueline, Jackeline, Jesse, José Antônio, Juliana, Karina, Kelly, Kevin, Laís Ribas, Laís, Liliane, Loiane, Luciene, Luiz Mateus, Marcelle, Maria Luiza, Mariana, Marina, Márjully, Michele, Paula, Remberto, Renan, Samia, Tatiana, Thayse, Valéria, Vanessa. Daisy, minha querida amiga, como você fez falta nesse laboratório. Mesmo de longe me apoiou. Obrigada pela amizade. Á todos os meus amigos e familiares Aos Docentes Aos docentes da Disciplina de Odontopediatria da Faculdade de Odontologia de Araçatuba, UNESP Prof. Alberto Carlos Botazzo Delbem, Prof. Célio Percinoto, Profª Cristiane Duque, Prof. Juliano Pelim Pessan, Prof. Robson Federico Cunha, Profª Sandra M. H. C. Ávila de Aguiar, pela agradável convivência e conhecimentos transmitidos. Todos muito dispostos a ensinar e ajudar. Professor Robson Federico Cunha, por seu empenho em transmitir seus ensinamentos clínicos, por sua preocupação com nosso aprendizado. Professora Doris Hissako Sumida, minha orientadora durante o mestrado, pela amizade, por me apoiar quando resolvi mudar de área, por deixar as portas do seu laboratório abertas, pelos empréstimos de materiais. Professora Sandra Helena Penha de Oliveira, sempre muito prestativa, obrigada por nos ajudar com empréstimos de materiais. Professor Antônio Augusto Ferreira de Carvalho (Professor Toninho), sempre muito amigo e prestativo. Obrigada pelo acolhimento e por emprestar a câmara escura da radiologia para que eu pudesse fazer parte dos experimentos. Professor Marcos Rogério Mendonça, pela agradável convivência e ensinamentos transmitidos. Aos funcionários Aos funcionários do departamento de Odontologia Infantil e Social, Maria Bertolina, Ilídio, Mario e Ricardo pela atenção dispensada. Aos funcionários da Seção de Pós-Graduação da Faculdade de Odontologia de Araçatuba - UNESP, Valéria Queiroz Marcondes Zagatto, Cristiane Regina Lui Mattos, Liliam Sayuri Mada pelo excelente trabalho, atenção dispensada, grande disposição em atender e ótimo relacionamento. Aos funcionários e estimados amigos da Biblioteca, Izamar da Silva Freitas, Ana Claudia Martins Grieger Manzatti, Claudio Hideo Matsumoto, Ana Paula Rimoli de Oliveira, Denise Haruyo Nakamura, Ivone Rosa de Lima Munhoz, Luis Claudio Sedlacek, Luzia Anderlini e Maria Claudia de Castro Benez, sempre prontos para nos ajudar. Às funcionárias da Diretoria Técnica Administrativa, Isabel Cristina Lui Poi e Célia Cristina Antonello Cunha, pela atenção dispensada. A todos os demais professores e funcionários da Faculdade de Odontologia de Araçatuba - UNESP, pela ajuda e atenção que me dispensaram desde a graduação. À Faculdade de Odontologia de Araçatuba Ao curso de Pós Graduação em Ciência Odontológica À Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) pela bolsa concedida e a Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) pelo auxílio-pesquisa (processo 2012/19235-5). Agradeço a todos aqueles que estiveram ao meu lado nessa jornada e de alguma forma a tornaram mais agradável pela companhia ou mais leve pela ajuda. Epígrafe “Talvez não tenha conseguido fazer o melhor, mas lutei para que o melhor fosse feito. Não sou o que deveria ser, mas graças a Deus, não sou o que era antes”. Marthin Luther King Resumo geral Colombo, NH. Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância. 2015. 101f. Tese (Doutorado em Ciência Odontológica) Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, Araçatuba, 2015. A cárie precoce da infância (CPI) é ainda um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento. Estudos têm sugerido a associação da ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança na etiologia da CPI. O objetivo deste estudo foi avaliar os aspectos microbiológicos e imunológicos associados ao desenvolvimento da cárie precoce da infância. Crianças com idade entre 36 e 60 meses foram selecionadas e divididas em três grupos: LC - livres de cárie, CPI e CPI-S (CPI-severa). Questionário sobre os aspectos socioeconômico-culturais, hábitos de higiene bucal e diários de dieta foram respondidos pelos responsáveis. Foram coletadas amostras de saliva e biofilme dental das crianças e processadas para subsequentes avaliações laboratoriais. Em seguida, os níveis de IgA salivar total e contra GbpB de S. mutans foram determinados por ELISA e Western blot, respectivamente, as concentrações salivares dos peptídeos catiônicos antimicrobianos (PCAM): defensinas hBD-2 e hBD-3, catelicidina LL-37 e histatina 5 (HTN-5) por ELISA e a presença e os níveis salivares de Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. e Scardovia wiggsiae por qRT-PCR, sendo que estes dados foram correlacionados com os níveis salivares e no biofilme dental de estreptococos mutans (SM) e Lactobacillus spp. por meio de cultivo em meios específicos. Os resultados mostraram que as crianças com CPI-S apresentaram menor renda familiar quando comparadas às crianças LC ou CPI. Contudo, a ingestão de açúcar não diferiu entre os grupos. O grupo CPI-S apresentou maior contagem de SM na saliva/biofilme em relação aos grupos LC e CPI. Houve uma correlação positiva entre a resposta de IgA contra GbpB e os níveis de SM, quando a população geral foi avaliada. Quando apenas crianças com altos níveis de SM foram comparadas, o grupo CPI-S mostrou redução significante na resposta do IgA contra GbpB em relação ao grupo LC. Esta alteração não foi observada para o grupo CPI. O presente estudo mostrou correlação positiva de hBD-2 e HTN-5 com os níveis salivares de SM. Além disso, houve uma correlação positiva entre hBD-2, LL- 37 e HTN-5, sugerindo uma ação combinada desses peptídeos na proteção do organismo. Contudo, as concentrações salivares de cada PCAM não diferiram entre os grupos. Os resultados do qRT-PCR mostraram que a frequência de detecção de S. mutans, Bifidobacterium spp. e S. wiggsiae aumentou com a severidade da cárie. Os níveis de S. mutans, S. sobrinus, S. wiggsiae e Bifidobacterium spp. foram maiores no grupo CPI-S em relação aos grupos LC e CPI. Não houve diferença nos níveis de Lactobacillus spp. e em sua frequência de detecção entre os grupos. Este estudo sugere que crianças com CPI-S apresentam resposta imunológica reduzida representada pelos níveis de IgA contra GbpB de S. mutans, mas não relacionadas com as concentrações salivares dos PCAM. Em relação aos aspectos microbiológicos, conclui-se que, além de S. mutans, outras espécies bacterianas como S. sobrinus, S. wiggsiae e Bifidobacterium spp., estão associadas com a severidade da cárie na infância. Palavras-chave: Cárie dentária. Sistema imunológico. Peptídeos catiônicos antimicrobianos. Bactérias. Scardovia wiggsiae. General Abstract Colombo, NH. Microbiological and immunological factors for the development of early childhood caries. 2015. 101f. Tese (Doutorado em Ciência Odontológica) Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, Araçatuba, 2015. Early childhood caries (ECC) is still a serious public health problem worldwide, especially in developing countries. Studies have been suggested the association among frequent intake of fermentable carbohydrates such as sucrose, high cariogenic microorganism’s counts and child's immune vulnerability in the etiology of ECC. The objective of this study was to evaluate the microbiological and immunological factors for the development of early childhood caries. 36 to 60 month- old children were selected and distributed into three groups: caries free (CF), ECC and S-ECC (severe-ECC). Questionnaires about socio-economic-cultural data, oral hygiene habits and food-frequency diary were completed by the parents. Saliva and dental biofilm were collected from children and processed for subsequent laboratorial tests. The following analyses were determined: total IgA and IgA response against S. mutans GbpB by ELISA and Western blot, respectively; salivary concentrations of antimicrobial peptides (AMPs): defensins hBD-2 and hBD-3, cathelicidin LL-37 and histatin 5 (HTN-5) by ELISA; salivary detection and quantification of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. and Scardovia wiggsiae by qRT-PCR, and these data were correlated with mutans streptococci (MS) and Lactobacillus spp. levels by culture in specific medium. Results showed that S-ECC children had reduced family income compared to ECC and CF. However, sugar intake did not differ among the groups. S-ECC group had higher MS count than CF/ECC groups. Positive correlations between salivary IgA response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, S-ECC group showed a significant decrease in IgA antibody levels against GbpB compared to CF group. This finding was not observed for ECC group. The present study showed positive correlations between salivary hBD-2 and HTN-5 with salivary mutans streptococci levels. In addition, results showed a positive correlation among hBD-2, LL-37 and HTN-5, suggesting a combined action of these peptides in the host protection. However, salivary concentration of AMPs did not differ among the groups. The results of qRT-PCR showed that the frequency of S. mutans, Bifidobacterium spp. and S. wiggsiae detection increased with caries severity. The levels of S. mutans, S. sobrinus, Bifidobacterium spp. and S. wiggsiae were significantly higher in S-ECC children compared to CF and ECC children. There was no statistical difference among the groups considering the levels or frequency of Lactobacillus spp. This study suggested that children with S-ECC have reduced immunological response, represented by the salivary IgA levels against S. mutans GbpB, but not with salivary concentrations of AMPs. In relation to microbiological aspects, it was concluded that, in addition to S. mutans, other bacterial species such as S. sobrinus, S. wiggsiae and Bifidobacterium spp., are associated with the severity of early childhood caries. Keywords: Dental caries. Immune system. Antimicrobial cationic peptides. Bacteria. Scardovia wiggsiae. Lista de Figuras Capítulo 1 Figure 1 Scatter plot showing correlation between levels of IgA antibody reactive with GbpB and mutans streptococci (MS). The analysis was carried out for the total population, regardless the caries status. Positive Pearson correlation (R=0.583, p=0.00) was observed for MS counts. 38 Figure 2 Box plots of the western blot reactivity of salivary IgA antibody with S. mutans GbpB among the groups of children, distributed according to caries status (CF, ECC and S-ECC) and mutans streptococci levels (LMS and HMS). Bars indicate minimum and maximum values. Black and white boxes indicate lower and upper quartiles, respectively. Line in the middle of boxes is median. 39 Capítulo 2 Figure 1 Levels of antimicrobial peptides (hBD-2, hBD-3, LL-37 and HTN-5) detected in saliva samples of children. *There was no statistical difference (p>0.05) among the groups, considering each AMP separately, according to Kruskal-Wallis tests. 56 Figure 2 Only significant correlations are shown. A. Relationship between hBD-2 and salivary mutans streptococci levels (Spearman correlation, r= 0.228, p=0.043). B. Relationship between HTN-5 and salivary mutans streptococci levels (Spearman correlation, r= 0.235, p=0.039). 57 Figure 3 Only significant correlations are shown. A. Relationship between LL-37 and hBD-2 (Spearman correlation, r= 0.831, p=0.000). B. Relationship between HTN-5 and LL- 37 (Spearman correlation, r= 0.765, p=0.000). C. Relationship between hBD-2 and HTN-5 (Spearman correlation, r= 0.796, p=0.000). D. Relationship between hBD-3 and LL-37 (Spearman correlation, r= 0.188, p=0.053). E. Relationship between hBD-3 and hBD-2 (Spearman correlation, r= 0.193, p=0.048). 58 Capítulo 3 Figure 1 Frequency (%) of microorganisms detected by qPCR. a Different lower case letters show statistical difference (p<0.05) among the groups, according to Chi-square test. 73 Lista de Tabelas Capítulo 1 Table 1 Comparative analysis between the severity of early childhood caries and related etiological factors. 37 Table 2 Medians (range) of total salivary IgA, distributed according to mutans streptococci levels. 38 Capítulo 2 Table 1 Comparative analysis between the severity of early childhood caries and related etiological factors. 55 Table 2 Relationship between antimicrobial peptides (hBD-2, hBD- 3, LL-37 and HTN-5) and your combinations and dmfs. 59 Capítulo 3 Table 1 Primer used for qRT-PCR analysis 70 Table 2 Comparative analysis between the severity of early childhood caries and related etiological factors. 71 Table 3 Microorganism levels [Means (medians) standard errors] obtained by qPCR (ng/µL) 72 Lista de Abreviaturas AMPs: Antimicrobial peptides; Peptídeos catiônicos antimicrobianos Ags: Antigens; Antígenos BHI: Brain Heart Infusion; Infusão de Cérebro e Coração ATCC: de American Type Culture Collection; Coleção Americana de tipos de cultura CF: Caries Free; Livres de cárie CFU: Colony-forming units; Unidades formadoras de colônia dmfs: Decayed Missing Filled Surfaces; Superficies cariadas, perdidas ou restauradas DNA: Deoxyribonucleic acid; Ácido desoxirribonucleico ECC: Early childhood caries; Cárie precoce da infância hBD1: Human β-defensin 1; β-defensina 1 humana hBD-2: Human β-defensin 2; β-defensina 2 humana hBD-3: Human β-defensin 3; β-defensina 3 humana HMS – high mutans streptococci count; alta contagem de estreptococos mutans hNP1-3: Human neutrophil defensins (α-defensin); α-defensina HTN-5: Human-Histatin 5; Histatina 5 humana GtfB: Glucosyltransferase B; Glicosiltransferase B GtfC: Glucosyltransferase B; Glicosiltransferase C GtfD: Glucosyltransferase B; Glicosiltransferase D GbpA: Glucan-binding proteins A; Proteína ligante de glicano A GbpB: Glucan-binding proteins B; Proteína ligante de glicano B GbpC: Glucan-binding proteins C; Proteína ligante de glicano C HMS: High Mutans streptococci count; Alta contagem de estreptococos mutans IgA: Immunoglobulin A; Imunoglobulina A KDa: Kilo Daltons; Quilo Daltons (unidade de massa atômica). LL-37: Human cathelicidin LL-37; Catelicidina humana LL-37 LMS: Low mutans streptococci count; Baixa contagem de estreptococos mutans MS: mutans streptococci MSA: Mitis Salivarius Agar; PCR: Polymerase Chain Reaction; Reação em cadeia da polimerase qRT-PCR: Quantitative Real-Time PCR; PCR quantitativo em tempo real S-ECC: Severe early childhood caries; Cárie severa da infância Sumário 1. INTRODUÇÃO GERAL 20 2. Capítulo 1- Relationship between the Immunoglobulin A Antibody Response to Streptococcus mutans Glucan-Binding Protein B and the Severity of Early Childhood Caries 2.1 SUMMARY 25 2.2 INTRODUCTION 26 2.3 METHODS 27 2.4 RESULTS 30 2.5 DISCUSSION 31 2.6 REFERENCES 34 3. Capítulo 2 - Salivary levels of the antimicrobial peptides in children with severe early childhood caries 3.1 ABSTRACT 42 3.2 INTRODUCTION 43 3.3 MATERIALS AND METHODS 44 3.4 RESULTS 46 3.5 DISCUSSION 47 3.6 REFERENCES 51 4. Capítulo 3 - Molecular bacterial detection and severity of early childhood caries 4.1 ABSTRACT 61 4.2 INTRODUCTION 62 4.3 MATERIALS AND METHODS 63 4.4 RESULTS 65 4.5 DISCUSSION 65 4.6 REFERENCES 68 ANEXOS 74 Natália Helena Colombo 20 Introdução geral A cárie dentária é uma doença infecciosa, causada por ácidos provenientes da fermentação microbiana dos carboidratos da dieta que, com o tempo, causam a desmineralização dos tecidos duros do dente (1). Quando ocorre em crianças com menos de 6 anos esta doença é chamada de cárie precoce da infância (CPI ou ECC do inglês early childhood caries). A Academia Americana de Odontopediatria (2008) (2) determina a severidade da cárie precoce da infância de acordo com idade da criança. Em crianças menores de 3 anos de idade, qualquer sinal de cárie em superfície dental lisa é indicativo de cárie severa da infância (CPI-S ou S-ECC). Entre os 3 e 5 anos de idade, uma ou mais superfícies lisas cavitadas, perdidas (devido à cárie) ou restauradas em dentes decíduos anteriores constitui CPI-S. Crianças apresentando ≥4 (aos 3 anos de idade), ≥5 (aos 4 anos de idade), ou ≥6 (aos 5 anos de idade) superfícies cariadas, perdidas ou restauradas também são classificadas com CPI-S. A cárie precoce da infância continua sendo um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento (3). No Brasil, a CPI apresenta prevalência entre 14,8% e 43,4% (4-6). Devido à rápida destruição dentária em curto período de tempo, tem sido sugerida a associação entre os seguintes fatores na etiologia da CPI: ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança (6-10). O grupo bacteriano considerado mais cariogênico é o dos estreptococos mutans (SM), especialmente Streptococcus mutans (9, 10). Embora a associação entre S. mutans e CPI pareça convincente, grande percentual das crianças colonizadas por essa espécie bacteriana não manifesta a doença (6, 8). Assim, outras espécies acidogênicas e acidúricas, incluindo estreptococos não mutans e Actinomyces, estão envolvidas com o início das lesões de cárie (11, 12). Van Ruyven et al.(13) detectaram também outras espécies bacterianas, entre elas, Lactobacillus e Bifidobacterium, em biofilmes dentários cobrindo lesões de mancha branca. Tanner et al. (14) avaliaram por PCR as espécies mais frequentes na saliva de crianças com CPI e notaram alta prevalência de bactérias da família Natália Helena Colombo 21 Bifidobacteriaceae, entre elas, Scardovia wiggsiae ou ainda grande associação entre Streptococcus mutans e bifidobactérias. Para se aderir à superfície dental, Streptococcus mutans sintetizam três tipos de proteínas: adesina Atg I/II; glucosiltransferases (Gtfs), e proteínas ligantes de glucano (Gbps A,B, C e D, referente à “glucan-binding proteins”) (15). Diversos estudos têm demonstrado que a indução de anticorpos específicos contra esses antígenos proteicos pode prevenir o desenvolvimento de cárie em modelos animais (16, 17) e em humanos (18). Na cavidade bucal, as imunoglobulinas salivares, principalmente IgA secretória, apresentam grande importância na resistência da mucosa às infecções. Nogueira et al. (19) mostraram que a resposta de IgA contra GbpB esteve relacionada ao atraso na infecção bucal por S. mutans. A intensidade dos padrões de IgA aos antígenos de S. mutans foi estudada por Parisotto et al.(7) que verificaram baixos níveis de anticorpos IgA contra GbpB associados com alto risco de cárie. Entretanto, para crianças com padrões típicos de cárie severa da infância, os níveis de IgA contra esses antígenos ainda não foram avaliados . O sistema imunológico apresenta diversas formas de defesa contra a microbiota. As mucosas, além de apresentarem a função de barreira física contra a entrada de organismos estranhos, são fonte de potentes peptídeos catiônicos antimicrobianos (PCAM). Os PCAM fazem parte da resposta imune inata, participando da primeira linha de defesa em vários locais do corpo (20). Na cavidade oral, PCAM estão presentes na saliva, epitélio gengival e fluido gengival (21) Alguns PCAM têm se destacado na literatura por sua atividade bactericida ou bacteriostática contra patógenos orais, entre eles estão as α- e β-defensinas, catelicidina humana LL-37 e histatinas (22). As defensinas são peptídeos pequenos, de 15 a 45 aminoácidos, que dependendo do padrão de pareamento de seus resíduos de cisteína, são subdivididas em duas principais subfamílias: α e β-defensinas. Foram identificadas seis α-defensinas em humanos, sendo que quatro são produzidas pelos neutrófilos e denominadas de peptídeo neutrofílico humano (HNP-1 a 4) e as outras duas são produzidas por células de Paneth nas criptas intestinais. As β-defensinas (hBDs) são produzidas por células epiteliais de diversos órgãos como olhos, pele, pulmão, rim, pâncreas, mucosa nasal e oral e embora tenham sido encontradas quase 40 regiões gênicas potenciais para hBDs, as mais bem caracterizadas são HBD- 1 a -4. As α e β-defensinas apresentam função imunomoduladora, modificando a migração e Natália Helena Colombo 22 maturação celular, induzindo citocinas e a liberação de histamina e prostaglandina A2 de mastócitos (23-25). O peptídeo catiônico humano (hCAP-18) é a única catelicidina identificada em seres humanos isolada primeiramente em grânulos de neutrófilos. hCAP-18 é produzida também por células epiteliais do pulmão, intestino, cavidade bucal e trato urogenital, sendo encontrada no plasma seminal e plasma sanguíneo. Após a secreção, ocorre a quebra de hCAP-18 pela ação de proteases em pequenos fragmentos de peptídeos RK-31 e KS-30 e em um peptídeo de cadeia longa LL-37, todos com ação antimicrobiana. Esse último fragmento do peptídeo hCAP-18, o LL- 37, é um modulador multifuncional da imunidade inata, envolvendo a função antibacteriana, estímulo de angiogênese, cicatrização cutânea e quimiotaxia de células inflamatórias e do sistema imune. Esse fragmento de peptídeo causa a formação de poros na membrana das bactérias e a lise celular, entretanto, em altas concentrações (>13µM) pode se tóxica para as células eucarióticas (23, 24). As histatinas são proteínas catiônicas ricas em histidina, produzidas pelas glândulas salivares, que apresentam ação bactericida e fungicida. Existem, pelo menos, 12 histatinas na saliva, como resultado de ligações ou proteólise das histatinas 1 e 3. O fragmento de 24 aminoácidos da porção N-terminal da histatina 3 é denominado de histatina 5. Esta é a histatina salivar com maior ação fungicida. As histatinas também podem inibir proteinases e prevenir a co-agreagação bacteriana (24). Alguns estudos relacionaram a presença de PCAM e cárie na infância. Tao et al (26) observaram que baixos níveis de HNP1-3 podem representar um fator biológico para a susceptibilidade à cárie, pois foram detectadas maiores concentrações de HNP1-3 em crianças livres de cárie. Davidopoulou et al. (27) avaliaram os níveis de LL-37 em crianças com dentição decídua, mista e permanente com ou sem lesões de cáries e gengivite e verificaram que crianças com dentição decídua tiveram concentrações significantemente menores do peptídeo que aquelas na dentição mista e permanente. O mesmo foi verificado para crianças com alta atividade de cárie quando comparadas às crianças com baixa ou moderada atividade de cárie. Natália Helena Colombo Capítulo 1 Natália Helena Colombo 24 Relationship between the Immunoglobulin A Antibody Response to Streptococcus mutans Glucan-Binding Protein B and the Severity of Early Childhood Caries Natália Helena Colombo1, Jesse Augusto Pereira1, Márjully Eduardo Rodrigues da Silva1, Laís Fernanda Fonseca Ribas1, Thaís Manzano Parisotto2, Renata de Oliveira Mattos-Graner3, Daniel J Smith4, Cristiane Duque1* 1 Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Univ Estadual Paulista (UNESP), Araçatuba, SP, Brazil 2 Laboratory of Microbiology and Molecular Biology, Sao Francisco University Dental School, Bragança Paulista, SP, Brazil 3 Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas (UNICAMP), Piracicaba, SP, Brazil. 4 Department of Immunology and Infectious Disease, Forsyth Institute, Cambridge, MA, USA. Running head: IgA antibody response to S. mutans GbpB and caries Keywords: IgA, Dental decay, Glucan-binding proteins, GbpB, Immunity *Correspondence: Dr Cristiane Duque, Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Univ Estadual Paulista (UNESP) Address: R. José Bonifácio, 1193, CEP: 16015-050, Araçatuba-SP, Brazil Tel: (+55) 1836363315 E-mail: cristianeduque@yahoo.com.br * According to guidelines for authors of Molecular Oral Microbioly (Anexo D). mailto:cristianeduque@yahoo.com.br Natália Helena Colombo 25 SUMMARY Lower levels of salivary IgA antibody against S. mutans GbpB have been associated with higher risk to develop dental caries. The objective of this study was to explore the associations between the severity of dental caries in childhood and putative etiological factors: socio-economic-cultural aspects, dietary habits, mutans streptococci (MS)/lactobacilli colonization and IgA antibody response against S. mutans GbpB. 36 to 60 month-old children were grouped into Caries-Free (n=19, CF), Early Childhood Caries (n=17, ECC) and Severe Early Childhood Caries (n=21, S-ECC). Questionnaires were completed by the parents, which assessed socio- economic-cultural data, oral hygiene habits and dietary patterns. Saliva was collected from children for microbiological analysis (MS and lactobacilli levels) by culture and for detection of salivary IgA antibody reactive with S. mutans GbpB in western blot. Results showed that S-ECC children had reduced family income compared to ECC and CF. There was a difference between CF/ECC and S-ECC children in MS counts. Positive correlations between salivary IgA antibody response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, the S-ECC group showed a significant decrease in IgA antibody levels to GbpB compared to the CF group. This finding was not observed with the ECC group. This study suggests that children with S-ECC have reduced salivary IgA immune responses to S. mutans GbpB, potentially compromising their ability to modify MS infection and its cariogenic potential. Furthermore, a reduced family income and high levels of SM was also associated with S-ECC. Natália Helena Colombo 26 INTRODUCTION Dental caries is an infectious disease that results from the dissolution of tooth mineral by acids derived from bacterial fermentation of sucrose and other dietary carbohydrates (Loesche, 1996). When occurring in young children less than 71 months of age, this disease is called early childhood caries (ECC). ECC remains a serious worldwide public health problem, particularly in developing countries. According to the American Academy of Pediatric Dentistry (2008), from ages three through five, one or more cavitated, missing (due to caries) or filled smooth surfaces in primary maxillary anterior teeth or a decayed, missing or filled score of greater than or equal to four (age 3), five (age 4) or six (age 5) surfaces constitutes severe ECC (S-ECC). The disease can progress and lead to destruction of the primary dentition affecting negatively children’s physical and mental health, as well as increasing the risk of new caries lesions in the permanent dentition (Ng & Chase, 2013, Isaksson et al., 2013). The mutans streptococci (MS) group, mainly Streptococcus mutans, is most strongly associated with the pathogenic process of ECC because of its high presence in the biofilms and saliva of the affected children (Parisotto et al., 2010b, Ge et al., 2008). Two important cell-associated antigens (Ags) are correlated directly with the ability of S. mutans to adhere and accumulate in the tooth surfaces forming dental biofilm: glucosyltransferases (GtfB, GtfC, GtfD) and glucan-binding proteins (GbpA, GbpB, GbpC) (Smith, 2002). Gtf catalyzes glucan synthesis and Gbps increase the binding of S. mutans to each other and to glucans deposited on tooth surfaces, contributing to the sucrose-dependent adherence to teeth (Smith, 2002). Several studies have demonstrated that induction of specific antibodies against these antigens can prevent the development of dental caries in animal models (Jespersgaard et al., 1999, Koga et al., 2002, Smith & Taubman, 1996) and modify infection in humans (Smith & Taubman, 1987). Salivary immunoglobulins, particularly secretory IgA have major importance in the resistance of the mucosa to oral infections. The secretory IgA immune response represents the first line of adaptive immune defense against mutans streptococci, blocking microbial adhesins and potentially reducing oral colonization with this cariogenic microflora. Salivary IgA has also been shown to enhance the activity of several enzymes such as lactoferrins and lysozymes (Law et al., 2007). A clinical Natália Helena Colombo 27 study showed that the IgA antibody response against S. mutans GbpB was predominant in the first year of life and frequently correlated with the delay in the oral infection with S. mutans (Nogueira et al., 2005). (Parisotto et al., 2011) studied the intensity of IgA patterns against antigens of S. mutans and found that lower levels of salivary IgA against GbpB were associated with higher caries risk. However, the relationship between IgA antibody levels against this antigen and different levels of caries has not yet been evaluated. The objective of this study was to explore the associations among the severity of ECC and caries-related etiological factors: socio- economic-cultural aspects, dietary habits, mutans streptococci/lactobacilli colonization and IgA response against S. mutans GbpB. METHODS Subjects The study population included 36- to 60-month-old children who attended the four public nursery schools in the city of Araçatuba, São Paulo, Brazil (21° 12′ 32″ S, 50° 25′ 58″ W). The city´s population has access to public water supplied with fluoride to a level of 0.7 ppm. Children’s parents as well as the preschools involved granted written permission for the study which had been previously approved by the Research Ethics Committee of Univ. Estadual Paulista (UNESP), Brazil (Certificate of Presentation for Ethical Consideration (CAAE) # 13079213.4.0000.5420). Seventy- five 36 to 60 month-old children were selected to participate in this study. Questionnaires were supplied to the parents to assess socio-economic-cultural data and oral hygiene habits. The socio-economic-cultural data analyzed were family income and mother’s education. Dietary data were obtained from a food-frequency diary filled out by parents for three days during the week. Clinical examination was carried out at the school by a single calibrated examiner using mouth mirror and probe under natural light. Children were separated into three groups according to their caries status: Caries-Free group (CF), Early Childhood Caries group (ECC) and Severe Early Childhood Caries group (S-ECC). ECC was defined for this study as the presence of 1-3 decayed tooth surfaces (cavitated lesions) and S-ECC was defined as the presence of decayed surfaces score of ≥ 4 (age 3) and ≥ 5 (age 4) (American Academy of Pediatric Dentistry, 2008). Children suffering from systemic diseases, using long-term medication or antibiotics less than one month before examination, or Natália Helena Colombo 28 children with mucosal breaks were excluded from the study. Children with restored or absent teeth when saliva was collected for microbiological and immunological analysis were also excluded. Thus, the final number of children who participated in this study was fifty-seven, distributed in the following groups: 19 (CF), 17 (ECC) and 21 (S-ECC). Children were encouraged and instructed on dental hygiene and received all other necessary oral care. Saliva samples Unstimulated whole saliva was collected from each subject into a 50 mL sterile falcon conical tube for 5-10 min. Collections were performed at least 1 h after feeding to avoid contamination with non salivary components. Tubes were transported on ice to the laboratory and processed within 1 h. After agitation, one hundred microliters of saliva were separated for microbiological procedures. The remaining saliva was clarified by centrifugation at 10000 rpm at 4ºC for 10 min. The supernatants were collected and 250 mM EDTA was added to minimize salivary IgA aggregation. Aliquots of 50µl of each saliva samples were frozen at −70°C until immunological analysis. Microbiological procedures Aliquots of saliva were homogenized by vortexing for 1min and the suspensions were serially diluted (10−1 to 10−7) in 0.9% saline solution. Each dilution was cultivated in triplicate on the surface of two selective media: Mitis Salivaris Agar (Difco Laboratories, Detroit, MI, USA) with sucrose and bacitracin for isolation of mutans streptococci and Rogosa agar (Oxoid, Basingstoke, Hampshire, England) for lactobacilli. All plates were incubated at 37°C for 48 h in 5% CO2 atmosphere. After 48h of incubation, the total number of colony-forming units (CFU) was counted from a representative area of each agar plate yielding 30–300 colonies using a stereoscopic microscope. Results were expressed as CFU/ml. Total salivary IgA level measurement The concentration of total IgA in saliva samples were determined by an enzyme- linked immunosorbent assay (ELISA) kit using a commercially available analysis kit (Mabtech Inc, Cincinnati OH, USA) and following the manufacturer’s instructions. Natália Helena Colombo 29 Western blot analysis of salivary antibody to S. mutans GbpB In order to analyze the influence of patterns of specificity of IgA response to S. mutans GbpB, levels of infection, and caries status, Western blot assays were performed using saliva samples from children and tested against Ags extracted from a standard S. mutans strain (ATCC 25175). For Ags preparation, colonies of S. mutans from fresh Brain Heart Infusion Agar (BHI, Difco) were inoculated in 5ml BHI broth and incubated for 18h. Bacterial cells were then harvested from 1 ml of cultures previously adjusted to an absorbance of 1.0 (A550nm). Cells were ressuspended in TE containing 100-μm-diameter zirconia/silica beads and mechanically disrupted using a Mini-BeadBeater (BioSpec) at maximum speed (2,800 rpm) for 1-min pulses, three times, with a 30-s rest on ice between pulses. Cell pellets were then boiled in Laemmli buffer for 5 min, and protein extracts were separated by centrifugation at 4°C (10000 rpm for 4 min). Protein concentrations were determined by the method of Bradford and a total of 16 µg of protein extract was used for Western blot analysis (Nogueira et al., 2005). Ags extracts were loaded per lane, separated by sodium dodecyl sulfate–6% polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. After transference, membranes were washed and blocked overnight at 4°C (in Tris-buffered saline– Tween, pH 7.5, 5% nonfat milk). Incubations with saliva samples diluted 1:100 were performed at room temperature for 2 h. As negative controls, membranes were incubated only with blocking buffer, and as positive controls, membranes were incubated with a standard saliva sample obtained from an adult subject whose pattern of reaction with S. mutans antigen extracts had been previously measured. The secondary antibody was HRP-Goat Anti-Human IgA (1:4000 dilution) (Invitrogen, Life Technologies, USA). Immunoreactive bands were detected by autoradiography using ECL chemiluminescent substrate reagent kit (Invitrogen, Life Technologies, USA) according to the manufacturer’s instructions. X-ray films were scanned in a transluminator using a White Light Converter Plate (UVP, LLC, Upland, CA, USA) and the patterns of antigen recognition, including the number and intensity of reactive bands were analyzed with UVP Image software. The molecular weight of S. mutans GbpB was about 60 kDa. Migration position of GbpB were determined in parallel western blot assays performed with specific polyclonal rat antiserum to GbpB (Smith & Taubman, 1996). Natália Helena Colombo 30 Statistical analysis The statistical analysis was performed using the three groups of children with dental caries (CF, ECC and S-ECC) as the dependent variables. The comparisons among the groups were performed according to data distribution. ANOVA and Tukey tests were applied for caries levels (dmfs), age and sugar intake. Kruskal-Wallis and Mann-Whitney tests were applied for gender comparison, family income, mother´s education level, mother’s helping with tooth brushing, artificial (bottle) feeding, mutans streptococci/lactobacilli counts and total IgA levels. Medians and ranges of bacterial counts were expressed as log (CFU +1) and the constant 1 was added to CFU counts, when the sample showed zero CFUs. Pearson correlation tests were conducted to compare IgA levels against S. mutans GbpB and bacterial counts for the entire population. The reactivities of salivary IgA antibody with S. mutans GbpB were compared with respect to caries severity (CF, ECC and S-ECC) and mutans streptococci levels (low mutans streptococci - LMS and high mutans streptococci - HMS) using Mann-Whitney tests. RESULTS With respect to caries status, there were no statistical differences among the groups in relation to age, gender, mother´s education level, mother´s help with tooth brushing and diet habits (artificial feeding and sugar intake) and total IgA levels (Table 1). Families of S-ECC children had reduced income compared to families of ECC and CF children showing a relationship between high levels of caries and economic condition of family. The colonization with caries-associated microbiota in saliva of children demonstrated statistical difference between CF or ECC and S-ECC children only for mutans streptococci (MS) counts. S-ECC children were heavily colonized by MS. There was a gradual increase in the MS count with respect to the severity of the disease (Table 1). Considering the total population, positive correlations between salivary IgA levels to GbpB and MS counts were found (Figure 1). For these reasons, children were paired according to levels of MS within each group. Considering the mean of MS counts of the population, the groups of children (CF, ECC and S-ECC) were subdivided as follows: LMS - low mutans streptococci count (with MS counts ≤ log 4 CFU/ml) and HMS - high mutans streptococci count (with MS counts ≥ log 5 CFU/ml). Comparing LMS and HMS, there was no significant Natália Helena Colombo 31 difference among the groups in relation to total IgA levels (Table 2). The levels of IgA antibody reactive with GbpB were lower in S-ECC compared to ECC and CF children, only for HMS (Figure 2). IgA antibody levels to GbpB increased for all groups, when compared with the same groups of children (CF, ECC and S-ECC) with LMS and HMS counts (Figure 2). However, S-ECC children with high mutans streptococci counts showed a significant decrease in IgA antibody levels to GbpB compared to the caries free group with HMS. No statistically significant differences were observed comparing ECC with the other groups with HMS (Figure 2). DISCUSSION Early childhood caries has a complex etiology with biological, behavioral, and socioeconomic influences (Arora et al., 2011, Ng & Chase, 2013). In this study, various factors determining oral health were analyzed. S-ECC children had a reduced family income compared to CF and ECC groups. This result is in accordance with the study of Oliveira et al. (2008) that demonstrated greater prevalence of dental caries in children with adverse socio-economic conditions and in children whose mothers had less than 8 years of education. However, our results showed no difference among groups in relation to mother's education level. These findings corroborated with the study of Parisotto et al. (2010a) that found no difference in mother’s education level between CF and caries group. This lack of difference probably occurred because children were selected from the schools with similar social characteristics. A strong association has been reported between high frequency of sugar exposure and occurrence of dental caries (Parisotto et al., 2010a, Kalsbeek & Verrips, 1994, Milgrom et al., 2000). However, the present study did not find a significant difference in sugar exposure among the groups. Our results are according to Ohlund et al. (2007) who found that caries experience was not correlated with intake frequency or total intake of sugary foods. One possible explanation for this finding in the present study is that the responses from questionnaires may have reflected present, not historical experience, which would have better revealed habits during the period when caries had started. Several previous studies found mutans streptococcus is a significant factor for the presence of ECC (Ohlund et al., 2007, Ge et al., 2008, Milgrom et al., 2000). Our Natália Helena Colombo 32 results showed that S-ECC children were highly colonized by mutans streptococci compared with CF and ECC children. Children with high level of S. mutans counts have 5 times more risk to develop dental caries than children with a lower level of these microorganisms (Milgrom et al., 2000). A recent systematic review and meta- analysis showed that there is scientific evidence of S. mutans transmission from mother to child especially when the mother is the primary caregiver (da Silva Bastos et al., 2015). Kozai et al. (1999) showed both mother and father can be sources of S. mutans transmission. However, 18% of bacterial strains were from an unknown source. The American Academy of Pediatric Dentistry recommended the reduction of mutans streptococci levels of mothers, primary caregivers and sibling(s), educating them on avoiding saliva-sharing behaviors (e.g., sharing spoons and other utensils, sharing cups, cleaning a dropped pacifier or toy with their mouth), to decrease the child's risk of ECC (American Academy of Pediatric Dentistry, 2008). The literature has shown that a better immune response to oral microorganisms, mainly Streptococcus mutans, may be a protective factor against the development of dental caries (Nogueira et al., 2005, Tao et al., 2005, Davidopoulou et al., 2012). This observation may be valid for both innate and adaptive immune responses. Reduced levels of some antimicrobial peptides, components of innate immune response, are associated with caries in childhood (Tao et al., 2005, Davidopoulou et al., 2012). The action of salivary IgA against specific surface proteins of cariogenic bacteria such as S. mutans has been the subject of many studies (Nogueira et al., 2005, Nogueira et al., 2007, Parisotto et al., 2011), with focus on GbpB and Gtf expression. These proteins may be targets in the development of vaccines against dental caries (Smith & Taubman, 1996, Kim et al., 2011). Smith & Taubman (1996) showed that the immunization of rats with GbpB induces an immune response that interferes with the accumulation of S. mutans and reduce the levels of dental caries. The caries protection resulting from immunization of rats with Gtf was lower than observed after immunization with S. mutans GbpB. The same study showed that saliva of sham-immunized/S. mutans infected rats contained antibody to GbpB in saliva at the end of the experiment, indicating that infection with S. mutans alone can induce an immune response to this antigen. Furthermore, Parisotto et al. (2011) showed that preschoolers with a lower baseline level of salivary IgA antibody reactive with GbpB had 7.5 higher risk to develop caries, but the study did not find differences between groups CF and ECC in Natália Helena Colombo 33 relation to salivary IgA against S. mutans antigens (GbpB, Gtf). Our results are in accordance with this study, we did not find a difference in IgA antibody levels against GbpB between CF and ECC children, regardless of their mutans streptococci levels. We only found a difference in anti-GbpB antibodies between CF and S-ECC children (with HMS), suggesting that reduced levels of IgA against GbpB may be related to severity of dental caries. Although IgA levels against GbpB were lower in the S-ECC (HMS) group, total salivary IgA levels did not differ between groups. Thus, heavily- infected S-ECC children have a reduced immune response, and this immunological failure could have contributed to the severity of caries status. Bolton & Hlava (1982) demonstrated that salivary IgA levels to S. mutans antigens were higher in a caries- free group than in caries group, a difference that persisted in children from 3 to 11 years old. A recent study comparing specific IgA levels in three-year old children revealed increased concentrations of anti-S.mutans IgA and anti-S. sanguinis IgA in children who were culture positive for S. mutans compared with those who were culture negative (Malcolm et al., 2014). Nogueira et al., (2005) showed that salivary IgA response to GbpB was often associated with a delay in infection with S. mutans, and this response may occur during the first year of life. The same group found that children infected with S. mutans showed a delay in the immune response to the S. mutans GbpB antigen (Nogueira et al., 2007). These studies paired children according to mutans streptococci infection and they were divided in two groups: infected or non-infected children. In the present study, children were paired according to the mutans streptococci (MS) counts (CFU/ml) because a positive correlation was observed for this variable and IgA response to S. mutans GbpB, for the total population. It is expected that with the increasing of bacterial infection, higher host immune response to pathogen will occur (Nogueira et al., 2007). The importance of GbpB for S. mutans viability has been studied by several investigators (Fujita et al., 2007, Matsumoto-Nakano et al., 2007, Duque et al., 2011). Using a GbpB-deficient mutant strain, authors suggested that GbpB may have an important role in cell-wall construction, as well as in the cell separation and cell-wall maintenance in S. mutans, similar to murein hydrolases (Fujita et al., 2007). Furthermore, a GbpB-deficient mutant was more sensitive to acid pH in the acid killing assays (Matsumoto-Nakano et al., 2007), had decreased autolysis, increased cell hydrophobicity, and increased sensitivity to antibiotics and osmotic and oxidative Natália Helena Colombo 34 stresses (Duque et al., 2011). These functions, associated with binding of S. mutans to glucans deposited on tooth surfaces, highlight the important role of GbpB in the biofilm formation and survival of S. mutans in the oral cavity (Matsumoto-Nakano et al., 2007). Thus a good strategy to control dental caries could be the interference in the virulence factors of S. mutans, such as GbpB, by means the development of vaccines to reduce its cariogenicity (Smith & Mattos-Graner, 2008). In conclusion, this study suggests that children with severe early childhood caries and high levels of mutans streptococci have reduced salivary IgA response to S. mutans GbpB showing that this parameter may influence the severity of caries status. Furthermore, a reduced family income and high levels of SM was associated with S-ECC. ACKNOWLEDGEMENTS This study was supported by grants (2012/19235-5; 2013/12167-7) from São Paulo Research Foundation (FAPESP), São Paulo, SP, Brazil and Coordination for the Improvement of Higher Education Personnel (CAPES). REFERENCES American Academy of Pediatric Dentistry, (2008) Policy on early childhood caries (ECC): unique challenges and treatment option. Pediatr Dent 30: 44-46. Arora, A., E. Schwarz & A.S. Blinkhorn, (2011) Risk factors for early childhood caries in disadvantaged populations. J Investig Clin Dent 2: 223-228. Bolton, R.W. & G.L. Hlava, (1982) Evaluation of salivary IgA antibodies to cariogenic microorganisms in children. Correlation with dental caries activity. J Dent Res 61: 1225-1228. da Silva Bastos, V.A., L.B. Freitas-Fernandes, T.K. Fidalgo, C. Martins, C.T. Mattos, I.P. de Souza & L.C. Maia, (2015) Mother-to-child transmission of Streptococcus mutans: A systematic review and meta-analysis. J Dent 43: 181-191. Davidopoulou, S., E. Diza, G. Menexes & S. Kalfas, (2012) Salivary concentration of the antimicrobial peptide LL-37 in children. Arch Oral Biol 57: 865-869. Duque, C., R.N. Stipp, B. Wang, D.J. Smith, J.F. Höfling, H.K. Kuramitsu, M.J. Duncan & R.O. Mattos-Graner, (2011) Downregulation of GbpB, a component of the VicRK regulon, affects biofilm formation and cell surface characteristics of Streptococcus mutans. Infect Immun 79: 786-796. Fujita, K., M. Matsumoto-Nakano, S. Inagaki & T. Ooshima, (2007) Biological functions of glucan-binding protein B of Streptococcus mutans. Oral Microbiol Immunol 22: 289- 292. Ge, Y., P.W. Caufield, G.S. Fisch & Y. Li, (2008) Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children. Caries Res 42: 444-448. Natália Helena Colombo 35 Isaksson, H., A. Alm, G. Koch, D. Birkhed & L.K. Wendt, (2013) Caries prevalence in Swedish 20-year-olds in relation to their previous caries experience. Caries Res 47: 234-242. Jespersgaard, C., G. Hajishengallis, Y. Huang, M.W. Russell, D.J. Smith & S.M. Michalek, (1999) Protective immunity against Streptococcus mutans infection in mice after intranasal immunization with the glucan-binding region of S. mutans glucosyltransferase. Infect Immun 67: 6543-6549. Kalsbeek, H. & G.H. Verrips, (1994) Consumption of sweet snacks and caries experience of primary school children. Caries Res 28: 477-483. Kim, M.A., Y.M. Yang, Y.R. So, Y.H. Ko, S.M. Lim, K.Y. Lee & J.G. Kim, (2011) Development of a monoclonal antibody against glucosyltransferase D of Streptococcus mutans GS 5. Hybridoma (Larchmt) 30: 375-380. Koga, T., T. Oho, Y. Shimazaki & Y. Nakano, (2002) Immunization against dental caries. Vaccine 20: 2027-2044. Kozai, K., R. Nakayama, U. Tedjosasongko, S. Kuwahara, J. Suzuki, M. Okada & N. Nagasaka, (1999) Intrafamilial distribution of mutans streptococci in Japanese families and possibility of father-to-child transmission. Microbiol Immunol 43: 99-106. Law, V., W.K. Seow & G. Townsend, (2007) Factors influencing oral colonization of mutans streptococci in young children. Aust Dent J 52: 93-100; quiz 159. Loesche, W.J., (1996) Microbiology of Dental Decay and Periodontal Disease. In: Medical Microbiology. e. Baron S (ed). Galveston (TX): University of Texas Medical Branch at Galveston, pp. Malcolm, J., A. Sherriff, D.F. Lappin, G. Ramage, D.I. Conway, L.M. Macpherson & S. Culshaw, (2014) Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque. Mol Oral Microbiol. Matsumoto-Nakano, M., K. Fujita & T. Ooshima, (2007) Comparison of glucan-binding proteins in cariogenicity of Streptococcus mutans. Oral Microbiol Immunol 22: 30-35. Milgrom, P., C.A. Riedy, P. Weinstein, A.C. Tanner, L. Manibusan & J. Bruss, (2000) Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children. Community Dent Oral Epidemiol 28: 295-306. Ng, M.W. & I. Chase, (2013) Early childhood caries: risk-based disease prevention and management. Dent Clin North Am 57: 1-16. Nogueira, R.D., A.C. Alves, W.F. King, R.B. Gonçalves, J.F. Höfling, D.J. Smith & R.O. Mattos-Graner, (2007) Age-specific salivary immunoglobulin A response to Streptococcus mutans GbpB. Clin Vaccine Immunol 14: 804-807. Nogueira, R.D., A.C. Alves, M.H. Napimoga, D.J. Smith & R.O. Mattos-Graner, (2005) Characterization of salivary immunoglobulin A responses in children heavily exposed to the oral bacterium Streptococcus mutans: influence of specific antigen recognition in infection. Infect Immun 73: 5675-5684. Ohlund, I., P.L. Holgerson, B. Backman, T. Lind, O. Hernell & I. Johansson, (2007) Diet intake and caries prevalence in four-year-old children living in a low-prevalence country. Caries Res 41: 26-33. Oliveira, L.B., A. Sheiham & M. Bönecker, (2008) Exploring the association of dental caries with social factors and nutritional status in Brazilian preschool children. Eur J Oral Sci 116: 37-43. Parisotto, T.M., W.F. King, C. Duque, R.O. Mattos-Graner, C. Steiner-Oliveira, M. Nobre- Dos-Santos & D.J. Smith, (2011) Immunological and microbiologic changes during caries development in young children. Caries Res 45: 377-385. Parisotto, T.M., C. Steiner-Oliveira, C. Duque, R.C. Peres, L.K. Rodrigues & M. Nobre-dos- Santos, (2010a) Relationship among microbiological composition and presence of dental plaque, sugar exposure, social factors and different stages of early childhood caries. Arch Oral Biol 55: 365-373. Parisotto, T.M., C. Steiner-Oliveira, C.M. Silva, L.K. Rodrigues & M. Nobre-dos-Santos, (2010b) Early childhood caries and mutans streptococci: a systematic review. Oral Health Prev Dent 8: 59-70. Natália Helena Colombo 36 Smith, D.J., (2002) Dental caries vaccines: prospects and concerns. Crit Rev Oral Biol Med 13: 335-349. Smith, D.J. & R.O. Mattos-Graner, (2008) Secretory immunity following mutans streptococcal infection or immunization. Curr Top Microbiol Immunol 319: 131-156. Smith, D.J. & M.A. Taubman, (1987) Oral immunization of humans with Streptococcus sobrinus glucosyltransferase. Infect Immun 55: 2562-2569. Smith, D.J. & M.A. Taubman, (1996) Experimental immunization of rats with a Streptococcus mutans 59-kilodalton glucan-binding protein protects against dental caries. Infect Immun 64: 3069-3073. Tao, R., R.J. Jurevic, K.K. Coulton, M.T. Tsutsui, M.C. Roberts, J.R. Kimball, N. Wells, J. Berndt & B.A. Dale, (2005) Salivary antimicrobial peptide expression and dental caries experience in children. Antimicrob Agents Chemother 49: 3883-3888. Natália Helena Colombo 37 Table 1 Comparative analysis between the severity of early childhood caries and related etiological factors. CF ECC S-ECC p value Dmfs (mean±SD) 0a 2 ± 1.06b 23.43 ± 17.17c 0.00 Age (months) Mean±SD 46.26 ± 5.05a 45.94 ± 9.83a 48.10 ± 8.59a 0.664 Gender (%) Female 47.37A 35.29A 52.38A 0.571 Male 52.63 64.71 47.62 Family income per month ≤ R$ 1448.00* 41.1A 25B 64.71C 0.05 Mother's education (%) Up to 8 years 37.50A 35.71A 47.62A 0.737 Mother’s help with tooth brushing (%) 82.35A 80.0A 65.0A 0.961 Bottle feeding (%) 82.36A 66.67A 85A 0.692 Sugar intake (mean±SD) liquid 4.21 ± 1.37a 4.02 ± 1.18a 4.56 ± 1.38a 0.465 solid 2.70 ± 0.90a 3.17 ± 1.43a 2.97 ± 1.40a 0.565 total 6.88 ± 1.62a 7.19 ± 1.66a 7.53 ± 2.32a 0.579 mutans streptococci median (range) (log UFC+1) 3.35 (0 - 7.29)A 3.74 (0 - 6.66)A 5.63 (3.15 - 8.3)B 0.012 total lactobacilli median(range) (log UFC+1) 0 (0 - 6.79)A 3.30 (0 - 6.47)A 1.97 (0 - 6.72)A 0.447 Total IgA Median (range) 99.15(24.52- 114.24)A 83.24 (32.27- 114.56)A 88.04 (18.17- 109.31)A 0.125 a Different lower case letters show statistical difference among the groups, according to ANOVA and Tukey tests. A Different upper case letters show statistical difference among the groups, according to Kruskal-Wallis and Mann-Whitney tests. CF – caries free, ECC – early childhood caries, S-ECC – severe early childhood caries * R$ - Brazilian real. 1 US$ ~ R$ 2.70 (2015, January). Natália Helena Colombo 38 Fig. 1 Scatter plot showing correlation between levels of IgA antibody reactive with GbpB and mutans streptococci (MS). The analysis was carried out for the total population, regardless the caries status. Positive Pearson correlation (R=0.583, p=0.00) was observed for MS counts. Table 2. Medians (range) of total salivary IgA, distributed according to mutans streptococci levels. MS levels*# LMS HMS p value CF 101.9 (24.52-114.24) 91.05 (60.04-107.43) 0.375 ECC 81.93 (32.27-107.43) 84.55 (48.02-114.56) 0.341 S-ECC 98.92 (18.17-104.14) 84.10 (26.01-109.31) 0.357 p value 0.380 0.243 * There was no statistical difference among groups of children (columns), considering each MS levels separately (LMS or HMS), using Kruskal-Wallis tests. # There was no statistical difference inside each group of children (rows), comparing MS levels (LMS x HMS), using Mann-Whitney tests. Natália Helena Colombo 39 Fig. 2 Box plots of the western blot reactivity of salivary IgA antibody with S. mutans GbpB among the groups of children, distributed according to caries status (CF, ECC and S-ECC) and mutans streptococci levels (LMS and HMS). Bars indicate minimum and maximum values. Black and white boxes indicate lower and upper quartiles, respectively. Line in the middle of boxes is median. A - Different upper case letters show statistical difference among groups of children considering each MS level separately (LMS or HMS), according to Mann-Whitney tests. For example: GbpB CF (LMS) x GbpB ECC (LMS) a - Different lower case letters show statistical difference inside each group of children, comparing MS level (LMS x HMS), according to Mann-Whitney tests. For example: GbpB CF (LMS) x GbpB CF (HMS). CF – caries free, ECC – early childhood caries, S-ECC – severe early childhood caries. LMS- Low mutans streptococcus levels HMS – High mutans streptococcus levels Natália Helena Colombo Capítulo 2 Natália Helena Colombo 41 Salivary levels of the antimicrobial peptides in children with severe early childhood caries Natália H. Colombo1, Laís F. F. Ribas1 Jesse A. Pereira1 Paula F. Kreling1 Christine A. Kressirer2,3 Anne C. R.Tanner2,3 Cristiane Duque1* 1 Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, University Estadual Paulista (UNESP), Araçatuba, SP, Brazil 2 Department of Microbiology, The Forsyth Institute, Cambridge, MA, USA 3 Harvard School of Dental Medicine, Harvard University, Boston, MA, USA Running head: Antimicrobial peptides and severe early childhood caries Correspondence: Dr. Cristiane Duque, Department of Pediatric Dentistry and Public Health, Araçatuba Dental School, Univ Estadual Paulista (UNESP) Address: R. José Bonifácio, 1193, CEP: 16015-050, Araçatuba-SP, Brazil Tel: (+55) 1836363315 E-mail: cristianeduque@yahoo.com.br, cduque@foa.unesp.br * According to guidelines for authors of Archives of Oral Biology (Anexo E). mailto:cristianeduque@yahoo.com.br, mailto:cduque@foa.unesp.br Natália Helena Colombo 42 Abstract Objective: Controversies are still exist regard the relationship between the concentrations of some antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine AMPs levels in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S- ECC) children, in order to determine if the levels of these salivary peptides isolated or combined could be related to caries severity and mutans streptococci levels. Design: 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n=29), ECC group (n=25) and S-ECC group (n=29). Saliva was collected from children for microbiological analysis by culture. Salivary concentration of cathelicidin LL-37, Human β-defensin 2 (hBD-2), Human β-defensin 3 (hBD-3) and Human-Histatin 5 (HTN-5) were determined by ELISA. Results: Salivary concentration of AMPs did not differ among CF, ECC and S-ECC groups. The present study showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlation also was found among hBD- 2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly with LL-37, were positively correlated with caries levels. Conclusions: Salivary concentrations of AMPs separately are not associated with the severity of early childhood caries. The stimulus of caries can trigger a biological response with a combined action of these peptides. Keywords: human cathelicidin LL-37, defensins, histatin-5, children, innate immunity, dental caries Natália Helena Colombo 43 Introduction Antimicrobial peptides (AMPs) constitute an important class of molecules belonging to the innate immune system, which participates in the first line defence reactions at various sites of the human body (1). AMPs are produced by epithelial tissues, phagocytic cells (2), parotid gland, gingival and lateral tongue tissue (3) and exhibit a broad spectrum of activity against bacteria, fungi, and viruses as well as chemotactic activities and induction of cytokine release (1). In the oral cavity, AMPs are present in saliva, gingival epithelia and gingival crevicular fluid (4). Among AMPs, cationic peptides, especially α- and β-defensins, human cathelicidin LL-37 and histatins, have been highlighted in the literature by their typical bactericidal and /or bacteriostatic activities against oral pathogens (5). Defensins (α- and β) are peptides with three disulfide bonds which differ from each other in the spacing and the pairing of the cysteine residues. Alpha-defensins are expressed in neutrophils and have been commonly identified in the gingival crevicular fluid. Beta-defensins (hBD1, hBD-2 and hBD-3) are expressed in epithelial cells of the oral cavity and are found in the gingival crevicular fluid and in the saliva. LL-37 is a long cationic alpha-helical peptide from human cathelicidin CAP18, expressed in neutrophils and epithelial cells and consequently present in saliva and gingival crevicular fluid. Histatins are expressed in salivary glands and found in saliva. Histatin 5 is derived from histatin 3 (1, 4, 5). Histatin 5 showed potent inhibition against C. albicans growth and bacterial co-aggregation (6). Ouhara et al.(7) evaluated antimicrobial activity of defensins and LL-37 and found that six Gram-positive bacteria, oral streptococci and L. casei, showed similar susceptibility to these peptides. Except for hBD1, all peptides demonstrated nearly 100% bactericidal activity with concentrations less than 10 mg/l. Some authors have suggested that the reduced concentration of some peptides could be associated with the presence of caries in children (8-10). The level of α-defensin (HNP1-3) was shown to be lower in saliva from caries-active children compared to caries-free children (8). The same was observed for the salivary levels of LL-37 in another study (9). Although the antimicrobial properties of β-defensins are proven, the literature does not show changes in the salivary concentrations of these peptides in caries-active compared to caries-free children (10). No studies were found revealing the salivary concentrations of histatin-5 in children yet. Natália Helena Colombo 44 Dental caries is an infectious disease that cause the dissolution of tooth mineral by acids derived from bacterial fermentation of dietary carbohydrates (11). When tooth decay affects children less than 71 months this disease is called early childhood caries (ECC) (12). If the disease progression is not interrupted, it may cause the destruction of several deciduous teeth, denominated as severe ECC (S- ECC), resulting in local, systemic, psychological and social consequences (13). Limited information can be found about the salivary antimicrobial peptides concentration in children and most of them evaluated children with mixed or permanent dentition. Since the salivary level of AMPs may contribute to caries susceptibility, these peptides could be a new and useful measure of the risk for caries in children. Thus, we aimed to examine the levels of hBD-2, hBD-3, LL-37 and HTN-5 in saliva of caries-free, ECC and S-ECC children, in order to determine if the levels of these salivary peptides could be related to caries severity. Furthermore, this study aimed to explore the associations among the severity of ECC and caries- related etiological factors: socio-economic-cultural aspects, dietary habits, mutans streptococci/lactobacilli colonization. Material and methods Subjects The study population included 36- to 60-month-old children who attended the four public nursery schools in the city of Araçatuba, São Paulo, Brazil. The city´s population has access to public water supply with fluoride level of 0.7 ppm. Children’s parents as well as the preschools involved granted written permission for the study which was previously approved by the Research Ethics Committee of Univ. Estadual Paulista (UNESP), Brazil (Certificate of Presentation for Ethical Consideration (CAAE) # 13079213.4.0000.5420). Questionnaires were applied to the parents to assess socio-economic-cultural data and oral hygiene habits. The socio-economic- cultural data analyzed were family income and mother’s education. Dietary data were obtained from a food-frequency diary filled out for three consecutive days during the workweek. The diet chart was filled during the workweek because in the weekend the diet can be modified. In addition to the diet data reported by parents, the preschools informed infant feeding in the school period, and many of them were in school full time (14). Clinical examination was carried out at the school by a single calibrated Natália Helena Colombo 45 examiner using mouth mirror and probe under natural light. Children suffering from systemic diseases, or using long-term medication or antibiotics less than one month before the examination and children with mucosal breaks were excluded from the study. Children with only presented filling or absent teeth were also excluded. Initially ninety children from both genders, 36-60 month-age were selected to participate to this study. Seven of the selected children did not attend for saliva collection. So, eighty-three children were divided into three groups according to oral health: caries- free group (CF) (n=29), early childhood caries group (ECC) (n=25) and severe early childhood caries group (S-ECC) (n=29). ECC was defined for this study as the presence of 1 through 3 decayed tooth surface (cavitated lesions), S-ECC was defined as the presence of decayed surfaces score of ≥ 4 (age 3 years), ≥ 5 (age 4 years), must also have at least one smooth-surface caries surface (15). Children were encouraged and instructed on dental hygiene and received all other necessary oral care. Saliva samples Unstimulated whole saliva was collected from each subject into a 50-ml sterile falcon conical tube for 5-10 min. Collections were performed at least 1 h after feeding to avoid contamination with no salivary components. Tubes were transported on ice to laboratory and processed within 1 h. After agitation, one hundred microliter of saliva was separated for microbiological procedures. The remaining saliva was clarified by centrifugation at 10000 rpm at 4oC for 10 min. The supernatants were collected and 250 mM EDTA was added to minimize salivary protein aggregation. Aliquots of 100µl of each saliva samples were frozen at −70°C until immunological use. The aliquots was frozen at −70°C until use for antimicrobial peptides level measurement by ELISA Microbiological procedures Aliquots of saliva were homogenized by vortexing for 1min and the suspensions were serially diluted (10−1 to 10−7) in 0.9% saline solution. Each dilution was cultivated in triplicate on the surface of two selective media: Mitis Salivaris Agar (Difco Laboratories, Detroit, MI, USA) with sucrose and bacitracin for isolation of mutans streptococci and Rogosa agar (Oxoid, Basingstoke, Hampshire, England) for lactobacilli. All plates were incubated at 37°C for 48 h in 5% CO2 atmosphere. After 48h of incubation, the total number of colony-forming units (CFU) was counted from a Natália Helena Colombo 46 representative area of each agar plate yielding 30–300 colonies using a stereoscopic microscope and the results were expressed as CFU/ml. Antimicrobial Peptides Level Measurement The concentration of antimicrobial peptides was determined by enzyme-linked immunosorbent assay (ELISA) using specific kits and according the manufacturer’s instructions. LL-37, Human β-defensin 2 (hBD-2) and Human histatin-5 (HTN-5) were purchased from MyBioSource Inc. (San Diego, CA, USA) and Human β-defensin 3 (hBD-3) from Assay Biotechnology Company Inc. (Sunnyvale, CA, USA). Statistical analysis The statistical analysis was performed considering three groups of children according to dental caries status (CF, ECC and S-ECC) as the dependent variables. The comparisons among the groups were performed according to data distribution. ANOVA/Tukey tests were applied for caries levels (dmfs), age, sugar intake. Kruskal- Wallis/Mann-Whitey tests were applied for gender, mother´s education level, adult’s help with tooth brushing, family income, breastfeeding, bottle feeding, mutans streptococci/lactobacilli counts and salivary levels of AMPs. Data were tested using Pearson or Spearman correlation tests. RESULTS The results did not show gender difference among the groups of children (Table 1) and neither gender difference in relation to the concentrations of salivary AMPs (Kruskal-Wallis test, p = 0.393). S-ECC presented a reduced family income compared to CF children. However, in this present study no significant difference in maternal education was found between groups. Sugar intake did not differ among the groups, but ECC and S-ECC children were breastfed for longer than CF children (Table 1). LL-37, hBD-2, hBD-3 and HTN-5 were detected in all saliva samples. Salivary concentration of AMPs does not differ among CF, ECC and S-ECC groups (Figure 1). The present study showed positive correlations between salivary hBD-2 and HTN- 5 with salivary MS levels and there was no relationship between MS levels and other AMPs tested (Figure 2). Lactobacilli counts could not be correlated with AMP levels Natália Helena Colombo 47 because of high number of children with counting zero. Other positive correlation was found between dmfs and LL-37 and dmfs and hBD-2 (Table 2). A combination among hBD-2, hBD-3, LL-37 and HTN-5 was correlated with dmfs too (Table 2). Positive correlation also was found among hBD-2, hBD-3 and LL-37. HTN-5 was only correlated with LL-37 and hBD-2 (Figure 3). DISCUSSION The present study shows that there was positive correlation between hBD-2 and HTN-5 with salivary mutans streptococci levels (Figure 2). Other positive correlation found was between hBD-2 and LL-37 levels and dmfs. This correlation was not found for hBD-3 and HTN-5 (Table 2). These results confirmed that innate immune system of children, represented by antimicrobial peptides, reacted to S. mutans aggression and their production is increased in the presence of dental caries. In addition, results showed a positive correlation between among hBD-2, hBD-3, LL- 37 and HTN-5 (except for the combination of HTN-5 and hBD-3), suggesting a combined action of these peptides in host protection. Malcolm et al. (16) showed that salivary concentrations of AMPs (hNP1–3 and LL-37) in 3-year-old children were positively correlated with detection of S. mutans by qPCR. Although the present study has shown that there was a positive correlation between some AMPs e dmfs, the results demonstrated that salivary concentration of any AMP studied differ among CF group, ECC group and S-ECC group. Our results are in agreement with the study of Tao et al.(8) and Phattarataratip et al. (10). Tao et al. (8) studied children between 11 and 15 years of age and showed no significant differences in LL-37 and hBD-3 levels in saliva of caries-free compared to caries-active subjects. Phattarataratip et al.(10) studied children with 13 years old and did not find statistically significant differences between salivary levels of LL-37, hBD-2, hBD-3 and hNP1-3 between caries-free and caries-active subjects. In contrast, there are studies showing that salivary concentration of hNP1-3 (8) and LL-37 (9) are lower in saliva from caries- active compared to caries-free children. Few studies have associated salivary peptides and caries experience and the majority of the available information in the literature refers to older children with mixed and permanent dentition (8-10). A recent study compared AMPs and bacteria- specific IgA levels in younger children (1-3 years old) and revealed higher Natália Helena Colombo 48 concentrations of hNPs 1–3, LL-37, anti- S. mutans IgA and anti-S. sanguinis IgA in children who had positive culture for mutans streptococci compared with those who had negative culture (16). In this present study, few children had negative culture for mutans streptococci making impossible the statistical analysis of the concentration of AMPs over this variable. Ribeiro et al. (17) evaluated protein composition of saliva from caries free children and children with caries experience (10-71 months age). Identification of molecular masses (chromatograms) suggested the presence of nine peptides, but only three of them were related to caries experience. The suggestive presence of α- defensin-3 and β-defensin-3 reduced the chances of caries experience, and the presence of proline-rich peptides IB-4 expressed a positive association with dental caries. The methodology (chromatograms) used in aforementioned study does not provide the concentrations of salivary peptides, only reveals its presence or absence. This present study provides the first investigation of salivary concentration of histatin 5 in children. The results showed that the salivary concentration of AMP in children is lower compared to that found in adults in the literature (18, 19). This difference was expected since the immune system is immature in young children. Johnson and co-workers (18) analyzed adults and elderly people and showed significant age-associated decrease for salivary histatin concentration. The authors suggested that the salivary concentration of histatin is compromised with the increasing of the age. Histatins are best known for their antifungal activity and the most of studies have concentrated on this aspect. Histatin 5 showed potent antifungal properties including inhibition of growth of C. albicans (6). Candida albicans is not only involved in fungal infections such as candidiasis. Several studies have demonstrated that this yeast presented a cariogenic potential (20-22). There is a significant association between the presence of C. albicans and early childhood caries. Carvalho et al. (23) showed that C. albicans was significantly more prevalent in children with early childhood caries than in caries-free group. Considering this information and anti-Candida activity presented by Histatin-5 we expected that the concentration of this peptide could be reduced in children with early childhood caries. However, no difference among the groups was identified. These data corroborate with the study of Dodds et al. (24) that evaluated a population of caries-active and caries-free young adults and no difference between the groups was found considering the histatin concentration from parotid saliva. In contrast, a recent study Natália Helena Colombo 49 showed increased levels of salivary histatin 5 in young adults (18 years old) with high caries activity compared to subjects with low caries activity (25) The literature has showed that caries activity may not be associated with salivary AMPs concentration, but associated with virulence characteristics of S. mutans strains identified in caries active children and to be more resistant to AMPs. S. mutans strains isolated from caries-active subjects showed greater resistance to salivary hNP-1-2, hBD-2, hBD-3 and LL-37 than those from caries-free subjects (10). Furthermore, S. mutans strains isolated from children with S-ECC have higher sucrose-dependent adhesion, water-insoluble glucan synthesis and gene expression levels of gtfB and gtfC than S. mutans strains isolated from caries-free children (26). These results showed that S. mutans strains from children with S-ECC can be more virulent than those found in caries-free children. Early childhood caries is multifactorial disease caused by oral bacteria and influenced by various social and behavioral factors (27-29). In the present study S- ECC group presented a reduced family income compared to CF group. This result is in accordance with study of Oliveira et al (30) and Corrêa-Faria et al (29) that demonstrated higher prevalence of dental caries in children with low-income family. Oliveira et al (30) also reported that children whose mothers had less than 8 years of education presented the highest prevalence of dental caries. However, in this present study no significant difference in maternal education was found between groups. High frequency of sugar exposure is a dietary habit strongly associated with dental caries (14, 31, 32). However, the present study did not found significant difference in sugar intake among the groups. Our results corroborate with the results obtained by Köhler et al (33) who demonstrated that caries experience was not correlated with sugar exposure. In the present study, the lack of difference among the groups in relation to sugar intake may be related to the time that the questionnaire was applied to the parents. The answers of parents about sugar intake reflected the present time, not historical experience, which would have better revealed habits during the period when caries had started. Moreover, information obtained from parents may be biased. Parents of children with ECC, who have some knowledge about the risk of sugary foods consumption in the development of dental caries, may not be ready to admit that they practiced habits that can increase the caries risk. Natália Helena Colombo 50 Despite eating habits are not different among the groups at the present, the question about breastfeeding habits revealed that ECC and S-ECC children were breastfed for longer than CF children. No statistical difference among the groups was observed for the bottle feeding time. This result provides evidence of the association of prolonged breastfeeding and early childhood caries. Our results are in accordance with study of Azevedo et al. (34) that demonstrated that breastfeeding after 1 year of age was associated with ECC. Kato et al (35), conducted a longitudinal study with more than 43,000 babies and found association between breastfeeding for at least 6 or 7 months and risk of dental caries at age 30 months. Prolonged breastfeeding may be associated with ECC because many mothers know the benefits of breast milk, such as the presence of antibodies (secretory IgA) for baby protection against a variety of diseases, but they often unaware that it also may cause tooth decay, and thus neglect oral hygiene of their babies after breastfeeding. Our results showed that S-ECC children had higher mutans streptococci levels in saliva and biofilm in relation CF and ECC children. High levels of mutans streptococci (MS) in the saliva of children, especially S. mutans, are considered a significant risk factor for the presence of ECC (32, 33, 36). Children with a high level of MS have 5 times more risk to develop dental caries than children with a lower level (32). The prediction of caries risk has been of long-standing interest and is very important for development of new strategies for caries prevention. This is especially significant for young children. In this present study, among caries-related etiological factors analyzed, a reduced family income and prolonged period of breastfeeding were associated with S-ECC. However, our results do not support the measurement of salivary levels of LL-37, hBD-2, hBD-3 and Histatin 5 as a reliable tool to predict caries risk. Thus, salivary concentrations of these AMPs are not associated with severity of early childhood caries. ACKNOWLEDGEMENTS This study was supported by grants (2012/ 19235-5) from the São Paulo Research Foundation (FAPESP), São Paulo, SP, Brazil and CAPES. Natália Helena Colombo 51 REFERENCES 1. Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3(9):710-720. 2. Zasloff M. Innate immunity, antimicrobial peptides, and protection of the oral cavity. Lancet 2002;360(9340):1116-1117. 3. Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, et al. Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun 1999;67(6):2740-2745. 4. Joly S, Maze C, McCray PB, Guthmiller JM. Human beta-defensins 2 and 3 demonstrate strain-selective activity against oral microorganisms. J Clin Microbiol 2004;42(3):1024-1029. 5. Gorr SU. Antimicrobial peptides of the oral cavity. Periodontol 2000 2009;51:152-180. 6. Oppenheim FG, Yang YC, Diamond RD, Hyslop D, Offner GD, Troxler RF. The primary structure and functional characterization of the neutral histidine-rich polypeptide from human parotid secretion. J Biol Chem 1986;261(3):1177-1182. 7. Ouhara K, Komatsuzawa H, Yamada S, Shiba H, Fujiwara T, Ohara M, et al. Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL-37, produced by human epithelial cells. J Antimicrob Chemother 2005;55(6):888-896. 8. Tao R, Jurevic RJ, Coulton KK, Tsutsui MT, Roberts MC, Kimball JR, et al. Salivary antimicrobial peptide expression and dental caries experience in children. Antimicrob Agents Chemother 2005;49(9):3883-3888. 9. Davidopoulou S, Diza E, Menexes G, Kalfas S. Salivary concentration of the antimicrobial peptide LL-37 in children. Arch Oral Biol 2012;57(7):865-869. 10. Phattarataratip E, Olson B, Broffitt B, Qian F, Brogden KA, Drake DR, et al. Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host antimicrobial peptides. Mol Oral Microbiol 2011;26(3):187- 199. 11. Loesche WJ. Microbiology of Dental Decay and Periodontal Disease. In: Baron S e, editor. Medical Microbiology, 4th edition ed. Galveston (TX): University of Texas Medical Branch at Galveston, 1996. Natália Helena Colombo 52 12. Dentistry AAoP, Pediatrics AAo, Affairs AAoPDCoC. Policy on early childhood caries (ECC): classifications, consequences, and preventive strategies. Pediatr Dent 2005;27(7 Suppl):31-33. 13. Losso EM, Tavares MC, Silva JY, Urban CeA. Severe early childhood caries: an integral approach. J Pediatr (Rio J) 2009;85(4):295-300. 14. Parisotto TM, Steiner-Oliveira C, Duque C, Peres RC, Rodrigues LK, Nobre- dos-Santos M. Relationship among microbiological composition and presence of dental plaque, sugar exposure, social factors and different stages of early childhood caries. Arch Oral Biol 2010;55(5):365-373. 15. American Academy of Pediatric Dentistry. Policy on early childhood caries (ECC): unique challenges and treatment option. Pediatr Dent 2008;30(7 Suppl):44- 46. 16. Malcolm J, Sherriff A, Lappin DF, Ramage G, Conway DI, Macpherson LM, et al. Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque. Mol Oral Microbiol 2014. 17. Ribeiro TR, Dria KJ, de Carvalho CB, Monteiro AJ, Fonteles MC, de Moraes Carvalho K, et al. Salivary peptide profile and its association with early childhood caries. Int J Paediatr Dent 2013;23(3):225-234. 18. Johnson DA, Yeh CK, Dodds MW. Effect of donor age on the concentrations of histatins in human parotid and submandibular/sublingual saliva. Arch Oral Biol 2000;45(9):731-740. 19. Torres SR, Garzino-Demo A, Meiller TF, Meeks V, Jabra-Rizk MA. Salivary histatin-5 and oral fungal colonisation in HIV+ individuals. Mycoses 2009;52(1):11-15. 20. Nikawa H, Yamashiro H, Makihira S, Nishimura M, Egusa H, Furukawa M, et al. In vitro cariogenic potential of Candida albicans. Mycoses 2003;46(11-12):471- 478. 21. Jin Y, Samaranayake LP, Samaranayake Y, Yip HK. Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 2004;49(10):789-798. 22. Falsetta ML, Klein MI, Colonne PM, Scott-Anne K, Gregoire S, Pai CH, et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun 2014;82(5):1968-1981. Natália Helena Colombo 53 23. de Carvalho FG, Silva DS, Hebling J, Spolidorio LC, Spolidorio DM. Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch Oral Biol 2006;51(11):1024-1028. 24. Dodds MW, Johnson DA, Mobley CC, Hattaway KM. Parotid saliva protein profiles in caries-free and caries-active adults. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;83(2):244-251. 25. Gornowicz A, Tokajuk G, Bielawska A, Maciorkowska E, Jabłoński R, Wójcicka A, et al. The assessment of sIgA, histatin-5, and lactoperoxidase levels in saliva of adolescents with dental caries. Med Sci Monit 2014;20:1095-1100. 26. Zhao W, Li W, Lin J, Chen Z, Yu D. Effect of sucrose concentration on sucrose-dependent adhesion and glucosyltransferase expression of S. mutans in children with severe early-childhood caries (S-ECC). Nutrients 2014;6(9):3572-3586. 27. Arora A, Schwarz E, Blinkhorn AS. Risk factors for early childhood caries in disadvantaged populations. J Investig Clin Dent 2011;2(4):223-228. 28. Ng MW, Chase I. Early childhood caries: risk-based disease prevention and management. Dent Clin North Am 2013;57(1):1-16. 29. Corrêa-Faria P, Martins-Júnior PA, Vieira-Andrade RG, Marques LS, Ramos- Jorge ML. Factors associated with the development of early childhood caries among Brazilian preschoolers. Braz Oral Res 2013;27(4):356-362. 30. Oliveira LB, Sheiham A, Bönecker M. Exploring the association of dental caries with social factors and nutritional status in Brazilian preschool children. Eur J Oral Sci 2008;116(1):37-43. 31. Kalsbeek H, Verrips GH. Consumption of sweet snacks and caries experience of primary school children. Caries Res 1994;28(6):477-483. 32. Milgrom P, Riedy CA, Weinstein P, Tanner AC, Manibusan L, Bruss J. Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children. Community Dent Oral Epidemiol 2000;28(4):295-306. 33. Ohlund I, Holgerson PL, Backman B, Lind T, Hernell O, Johansson I. Diet intake and caries prevalence in four-year-old children living in a low-prevalence country. Caries Res 2007;41(1):26-33. 34. Azevedo TD, Bezerra AC, de Toledo OA. Feeding habits and severe early childhood caries in Brazilian preschool children. Pediatr Dent 2005;27(1):28-33. Natália Helena Colombo 54 35. Kato T, Yorifuji T, Yamakawa M, Inoue S, Saito K, Doi H, et al. Association of breast feeding with early childhood dental caries: Japanese population-based study. BMJ Open 2015;5(3):e006982. 36. Ge Y, Caufield PW, Fisch GS, Li Y. Streptococcus mutans and Streptococcus sanguinis colonization correlated with caries experience in children. Caries Res 2008;42(6):444-448. Natália Helena Colombo 55 Table 1. Comparative analysis between the severity of early childhood caries and related etiological factors. CF ECC S-ECC p value Dmfs (mean±SD) 0a 2.36 ± 0.95b 19.34 ± 14.97c 0,000 Dmfs + white spot (mean±SD) 0a 2.76 ± 1.23b 24.41 ± 17.94c 0,000 Age in months (mean±SD) 48.31 ± 8.59 49.6 ± 7.78 50.21 ± 9.93 0,664 Gender (%) Female 55.2 44 51.7 0,593 Male 44.8 56 48.3 0,593 Family income per month (%) < R$ 1448.00* 59.3A 82.6A 92.6B 0.016 Mother's education (%) Up to 8 year 20.7 24 31 0,516 Adult’s help with tooth brushing (%) 72.4 68 69 0,851 Sugar exposure (mean±SD) Liquid 4.4 ± 1.62 5.12 ± 3.04 4.96 ± 1.56 0,729 Solid 3.71 ± 1.34 3.64 ± 1.33 3.27 ± 1.80 0,227 Total 8.12 ± 2.20 8.76 ± 3.01 8.23 ± 2.30 0,534 Breast feeding (%) 79.3 88 96.4 0,169 Bottle feeding (%) 96.6 96 92.9 0,524 Breastfeeding duration in months (mean±SD) 5.62 ± 6.68A 13.68 ± 15.77B 17.21 ± 12.80B 0,000 Bottle feeding duration in months (mean±SD) 37.9 ± 15.96 31.67 ± 16.72 31.46 ± 16.77 0,178 Bottle early age (mean±SD) 5.69 ± 5.52A 10.33 ± 9.83B 13.00 ± 10.03B 0,015 Mutans streptococci count (log CFU+1) median (range) saliva 4.08 (0 - 6.62)A 4.05 (0 - 6.41)A 5.08 (0 - 8.30)B 0,034 biofilm 4.08 (0 - 6.23)A 4.08 (0 - 6.10)A 4.71 (0 - 6.72)B 0,026 Lactobaccili count (log CFU+1) median (range) saliva 0 (0 - 7.08) 3 (0 - 6.47) 3.7 (0 - 6.88) 0,335 biofilm 0 (0 - 5.29) 0 (0 - 7.05) 0 (0 - 6.33) 0,887 a Different lower case letters show statistical difference (p<0,05) among the groups, according to ANOVA and Tukey tests. A Different upper case letters show statistical difference (p<0,05) among the groups, according to Kruskal-Wallis and Mann-Whitney tests. CF – caries free, ECC – early childhood caries, S-ECC – severe early childhood caries * R$ - Brazilian real. 1 US$ ~ R$ 2.70 (2015, January). Natália Helena Colombo 56 Figure 1. Levels of antimicrobial peptides (hBD-2, hBD-3, LL-37 and HTN-5) detected in saliva samples of children. *There was no statistical difference (p>0.05) among the groups, considering each AMP separately, according to Kruskal-Wallis tests. A B C D Natália Helena Colombo 57 Figure 2. Only significant correlations are shown. A. Relationship between hBD-2 and salivary mutans streptococci levels (Spearman correlation, r= 0.228, p=0.043). B. Relationship between HTN-5 and salivary mutans streptococci levels (Spearman correlation, r= 0.235, p=0.039). Natália Helena Colombo 58 Figure 3. Only significant correlations are shown. A. Relationship between LL-37 and hBD-2 (Spearman correlation, r= 0.831, p=0.000). B. Relationship between HTN-5 and LL-37 (Spearman correlation, r= 0.765, p=0.000). C. Relationship between hBD- 2 and HTN-5 (Spearman correlation, r= 0.796, p=0.000). D. Relationship between hBD-3 and LL-37 (Spearman correlation, r= 0.188, p=0.053). E. Relationship between hBD-3 and hBD-2 (Spearman correlation, r= 0.193, p=0.048). Natália Helena Colombo 59 Table 2. Relationship between antimicrobial peptides (hBD-2, hBD-3, LL-37 and HTN-5) and your combinations and dmfs. dmfs Pearson Correlation p LL-37 0,297 0,007 hBD-2 0,268 0,015 hBD-2 e LL-37 0,307 0,005 hBD-3 e LL-37 0,293 0,008 LL-37 e HTN-5 0,238 0,031 hBD-2, hBD-3 e LL-37 0,295 0,007 hBD-2, LL-37 e HTN-5 0,24 0,030 hBD-3, LL-37 e HTN-5 0,231 0,037 AMPs 0,232 0,036 Only significant correlations are shown. Natália Helena Colombo 60 Molecular bacterial detection and severity of early childhood caries Natália H. Colombo1 Paula