Infants born prematurely are a vulnerable population with diverse nutritional needs to battle their increased risk of gastrointestinal (GI) diseases. Human milk is considered the 'gold standard' of infant nutrition. Human milk not only provides nutrition for newborn growth, but contains bioactive components which contribute to GI maturation, immune protection and neurological development. Among these bioactive components are extracellular vesicles known as exosomes. Exosomes are double-lipid membrane vesicles containing mRNA, microRNA and proteins, secreted by cells as a form of cell-to-cell communication. Human milk exosomes contain immune-related microRNA and proteins that withstand in vitro simulated human digestion, suggesting that signals are being delivered to the cells residing in the GI tract of a newborn. In premature birth, disruption of GI tract maturation predisposes the infant to increased susceptibility of GI inflammatory diseases. To prevent inflammation, immune tolerance in the GI tract of premature infants should be promoted and I hypothesized that exosomes differ between preterm and term milk, and may contribute to the anti-inflammatory effects of human milk. Human milk exosomes from mothers who gave birth to term or preterm infants were characterized based on size, surface protein markers and total protein. Preterm milk exosomes contained a more diverse protein profile. The effects of milk exosomes on intestinal epithelial cells were observed in an in vitro model using Caco-2/15 cells. Milk exosomes were able to attenuate the inflammatory response induced by heat-killed bacteria as measured by the transcription of pro-inflammatory cytokines.

INTRODUÇÃO: A lactoferrina disponível no leite materno desempenha função imunológica e protege recém-nascidos de infecções por se ligar ao ferro e privá-lo de bactérias patogênicas, o que resulta em atividade bacteriostática contra organismos patogênicos ferro dependentes. A utilização de aditivo de leite materno suplementado com ferro poderia prejudicar os efeitos protetores da lactoferrina e aumentar os riscos de infecção em recém-nascidos. OBJETIVO: Comparar o crescimento bacteriano no colostro puro versus colostro com aditivo de leite materno suplementado com ferro. MÉTODO: O crescimento bacteriano de Escherichia coli, Staphylococcus aureus e Pseudomonas aeruginosa foi comparado em 78 amostras de colostro puro ou colostro com aditivo do leite humano suplementado com ferro. Para análise qualitativa, discos de papel filtro foram imergidos nas amostras de leite materno puro ou leite materno com aditivo suplementado de ferro e incubados por 48 horas em placas de Petri contendo 101 Unidades Formadoras de Colônia por ml (UFC/ml) de cada cepa de bactérias. Para a análise quantitativa, 1ml de cada cepa de bactérias contendo 107 UFC/ml foi homogeneizado com 1ml de colostro puro ou colostro com aditivo do leite humano suplementado com ferro e semeado em placa de Petri. O número de UFC/ml foi contado após 24 horas de incubação a 37oC. RESULTADOS: A análise qualitativa não mostrou diferença no crescimento bacteriano. Na avaliação quantitativa, o crescimento de Escherichia coli no colostro puro foi de 29.4 ± 9.7 x 106CFU/ml e no colostro com aditivo de leite materno suplementado de ferro foi de 31.2 ± 10.8x 106CFU/ml, com diferença na média de crescimento de 1.9 ± 4.9 x 106CFU/ml (p = 0,001). O crescimento bacteriano nas cepas de Staphylococcus aureus e Pseudomonas aeruginosa no colostro puro e no colostro com aditivo de leite materno não apresentou diferença estatística. CONCLUSÃO: O acréscimo de aditivo de leite materno suplementado com ferro nesta concentração reduziu a ação bacteriostática contra Escherichia coli / BACKGROUND: Lactoferrin in human breast milk has been shown to protect newborns from infection by binding to iron and depriving it from pathologic bacteria that need iron to proliferate. If iron-enriched fortifier is added to breast milk, it might impair the protective effect of lactoferrin and increase the risk of infection in newborns. OBJECTIVE: To compare bacterial growth in pure colostrum versus colostrum with human milk fortifier containing iron. METHODS: The growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in 78 samples of pure colostrum or of colostrum with added human milk fortifier containing iron was compared. For qualitative analysis, filter paper discs were immersed in samples from each group and incubated for 48 hours with 101Colony Forming Units/ml of each strain. For quantitative assessment, 1 ml of each strain containing 107Colony Forming Units/ml was homogenized with 1 ml of either colostrum or colostrum with human milk fortifier, seeded into a Petri dish, and incubated at 37oC. Twenty-four hours later the number of Colony Forming Units was counted. RESULTS: Qualitative analysis showed no difference in bacterial growth. In the quantitative evaluation, Escherichia coli growth in the pure colostrum group was 29.4 ± 9.7 x 106CFU/ml while in the human milk fortifier group it was 31.2 ± 10.8x 106CFU/ml; the difference between average growth was 1.9 ± 4.9 x 106CFU/ml (p = 0.001). There were no differences in Staphylococcus aureus and Pseudomonas aeruginosa growth. CONCLUSION: Addition of iron at this concentration reduced breast milk bacteriostatic action against Escherichia coli

Objetivo: Verificar a associação entre os níveis de leptina do leite materno maduro e os escores de dobras cutâneas aos três e seis meses de vida de crianças expostas a diferentes ambientes intrauterinos. Metodologia: Trata-se de um estudo observacional longitudinal, para o qual foram recrutados pares de mães e crianças residentes em Porto Alegre. Foram excluídas puérperas com teste positivo para HIV, doenças hipertensivas específicas da gravidez, hipertensão arterial sistêmica crônica ou gestacional, crianças gemelares, prematuras, com doenças congênitas ou necessidade de internação hospitalar. Os pares foram divididos, conforme a exposição ao ambiente intrauterino, em grupo Diabetes Mellitus (DM), Tabaco, Pequeno para Idade Gestacional (PIG) e Controle. As entrevistas foram realizadas um, três e seis meses após o parto, e foram coletados dados antropométricos da mãe e da criança, além de informações sobre a alimentação desta. Na entrevista de um mês de vida, foi coletada amostra de leite materno, analisada para quantificação da leptina. Quanto aos procedimentos estatísticos, foi considerado o nível de significância de 5%. Para comparação entre variáveis categóricas e contínuas, quando paramétricas, foi utilizado o teste ANOVA com post hoc de Tukey, e para variáveis não paramétricas, foi utilizado o teste de Kruskal-Wallis com post hoc de Dunn. Na comparação entre variáveis categóricas, foi utilizado o teste de qui-quadrado de Pearson ou Exato de Fisher. Ao analisar variáveis contínuas, foi aplicada a correlação de Spearman. Resultados: Foram analisados os dados de 127 pares mãe-criança, sendo 18 no grupo DM, 34 no grupo Tabaco, 16 no grupo PIG e 59 no grupo Controle. A quantidade de leptina no leite materno não diferiu entre os grupos (p = 0,59). Houve correlação significativa positiva entre a quantidade de leptina do leite e o Índice de Massa Corporal (IMC) materno, a dobra cutânea tricipital (DCT) e a dobre cutânea subescapular (DCSE) maternas (p < 0,001). Sem considerar o tipo de alimentação, não houve correlação entre a leptina e as variáveis antropométricas da criança (p > 0,05). Dentre as crianças amamentadas exclusivamente aos três meses de vida, a leptina do leite materno correlacionou-se negativamente com o escore Z da DCT aos três meses (r = -0,447; p = 0,032) e seis meses (r = -0,499; p = 0,015), além do peso aos três (r = -0,408; p = 0,043) e seis meses de vida (r = -0,443; p= 0,034). Também houve correlação negativa com o ganho de peso do nascimento aos seis meses (r = -0,472; p = 0,023) e de um a três meses (r = -0,396; p = 0,050). Conclusão: A leptina, oferecida através do aleitamento materno exclusivo, atua na regulação do balanço energético corporal da criança, influenciando precocemente variáveis antropométricas. / Objective: Assess the association between mature breast milk leptin and skinfold thickness Z scores at 3 and 6 months of life of infant exposed to different intrauterine environments. Methods: This is a longitudinal study, for which mother and newborn pairs were recruited from 24-48 hours postpartum in three public hospitals in Porto Alegre city, Brazil. Exclusion criteria were mothers who test positive for HIV, with hypertensive disorders, newborn from twin gestation, preterm (gestational age <37 weeks), with malformations at birth, or requiring hospitalization. Pairs were divided as exposure to intrauterine environment in Diabetes Mellitus (DM), Tobacco, Small for Gestational Age (SGA) and Control group. Interviews occurred in postpartum, 1, 3 and 6 months of infant's life, where mother and infant anthropometric data were collected and information about infant’s complementary food was asked. The breast milk sample was collected in the 1 month interview. About statistical procedures, a significance level of 5% was considered. To compare categorical and continuous variables, was used the ANOVA test with Tukey post hoc, when parametric. For nonparametric variables, was used Kruskal -Wallis test with Dunn post hoc. When comparing categorical variables, was used the chi-square of Pearson or Fisher's exact test. To analyze continuous variables, the Spearman correlation was applied. Results: Data of 127 pairs were analyzed, 18 in DM group, 34 Tobacco, 16 SGA and 59 Control. Value of leptin in breast milk did not differ between groups (p = 0.59). There was a significant positive correlation between leptin milk and maternal anthropometric variables in the overall sample (p < 0.001) and between the groups (p< 0.05). Without considering infant's complementary feeding, there was no correlation between leptin and infant anthropometric variables (p > 0.05). However, among the infants exclusively breastfed at 3 months, leptin significantly negative correlated with Z score of triceps skinfold thickness at 3 (r = -0.447; p = 0.032) and 6 months (r = -0.499; p=0.015), infant weight at 3 (r = -0.408; p = 0.043) and 6 months (r = -0.443; p = 0.034), weight gain from birth to 6 months (r = -0.472; p = 0.023) and from 1 to 3 months (r = -0.396; p = 0.050) in the overall sample. Conclusion: Among the infants exclusively breastfed, leptin in mature milk acts in body energy balance and may impact early infant anthropometric variables.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-14T20:45:25Z No. of bitstreams: 1 HerminioMendes.pdf: 6465933 bytes, checksum: b67849d7574d0171fe7eab9850a0985a (MD5) / Made available in DSpace on 2017-09-14T20:45:25Z (GMT). No. of bitstreams: 1 HerminioMendes.pdf: 6465933 bytes, checksum: b67849d7574d0171fe7eab9850a0985a (MD5) Previous issue date: 2017-06-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / The use of food for probiotic bacteria has recently been considered a possibility of prevention against the installation or attenuation of several infectious processes. Species belonging to the genera Bifidobacterium and Lactobacillus predominate in the gastrointestinal microbiota and are widely used in functional foods. In general, a selection of new probiotic strains for use in humans has as its source a human intestinal flora and breast milk. Thus, the objective of this study was to isolate and identify new species with potential probiotic from samples of breast milk and feces of infants exclusively breastfed, in addition to evaluating their probiotic potential. Samples of breast milk from two different lactation periods (colostrum and transitional milk) and samples of fecal material from infants were used. Isolation was used using MRSC and for Beerens for Lactic-Acid Bacteria and Bifidobacterium spp. respectively. Probiotic potential was assessed by physical pH and bile salt tolerance tests. Verified its ability to adhere to mucin and human intestinal cells (HT-29) and inhibit adhesion of different diarrheogenic strains of E. coli. The identification of the promising isolates was performed through the 16S rDNA region. Five isolates had acid-bile resistance, with CLM0109 insulation having 90.2% and 64.6% resistance, pH 3.0 and pH 2.0, respectively. These isolates have an ability to adhere to mucin and HT-29 cells, the isolate FEB0308 showed the highest level of adherence to HT-29 cells. The adhesion assay demonstrated that all isolates were absorbed by adhesion of diarrheogenic E. coli (DECs). Antimicrobial susceptibility analysis showed that 3 isolates showed resistance to one or two antibiotics (kanamycin and streptomycin). The molecular analysis allowed identifying the isolates as belonging to the species Lactobacillus casei. The results show that the five isolates present probiotic potential especially for use in the prevention and treatment of infections caused by DEC and demonstrate that milk and stool samples from infants can act as a source of potential probiotic bacteria. / O uso de alimentos contendo bactérias probióticas foi recentemente considerado uma possibilidade de prevenção contra a instalação ou atenuação de diversos processos infecciosos. Espécies pertencentes aos gêneros Bifidobacterium e Lactobacillus predominam na microbiota gastrintestinal humana e são amplamente utilizadas em alimentos funcionais. Em geral, a seleção de novas cepas probióticas para uso em humanos, tem como fonte a microbiota intestinal humana e o leite materno. Dessa forma, o objetivo deste estudo foi isolar e identificar novas estirpes com potencial probiótico a partir de fezes de lactentes amamentados exclusivamente com leite materno, além de avaliar seu potencial probiótico. Foram utilizadas amostras de material fecal de bebes alimentados exclusivamente com leite materno. O isolamento foi realizado utilizando-se ágar MRSC e ágar Beerens para bactérias ácido-lácticas (BAL) e Bifidobacterium spp. respectivamente. O potencial probiótico foi avaliado pelos testes de tolerância ao pH gástrico e sais biliares. Além disso, foi verificado sua capacidade de aderir à mucina e as células intestinais humanas (HT-29), bem como a capacidade de inibir adesão de distintas linhagens diarreiogênicas de E. coli além de sensibilidade a antimicrobianos. A identificação dos isolados promissores foi realizada através da região 16S rDNA. Cinco isolados apresentaram resistência ácido-biliar, com destaque para o isolado CLM0109 que apresentou 90.2% e 64.6% de resistência a pH 3.0 e pH 2.0 respectivamente. Estes isolados tiveram a capacidade de aderir à mucina e células HT-29, o isolado FEB0308 apresentou o maior nível de aderência a células HT-29. O ensaio de inibição da adesão demonstrou que todos os isolados foram capazes de inibir adesão de E. coli diarreiogênicas (DECs). Além disso, a análise de sensibilidade a antimicrobianos demonstrou que 3 isolados apresentaram resistência a um ou dois antibióticos (canamicina e estreptomicina). A análise molecular identificou os isolados como Lactobacillus casei. Concluímos que os cinco isolados apresentam potencial probiótico especialmente para uso na prevenção e tratamento de infecções causadas por DEC e demonstra que as amostras fecais de lactentes alimentados exclusivamente com leite materno, podem funcionar como fonte de bactérias com potencial probiótico.

As manifestações do comportamento da criança e a capacidade de amamentar são fatores reconhecidos, pela mulher, como determinantes do bem estar do filho. A decisão adotada pelas mulheres diante do curso da amamentação, particularmente sobre sua capacidade de amamentar, se baliza em percepções próprias relacionadas aos atributos do leite produzido e às respostas de satisfação do filho. Este estudo teve como objetivos: caracterizar a prática da amamentação realizada pelas mulheres com base nos indicadores da OMS; caracterizar a prática do aleitamento materno exclusivo (AME) com base nos parâmetros: freqüência e duração das mamadas, intervalo entre as mamadas, término da mamada, alternância e esvaziamento das mamas; identificar a percepção materna sobre o leite produzido e sua relação com a duração, condução do AME e percepção sobre a saciedade da criança; identificar os problemas percebidos pelas mulheres durante a amamentação e sua relação com a duração do AME. Trata-se de um estudo epidemiológico, transversal, descritivo exploratório, que foi desenvolvido na UBDS Vila Virgínia, em Ribeirão Preto-SP. A amostra constituiu-se de 231 mulheres que acompanharam seus filhos de 0 a 4 meses, na consulta de puericultura ou para realização das vacinas, de fevereiro a agosto de 2008. Os dados foram coletados utilizando-se um questionário sobre a alimentação oferecida à criança, contendo 52 questões. Os dados foram processados e analisados no programa Statistical Analysis System (SAS), versão 9.0. Na análise realizou-se distribuição de freqüências, médias, desviospadrões, medianas, comparação entre médias, teste exato de Fisher e regressão logística. No momento da coleta de dados, 58,9% das crianças estavam recebendo AME e, para as outras crianças esta prática durou em média 47 dias (desvio-padrão = 35,4 dias). A média de mamadas foi de 9,4 por dia, 27 minutos cada mamada e intervalo de 1 hora e 54 minutos entre as mamadas. 88,3% das participantes ofereciam as mamas em livre demanda e o término da mamada era realizado pela criança em 87,0% dos casos. Após a mamada, 71,4% das mulheres sentiam a mama vazia. A percepção materna sobre o leite produzido apresentou resultado estatisticamente significativo quando associado com a saciedade da criança, indicando que as mulheres que perceberam a criança insatisfeita após a mamada têm 32 vezes a chance de apresentar uma percepção ruim sobre seu leite (p<0,01; OR ajustado = 32,70 e IC 95% [14,73 72,59]). Foi verificada diferença estatística significativa entre os intervalos de mamadas feitos pelas mulheres que tinham uma boa percepção sobre o seu leite e aquelas que tinham uma percepção ruim. (p<0,01, IC 95% [0,10 0,39]). Além disso, as mulheres que não apresentaram problemas durante a amamentação tem 2,53 vezes a chance de manterem o AME até os 4 meses de vida da criança (p<0,01; OR = 2,53 e IC 95% [1,48 4,34]). Concluise que, para as participantes deste estudo, o indicador mais utilizado para avaliar o seu leite produzido foi a satisfação da criança, mostrando que as ações pró-amamentação devem ser revistas, no sentido de contemplar as necessidades das mulheres e incentivar a sua confiança na capacidade de boa produção láctea. / Childrens behavior and the capacity to breastfeeding are factors recognized by woman, as determinants of her childs well being. The womens decision to breastfeeding, particularly concerned about their capacity to breastfeed, is based on their own perceptions about the attributions given to the human milk and the signs of childs satisfaction. This study aimed: to characterize the breastfeeding practice by women based on World Health Organizations indicators; to characterize exclusive breastfeeding (EBF) practice based on the following parameters: frequency and length of time, interval between breastfeed, how breastfeed ends, breasts interchange and breast emptying; to identify womens perception about the milk produced and its relation with the breastfeeding maintenance, exclusive breastfeeding behave and the perception of child satiation; to identify problems perceived by women during breastfeeding and their relation with the exclusive breastfeeding lastingness. This is a crosssectional descriptive study, developed in one Basic and District Health Unit in Ribeirão Preto SP, Brazil. The sample was composed by 231 women that were previously schedule in the child care service or immunization for the children with ages from 0 to 4 months, from February to August 2008. Data collection was performed using a specific questionnaire, with 52 items related to nutrition offered to the children. Data were analyzed using Statistical Analysis System (SAS), vs. 9.0. In the data analysis, frequency distribution, mean, standard deviation, median, means comparison, Exact Fisher Test and logistic regression were used. When data were collected, 58,9% of children were receiving EBF and, for other children this practice lengthen 47 days (standard deviation = 35,4 days). Daily breastfeeding mean was 9,4, 27 minutes each breastfeed and interval of 1,54 hours. 88,3% of participants offered ondemand breastfeeding and the breastfeeding end was lead by child in 87,0% of occurrences. After breastfeeding, 71,4% of women felt the breast empty. Maternal perception about produced milk showed statistically significant result when associated with childs satiation, indicating that women who perceived the child insatiate after breastfeeding had 32 times the chance to have a bad perception about their own milk (p<0,01; adjusted OR=32,70 and CI 95% [14,73-72,59]. Statistically significant difference was verified between the breastfeeding interval adopted by women who had good perception about their milk and by those who had a bad perception (p<0,01, CI 95% [0,10 0,39]). Besides, women who do not presented problems during breastfeeding have 2,53 times chance to keep EBF until 4 months of childs life (p<0,01; OR = 2,53 e CI 95% [1,48 4,34]). In conclusion, for the participants, the most used indicator to evaluate their own milk was child satisfaction, pointing that, actions probreastfeeding must be reviewed, in order to observe womens need and improve their confidence in their capacity to produce good milk.

Introdução: Recém-nascidos (RN) pré-termo tardios são submetidos repetidamente a lancetagem de calcâneo para controle glicêmico nas primeiras horas de vida. A lancetagem, embora dolorosa, raramente é acompanhada de medida analgésica. Objetivo: Comparar a eficácia do leite materno ordenhado com glicose 25% nos escores da dor em recém-nascidos pré-termo tardios, submetidos à lancetagem de calcâneo. Método: Ensaio clínico randomizado, de não inferioridade, conduzido no Berçário Anexo à Maternidade do Instituto da Criança da Faculdade de Medicina da Universidade de São Paulo (BAM-ICrFMUSP) no período de agosto de 2009 a maio de 2010. A amostra foi de 113 RN, dos quais 56 randomizados no grupo experimental (GE) e 57, no grupo controle (GC). Dois minutos antes de serem submetidos à lancetagem de calcâneo, o GE recebeu 2mL de leite materno ordenhado (LMO) por via oral e os do GC, 2mL de glicose 25% por via oral. O desfecho primário foi o escore da dor obtido com a escala Premature Infant Pain Profile (PIPP) nos primeiros 180 segundos após a lancetagem (T30, T60, T90, T120, T150 e T180). Os desfechos secundários foram alterações relacionadas à mímica facial (sobrancelhas salientes, olhos espremidos, sulco nasolabial), frequência cardíaca (FC), saturação de oxigênio (SatO2), choro e eventos adversos. O projeto de pesquisa recebeu aprovação do comitê de ética da instituição, local do estudo, e foi registrado no Australian and New Zealand Clinical Trials Registry. Resultados: Pela ausência ou volume insuficiente de LMO, 25 neonatos não receberam a intervenção, mas foram incluídos na análise por intenção de tratar. Os grupos foram homogêneos em relação ao tipo de parto, Apgar no 5º minuto de vida, sexo, idade gestacional, idade em horas, intervalo entre a última mamada e a coleta de dados e glicemia capilar. Observou-se diferença estatística significante quanto ao peso do nascimento (p=0,013) e peso na data do procedimento (p=0,017). A análise de variância (ANOVA) indicou escores de dor inferiores no GC: houve diferença significativa entre os grupos de alocação, independente do tempo (p<0,001) e, ao longo do tempo, independente da intervenção (p<0,001). O GC apresentou maior freqüência de escores PIPP indicativos de dor mínima ou ausência de dor (p=0,002 e, na análise por intenção de tratar, p=0,003). A avaliação da mímica facial demonstrou menor porcentagem média de tempo de ocorrência de sobrancelhas salientes, olhos espremidos e sulco nasolabial para o GC em relação ao GE. Para sobrancelhas salientes, observou-se diferença estatisticamente significante entre os grupos (p<0,001) e ao longo do tempo (p<0,001). Com relação ao olhos espremidos, a ANOVA indicou interação (p=0,001). Para os neonatos que receberam leite materno, houve diferença significativa na ocorrência de olhos espremidos no T30 e nos demais intervalos, com redução da ocorrência da mímica ao longo do tempo (p_0,0014). No GC, foi observada redução significativa na ocorrência de olhos espremidos, em relação ao T30, no T90 (p=0,0155), no T150 (p=0,0261) e no T180 (p=0,0122). Quanto ao sulco nasolabial, observou-se interação (p<0,001). No GE, houve diferença estatisticamente significativa entre o T30 e todos os demais períodos de avaliação (p<0,001), exceto entre o T30 e T60. Para o GC, observou-se redução significativa só entre T30 e T90 (p=0,022) e entre T30 e T180 (p=0,0175). Houve interação entre as intervenções e ao longo do tempo na avaliação da FC (p=0,027). Não se observou diferença estatisticamente significante na FC ao longo do tempo para o GE (p=0,563) e para o GC (p=0,955). Para SatO2, observou-se diferença significativa entre os grupos (p=0,004) e ao longo do tempo (p=0,017). Foi observado choro pós-procedimento em 33 neonatos no GE e em 19 no GC (p=0,001 e na análise por intenção de tratar, p=0,001). A porcentagem média de tempo de choro foi maior no GE (p=0,014). Os eventos adversos foram benignos e autolimitados, sem diferença estatisticamente significante entre os grupos (p=0,736 e na análise por intenção de tratar, p=0,637). A margem de não inferioridade estabelecida foi de 2 pontos e, para o teste da hipótese, o intervalo de confiança de 95% foi de 1,507 a 4,483. Conclusão: O teste da hipótese de não inferioridade do efeito do LMO em relação ao efeito da solução de glicose 25% no escore da dor PIPP em neonatos pré-termo tardios submetidos à punção capilar em calcâneo foi inconclusivo. / Background: Late preterm neonates undergo repeated heel lancing during their first hours of life due to capillary blood glucose monitoring. Heel lancing is a painful procedure however pain relief strategies are rarely implemented prior to the lances. Aim: To compare the efficacy of expressed breast milk and 25% glucose on pain scores of late preterm infants during heel lancing. Methods: This is a noninferiority randomized controlled trial, conducted at the Berçário Anexo à Maternidade do Instituto da Criança da Faculdade de Medicina da Universidade de São Paulo (BAM-ICrFMUSP) between August 2009 and May 2010. A total of 113 newborns were randomized: 56 comprised the experimental group (EG) and 57, the control group (CG). Two minutes before heel lancing, EG received 2mL of expressed breast milk (EBM) orally and CG received 2mL of 25% glucose orally. The Premature Infant Pain Profile (PIPP) score was the primary outcome and it was assessed within 180 seconds after lancing (T30, T60, T90, T120, T150 and T180). Secondary outcomes were: grimacing (brow bulging, eye squeezing, nasolabial furrowing), changes in heart rate (HR), and oxygen saturation (SpO2), crying, and adverse events. The research project was approved by the local ethics committee and was registered at the Australian and New Zealand Clinical Trials Registry. Results: Due to absence or insufficient volume of EBM, 25 neonates did not receive the intervention, but were included in the intention to treat analysis. The groups were similar with respect to type of delivery, Apgar score at 5 minutes, sex, gestational age, postnatal age in hours, interval between last feeding and data collection, and capillary blood glucose. There were statistically significant differences amongst the groups regarding birth weight (p=0.013) and weight at the time of the procedure (p=0.017). The analysis of variance (ANOVA) indicated lower pain scores in the CG: there was a significant difference between the interventions, regardless time (p<0.001), and main effect of the time, independent of the intervention (p<0.001). The CG had a higher frequency of PIPP scores indicative of minimal pain or absence of pain (p= 0.002 and p=0.003 on intention to treat analysis). Lower mean percentage of time of brow bulging occurrence, eye squeezing and nasolabial furrowing was observed for CG in comparison to EG. There was a statistically significant difference on brow bulging occurrence amongst the groups (p<0.001) and over the time (p<0.001). ANOVA indicated interaction between interventions and time (p=0.001) for eye squeezing. Infants who received breast milk demonstrated significant reduction on eye squeezing occurrence when comparing T30 and the remaining intervals (p_0.0014). In the CG, a significant reduction on eye squeezing were observed when comparing T30 to T90 (p=0.0155), to T150 (p=0.0261) and to T180 (p=0.0122). As for nasolabial furrowing, there was interaction (p<0.001). Statistically significant differences between the T30 and all other periods (p<0.001) were observed for EG, except when comparing T30 and T60. For neonates who received glucose, there were significant reductions in nasolabial furrowing occurrence between T30 and T90 (p=0.022) and T30 and T180 (p=0.0175). There was interaction in the evaluation of HR (p=0.027). No statistically significant differences were observed in HR over time for EG (p=0.563) and CG (p=0.955). Regarding SpO2, there were significant differences between intervention groups (p=0.004) and over the time (p=0.017). Of the infants who received EBM, 33 cried after lancing; in the CG, 19 neonates cried (p=0.001 and p=0.001 on intention to treat analysis). Longer mean percentage of duration of crying was observed in EG (p=0.014). Adverse events were benign and self-limited and there was no statistically significant difference among the groups (p=0.736 and p=0.637 on intention to treat analysis). Noninferiority margin was established in 2 points and the hypothesis test indicated a 95% confidence interval 1.507 to 4.483. Conclusion: The hypothesis test of noninferiority of EBM as compared to 25% glucose solution on reducing PIPP score in late preterm neonates undergoing heel lancing in was inconclusive.

INTRODUÇÃO: A lactoferrina disponível no leite materno desempenha função imunológica e protege recém-nascidos de infecções por se ligar ao ferro e privá-lo de bactérias patogênicas, o que resulta em atividade bacteriostática contra organismos patogênicos ferro dependentes. A utilização de aditivo de leite materno suplementado com ferro poderia prejudicar os efeitos protetores da lactoferrina e aumentar os riscos de infecção em recém-nascidos. OBJETIVO: Comparar o crescimento bacteriano no colostro puro versus colostro com aditivo de leite materno suplementado com ferro. MÉTODO: O crescimento bacteriano de Escherichia coli, Staphylococcus aureus e Pseudomonas aeruginosa foi comparado em 78 amostras de colostro puro ou colostro com aditivo do leite humano suplementado com ferro. Para análise qualitativa, discos de papel filtro foram imergidos nas amostras de leite materno puro ou leite materno com aditivo suplementado de ferro e incubados por 48 horas em placas de Petri contendo 101 Unidades Formadoras de Colônia por ml (UFC/ml) de cada cepa de bactérias. Para a análise quantitativa, 1ml de cada cepa de bactérias contendo 107 UFC/ml foi homogeneizado com 1ml de colostro puro ou colostro com aditivo do leite humano suplementado com ferro e semeado em placa de Petri. O número de UFC/ml foi contado após 24 horas de incubação a 37oC. RESULTADOS: A análise qualitativa não mostrou diferença no crescimento bacteriano. Na avaliação quantitativa, o crescimento de Escherichia coli no colostro puro foi de 29.4 ± 9.7 x 106CFU/ml e no colostro com aditivo de leite materno suplementado de ferro foi de 31.2 ± 10.8x 106CFU/ml, com diferença na média de crescimento de 1.9 ± 4.9 x 106CFU/ml (p = 0,001). O crescimento bacteriano nas cepas de Staphylococcus aureus e Pseudomonas aeruginosa no colostro puro e no colostro com aditivo de leite materno não apresentou diferença estatística. CONCLUSÃO: O acréscimo de aditivo de leite materno suplementado com ferro nesta concentração reduziu a ação bacteriostática contra Escherichia coli / BACKGROUND: Lactoferrin in human breast milk has been shown to protect newborns from infection by binding to iron and depriving it from pathologic bacteria that need iron to proliferate. If iron-enriched fortifier is added to breast milk, it might impair the protective effect of lactoferrin and increase the risk of infection in newborns. OBJECTIVE: To compare bacterial growth in pure colostrum versus colostrum with human milk fortifier containing iron. METHODS: The growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in 78 samples of pure colostrum or of colostrum with added human milk fortifier containing iron was compared. For qualitative analysis, filter paper discs were immersed in samples from each group and incubated for 48 hours with 101Colony Forming Units/ml of each strain. For quantitative assessment, 1 ml of each strain containing 107Colony Forming Units/ml was homogenized with 1 ml of either colostrum or colostrum with human milk fortifier, seeded into a Petri dish, and incubated at 37oC. Twenty-four hours later the number of Colony Forming Units was counted. RESULTS: Qualitative analysis showed no difference in bacterial growth. In the quantitative evaluation, Escherichia coli growth in the pure colostrum group was 29.4 ± 9.7 x 106CFU/ml while in the human milk fortifier group it was 31.2 ± 10.8x 106CFU/ml; the difference between average growth was 1.9 ± 4.9 x 106CFU/ml (p = 0.001). There were no differences in Staphylococcus aureus and Pseudomonas aeruginosa growth. CONCLUSION: Addition of iron at this concentration reduced breast milk bacteriostatic action against Escherichia coli

Infectious diseases e.g. gastric ulcer, caries and perodontitis, are caused by bacteria in a biofilm. Adhesion of bacteria to host ligands e.g. proteins, polypeptides and glycoproteins, is a key event in biofilm formation and colonization of surfaces such as mucosa and tooth tissues. Thus, host ligands could contribute to the susceptibility to infectious diseases. The general aim of this doctoral thesis was to study the effect of phosphorylated polypeptides and gp-340 in saliva and milk on oral bacterial adhesion and aggregation. Statherin is a non-glycosylated, phosphorylated polypeptide in saliva. The polypeptide inhibits precipitation and crystal growth of calcium phosphate and mediates adhesion of microorganisms. By using a hybrid peptide construct, the domain for adhesion of Actinomyces isolated from human infections and from rodents was found to reside in the C-terminal end, and the adhesion was inhibitable. With alanine substitution the peptide recognition epitope in the C-terminal end was delineated to Q and TF, where QAATF was an optimal inhibitory peptide. In contrast, human commensal Actinomyces bound to the middle region in a non-inhibitable fashion. Gp-340 is another protein in saliva, and it is a large, multifunctional glycoprotein. Four novel size variants (I-IV) of salivary gp-340 were distinguished within individuals, and their glycoforms were characterized. All four size variants were identical in the N-terminal amino acid sequence and shared core carbohydrates. Low-glyco lung gp-340, high-glyco saliva gp-340, and size variants I-III aggregated bacteria differently. Human milk, which shares many traits with saliva, could inhibit adhesion of Streptococcus mutans to saliva-coated hydroxyapatite (s-HA), a model for teeth, in an individually varying fashion. Human milk caseins, lactoferrin, secretory IgA, and IgG inhibited the binding avidly. By using synthetic peptides the inhibitory epitope in b-casein was mapped to a C-terminal stretch of 30 amino acids. Inhibition by human milk, secretory IgA and the b-casein-derived inhibitory peptide was universal among a panel of mutans streptococci. The main conclusions are: (i) statherin mediates differential binding of commensal versus infectious Actinomyces strains with small conformation-dependent binding epitopes, (ii) salivary gp-340 has individual polymorphisms that at least affect binding of bacteria, (iii) human milk inhibits S. mutans adhesion to s-HA in an individually varying fashion, and the C-terminal end of human milk β-casein is one inhibitory component. Together these results suggest that the studied host ligands can influence the composition of the oral biofilm. Statherin may protect the host from colonization of bacteria associated with infections. Gp-340 size variants may affect functions related to host innate immune defences such as interactions with a wide array of bacteria, and human milk may have a protective effect in infants from colonization of mutans streptococci.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.