Дисертації з теми "Necrotizing enterocoliti"

Necrotizing enterocolitis (NEC), a severe inflammatory disorder of the gastrointestinal tract with high morbidity and mortality, affects primarily preterm infants. The diagnosis represents a challenging task, and no biomarker has been found to aid early diagnosis with high accuracy. Microdialysis has been widely used to detect metabolites of anaerobic metabolism, enabling a local and early detection of ischemia. This thesis aims to evaluate the possibility of detecting intestinal ischemic stress in experimental and clinical  NEC, by use of rectal intraluminal microdialysis. Intraluminal rectal microdialysis was performed on rats subjected to total intestinal ischemia. Metabolites of ischemia were detectable in both ileum and rectum, with raised glycerol concentrations and lactate/pyruvate ratios. Elevated concentrations of glycerol correlated to increasing intestinal histopathological injury. Experimental early NEC was induced in newborn rat pups, by hypoxia/re-oxygenation treatment. Development of NEC was confirmed by histopathology. Elevated glycerol concentrations were detected by rectal microdialysis. The genetic alterations following experimental NEC in rat pups were studied with microarray. Immunohistochemistry staining was performed for tight junction proteins claudin-1 and claudin-8. Several genes were altered in experimental NEC, mainly genes regulating tight junctions and cell adhesion. Immunohistochemistry revealed reduced expression of claudin-1. A prospective study was conducted on preterm infants with a gestational age of less than 28 weeks. The infants were admitted to a neonatal intensive care unit, and observed during a 4-week period. Rectal microdialysis was performed twice a week, and blood was drawn for analysis of I-FABP. A total of 15 infants were included in the study, whereof four infants developed NEC, and 11 served as controls. Rectal glycerol and I-FABP displayed high concentrations, which varied considerably during the observation periods, both in NEC and controls. No differences in either glycerol or I-FABP concentrations were seen in the NEC-group vs. the controls. In conclusion, rectal microdialysis can detect metabolites of intestinal ischemia, both in experimental and clinical NEC. Rectal microdialysis is safe and could provide a valuable non-invasive aid to detect hypoxia-induced intestinal damage or ischemic stress in extremely preterm infants. In this study however, it was not possible to predict the development of clinical NEC using microdialysis or I-FABP.

In extremely preterm infants, acute abdominal emergencies are fortunately less common with improving care. Spontaneous intestinal perforation and necrotizing enterocolitis are conditions where emergency surgery is most often needed. Conservative medical management and placement of temporary drain are often used in the initial management. Internal hernia (IH) is an uncommon cause of bowel obstruction in neonates, is difficult to diagnose and unfortunately are found only at autopsy. The presentation in preterm infants, distinction between these conditions, and the need for early diagnosis of IH are discussed.

Necrotizing enterocolitis (NEC) is a costly and deadly disease in neonates. Composite risk for NEC is poorly understood and consensus has not been established on the relevance of risk factors. This two-phase study attempted to validate and test a neonatal NEC risk index, GutCheck(NEC). Phase I used an E-Delphi methodology in which experts (n=35) rated the relevance of 64 potential NEC risk factors. Items were retained if they achieved predefined levels of expert consensus or stability. After three rounds, 43 items were retained (CVI=.77). Qualitative analysis revealed two broad themes: individual characteristics of vulnerability and the impact of contextual variation within the NICU on NEC risk. In Phase II, the predictive validity of GutCheck(NEC) was evaluated using a sample from the Pediatrix BabySteps Clinical Data Warehouse (CDW). The sample included infants born<1500 grams, before 36 weeks, and without congenital anomalies or spontaneous intestinal perforation (N=58,818, of which n=35,005 for empiric derivation and n=23,813 for empiric validation). Backward stepwise likelihood-ratio method regression was used to reduce the number of predictive factors in GutCheck(NEC) to 11 and derive empiric weights. Items in the final GutCheck(NEC) were gestational age, history of a transfusion, NICU-specific NEC risk, late onset sepsis, multiple infections, hypotension treated with Inotropic medications, Black or Hispanic race, outborn status, metabolic acidosis, human milk feeding on both day 7 and day 14 (reduces risk) and probiotics (reduces risk).Discrimination was fair in the case-control sample (AUC=.67, 95% CI .61-.73) but better in the validation set (AUC=.76, 95% CI .75-.78) and best for surgical NEC (AUC=.84, 95% CI .82-.84) and infants who died from NEC (AUC=.83, 95% CI .81-.85). A GutCheck(NEC) score of 33 (range 0-58) yielded a sensitivity of .78 and a specificity of .74 in the validation set. Intra-individual reliability was acceptable (ICC (19) =.97, p<.001). Future research is needed to repeat this procedure in infants between 1500 and 2500 grams, complete psychometric testing, and explore unit variation in NEC rates using a comprehensive approach.

Neonatal necrotizing enterocolitis (NEC) is one of the most severe digestive tract emergencies in neonates, involving bowel edema, hemorrhage, and necrosis, and can lead to serious complications including death. Since it is difficult to diagnose early, the morbidity and mortality rates are high due to severe complications in later stages of NEC and thus early detection is key to the treatment of NEC. In this thesis, a novel automatic image acquisition and analysis system combining a color and depth (RGB-D) sensor with an infrared (IR) camera is proposed for NEC diagnosis. A design for sensors configuration and a data acquisition process are introduced. A calibration method between the three cameras is described which aims to ensure frames synchronization and observation consistency among the color, depth, and IR images. Subsequently, complete segmentation procedures based on the original color, depth, and IR information are proposed to automatically separate the human body from the background, remove other interfering items, identify feature points on the human body joints, distinguish the human torso and limbs, and extract the abdominal region of interest. Finally, first-order statistical analysis is performed on thermal data collected over the entire extracted abdominal region to compare differences in thermal data distribution between different patient groups. Experimental validation in a real clinical environment is reported and shows encouraging results.

This honors thesis analyzes the methodology and results of ongoing research on the potential use of bile acid concentrations in neonatal stool to be used as an indicator for the onset of the disease necrotizing enterocolitis (NEC). NEC is a serious complication in a premature infant with the potential for death. Twenty-two patients were enrolled in the study over the period of eight months, however, no patient developed NEC. Control patients were analyzed for trends in bile acid concentrations. Elevation of in bile acid concentration were seen following each feeding. Previous inclinations were also proven in regards to that mother's milk is more easily digested and remains a more nutritionally sound form of feeding than formula milk.

Background/Aim: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in neonates. The aim of this study was to: 1) establish and validate a neonatal rat model of NEC; 2) investigate the use of Captopril, an angiotensinconverting enzyme (ACE) inhibitor, in this model; 3) explore stem cells in this model as a new therapeutic strategy for NEC. Methods: 1) Various stress factors were employed to reproduce experimental NEC. Rats were assessed using old and new parameters of evaluation, including weight, survival, clinical conditions and behaviour, macroscopic and microscopic gut appearance. 2) Rats were administered Captopril and assessed for clinical status, body weight and survival rate. Resected intestine was evaluated for gut vessel dilatation and histology. 3) Rats intra-peritoneally injected with Amniotic Fluid Stem (AFS) cells and their controls were analysed for survival, gut macroscopic appearance and histology, immunofluorescence for AFS cell detection, bowel absorption and motility, degree of gut inflammation, and enterocyte apoptosis and proliferation. Results: 1) A neonatal rat model of NEC was established using hyperosmolar formula, hypoxia, and oral lipopolysaccharide. 2) Captopril reduced the severity of gut damage and the incidence of NEC via dilatation of the intestinal vasculature. 3) AFS cells integrated in the bowel wall and improved rat survival and clinical conditions, decreased NEC incidence and macroscopic gut damage, improved intestinal function, decreased bowel inflammation, increased enterocyte proliferation, and reduced apoptosis. The beneficial effect was achieved via modulation of stromal cells expressing COX-2 in the lamina propria, as shown by survival studies using selective and non-selective COX-2 inhibitors. Conclusions: In experimental NEC, both Captopril and AFS cells reduce the severity of intestinal damage and NEC incidence, ameliorating rat clinical conditions. However, AFS cells have the advantage of a powerful effect on mortality. Stem cell therapy may represent a new therapeutic option for infants with NEC.

Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease in premature babies. Despite significant morbidity and mortality, the cause of this disease remains unclear and there are no preventative treatments available. Prematurity and enteral feeding of infant formula are considered to be the primary risk factors for development of NEC. Interestingly, the incidence of NEC is six to ten times lower in breast-fed babies compared to those that were formula-fed. The factors responsible for the protective effect of breast milk against NEC have not been identified, but epidermal growth factor (EGF) is one of the most promising candidates. EGF is found at high concentrations in human milk, but is not present in any commercial formula. Mothers with extremely premature babies have 50-80% higher levels of EGF in their breast milk compared to mothers with full term infants. This suggests that EGF plays an important role in the development of premature infants. Our studies have shown that supplementation of EGF into formula significantly reduces the incidence of NEC in a neonatal rat model. However, the mechanisms underlying this EGF-mediated reduction of NEC are not understood. The overall hypothesis of this dissertation is that the protective effect of EGF in NEC pathogenesis is mediated via increased expression of pro-survival genes and strengthening of the mucosal barrier. The results of the studies within this dissertation demonstrate that treatment with EGF significantly decreases intestinal epithelial cell apoptosis at the site of NEC injury by up-regulating anti-apoptotic genes and down-regulating pro-apoptotic genes. Furthermore, supplementation of formula with EGF strengthens the mucosal barrier by inducing accelerated maturation of ileal goblet cells and mucin-2 production. In addition, EGF treatment normalizes expression of crucial tight junction proteins in the ileum. Consequently, EGF treatment results in a significant decrease in intestinal paracellular permeability and improved barrier function. Results from these studies will provide significant contributions to the understanding of EGF-mediated reduction of NEC, which may lead to development of therapeutic strategies for the treatment of human NEC.

Актуальність: Некротичний ентероколіт (НЕК) - це запалення тонкої та/або товстої кишки, в основі якого лежить ішемічне ураження кишечної стінки з наступним розвитком геморагічних та виразково-некротичних змін. Захворюваність на НЕК становить 2,4 на 1000 новонароджених. Середні показники летальності при НЕК складають 10-45% і залежать, крім ступеня зрілості, також від стадії і поширеності процесу. В даний час НЕК розглядають як поліетіологічне захворювання. До факторів ризику розвитку НЕК відносять недоношеність, гіпоксію (асфіксію) при народженні, бактеріальну колонізацію кишечника патогенною мікрофлорою. Мета та завдання: Аналізування усіх випадків захворювання новонароджених на некротичний ентероколіт на базі Вінницької обласної дитячої клінічної лікарні за період з 2011 по 2013 роки.

Necrotizing enterocolitis is a severe condition of the neonatal intestine characterised by intestinal inflammation and necrosis. Although many risk factors for this condition have been identified, the pathogenesis remains unclear. The identification of a large number of potential risk factors for this disease have led authors to speculate that the pathogenesis is likely to be multifactorial. • In this thesis we have examined the effects of necrotizing enterocolitis on the neonatal intestine, with particular reference to the supramucosal defence. In addition we have assayed the impact ofnecrotizing enterocolitis on the levels of faecal calprotectin levels in an attempt to develop a new tool for the early diagnosis ofnecrotizing enterocolitis. • These studies demonstrate that necrotizing enterocolitis is associated with changes in expression of components ofthe supramucosal barrier. In particular necrotizing enterocolitis appears to be associated with a reduction in expression ofTFF3. These changes were not seen in foetal material or in normal neonatal controls. There are also a number ofmore subtle alterations in gene expression demonstrable in necrotizing enterocolitis. However, it remains unclear as to whether these changes are as a result of tissue destruction and cellular damage, 2 Supplied by The British Library - 'The world's knowledge' fI or whether these changes pre-date the physiological insult responsible for the subsequent development ofnecrotizing enterocolitis. We have studied the expression of components ofthe supramucosal barrier in the developing gastrointestinal tract, to determine whether deficiencies in component of the supramucosal barrier as a result ofprematurity may represent an important component in the pathogenesis of necrotizing enterocolitis. The results of this work confirm the findings of other authors that both the mRNA signal and formed protein product of components of the supramucosal barrier are present in the developing human gastrointestinal tract from the end of the first trimester. It is unlikely that absolute failures ofproduction of individual components ofthe supramucosal barrier are solely responsible for the development of necrotizing enterocolitis. More subtle alterations may result in failure to produce of an effective supramucosal barrier, which may be important in contributing to the pathogenesis ofnecrotizing enterocolitis, but are beyond the scope ofthis experimental work. We have also demonstrated that necrotizing enterocolitis is associated with changes in faecal calprotectin levels in infants when compared to age-matched controls. This may prove an important observation, allowing for the utilization of faecal calprotectin as a diagnostic tool in the early diagnosis of necrotizing enterocolitis.

Objective: To identify statistical associations with necrotizing enterocolitis (NEC) severity as dichotomized into cases with Bell stage II and III disease. Study Design: We conducted a retrospective study using eight consecutive years of data from a multihospital healthcare system analyzed NEC severity (Bell stage II vs. III). Results: We identified 220 neonates with stage ≥ II who had 225 separate episodes of NEC (157 stage II and 68 stage III). Those with stage III were born at earlier gestational age (P<0.0001) and lower birth weight (P<0.0001). Diagnosis of NEC occurred on about the same day of life in stage II and stage III cases. Those who developed stage III had significantly higher C-reactive protein (P<0.0001), I/T ratio (P= 0.0005), mean platelet volume (MPV) (P= 0.0001) and lower pH (P<0.0001) and platelet counts (P<0.0001). Transfusions were more common to those who progressed to stage III (P<0.0001). Regression analysis indicated higher odds of stage III in relationship to the volume of RBC transfusions (OR 2.41, {CI 1.85 to 3.11}, P<0.0001) and pasteurized human milk (PHM) (OR 1.32, {CI 1.07 to 1.62}, P = 0.0089). In contrast, feeding early mother's own milk (colostrum) for five days reduced the odds for stage III (OR 0.802, {CI 0.67 to 0.96}, P=0.0170). Those with small bowel resection were less likely to have received mother's own milk before NEC (OR 0.94, {CI 0.89 to 0.99}, P = 0.019) and factors predicting death from NEC were a low pH (OR 2.21, {CI 1.27 to 3.85}, P = 0.0005) and less colostrum (OR 0.96, {CI 0.94 to 0.99}, P = 0.003). Conclusions: RBC transfusions and PHM increased the odds for stage III NEC, whereas early mother's own milk five days reduced the odds. Mother's own milk with PHM decreased the risk for small bowl resection and early mother's milk decreased the odds for mortality from NEC. Future research and prospective randomized controlled studies are needed to quantify any reduction in NEC severity on the basis of decreasing RBC transfusions and increasing early mother's own milk or colostrum.

Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel disease affecting premature infants. The disease can progress rapidly and is associated with high mortality. Early diagnosis can be difficult as the early manifestations of NEC can be very similar to sepsis. There is currently no pre-symptomatic screening method available. The aim of the work reported in this thesis was to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants. Methods: Longitudinal (≥ daily) faecal samples and daily clinical data from throughout their neonatal intensive care unit stay were collected from 369 premature infants born at < 32 weeks gestation over a two-year period. The faecal microbiota of 12 infants with definite/severe NEC, 8 with suspected NEC, and 44 control infants was characterised using next-generation sequencing of 16S ribosomal RNA gene regions. Clinical characteristics and operational taxonomical units (OTUs) that discriminated cases from controls were identified using logistic regression. Bacteria isolated from stored faecal samples underwent selective culture, and the resulting isolates were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight. Isolates identified as Clostridium perfringens were additionally typed and screened for the presence of toxin genes. Results: Faecal samples collected from four NEC infants just prior to diagnosis were found to contain a higher abundance of a clostridial OTU compared to matched control samples. Culture investigation identified this OTU as Clostridium perfringens. No two Clostridium perfringens isolates were found to be identical, as determined by fluorescent amplified fragment length polymorphism. Infants with NEC who did not have an over-abundance of the clostridial OTU (n=7) prior to diagnosis had a faecal microbiota dominated by a Klebsiella OTU. Only one infant did not have an abundance of either the clostridial OTU or the Klebsiella OTU. The use of prolonged continuous positive airway pressure therapy with supplemental oxygen (CPAP oxygen) was also found to be associated with NEC risk. Conclusions: Two OTUs (Clostridium and Klebsiella) were identified at high levels in faecal samples collected from infants prior to NEC diagnosis. A screening tool incorporating these biomarkers with the duration of CPAP oxygen use may aid the clinician in making an early diagnosis of NEC.

Objetivo: Avaliar a influência da localização da enterocolite necrosante neonatal na mortalidade de recém-nascidos (RN) submetidos à laparotomia exploradora. Métodos: Estudo de coorte prospectiva de 141 recém-nascidos com ECN submetidos consecutivamente à laparotomia exploradora no período de novembro de 1991 a dezembro de 2005. Foram avaliados dados epidemiológicos, localização e extensão da doença, crescimento intra-uterino e o número de óbitos no período de 60 dias após a cirurgia. Resultados: Setenta e quatro (52,5%) crianças eram do sexo masculino, com peso médio de nascimento de 1.589 ± 665 gramas, com idade gestacional média de 33,6 ± 2,9 semanas. Prematuridade ocorreu em 84,4% (119/141) dos RN. Cinqüenta e sete (40,4%) eram pequenos para a idade gestacional. Óbito ocorreu em 68 crianças (48,2%). Na análise bivariada, observou-se que o comprometimento do jejuno-íleo foi associado com alta mortalidade (20 óbitos - 76,9%; OR = 20; intervalo de confiança de 95% = 4,6 - 96,3; p < 0,001) e que a doença no jejuno estava associada à maior extensão da ECN. Entretanto, no modelo de regressão logística múltipla com controle individual de cada variável, a doença no jejuno-íleo (OR = 0,61; intervalo de confiança de 95% = 0,06 - 6,14; p = 0,68) e no intestino grosso (OR = 2,91; intervalo de confiança de 95% = 0,81 - 10,50; p = 0,10) não foram consideradas fatores de risco para o óbito. Conclusões: Em análise adequada, com controle isolado de cada variável estudada, a mortalidade foi independente da localização da ECN no intestino delgado ou no intestino grosso. Porém, a localização da doença no jejuno foi um marcador de maior extensão da ECN e, conseqüentemente, de pior prognóstico. Extensão difusa da doença e recém-nascidos PIG foram os mais importantes fatores de risco de ocorrência de óbito nesses recém-nascidos submetidos à cirurgia.
Aim of the study: To evaluate the effect of disease site on the mortality rate of newborns with necrotizing enterocolitis (NEC) undergoing exploratory laparotomy. Methods: Prospective cohort of 141 consecutive newborns with NEC who underwent laparotomy from November 1991 to December 2005. The study variables included epidemiologic data, disease site and extent, intrauterine growth, and number of deaths in the 60 days after operation. The protocol was approved by the institution’s Research Ethics Committee. Main results: Seventy-four (52.5%) infants were male. Mean birth weight was 1,589 ± 665 g, and mean gestational age was 33.6 ± 2.9 weeks. One-hundred and nineteen (84.4%) newborns were premature. Small for gestational age was observed in 57 (40.4%). Sixty-eight (48.2%) infants died. Bivariate analysis revealed that involvement of the jejunum and ileum was associated with high mortality rates (20 deaths, 76.9%; OR = 20; 95% 95% CI = 4.6 – 96.3; p < 0.001), and that involvement of the jejunum was associated with greater disease extent. After controlling for individual variables, logistic regression showed that the mortality associated with jejunum and ileum involvement (OR = 0.61; 95% CI = 0.06 - 6.14; p = 0.68) did not differ from that associated with large bowel involvement (OR = 2.91; 95% CI = 0.81 – 10.50; p = 0.10); however, jejunum involvement remained significantly associated with disease extent. Conclusions: NEC-related mortality in newborns undergoing laparotomy was not influenced by disease site (small or large bowel). However, jejunum involvement was a marker of greater disease extent and therefore of poor prognosis. Diffuse disease extent and small for gestational age were the most important markers of risk of death in NEC newborns submitted to surgery.

Neonatal necrotizing enterocolitis (NEC) is the gastrointestinal disease responsible for the most deaths of premature infants. This honors thesis looks at the role of bile acids in NEC through analyzing bile acid concentration in neonatal stool samples. Subjects were enrolled from the neonatal intensive care unit at the Banner University Medical Center in Tucson, Arizona, their fecal samples were collected and then analyzed using Diazyme Total Bile Acids Assay Kit. There was a statistically significant difference in coefficients of variation between infants with and without NEC, suggesting that bile acid levels could be used clinically to predict the onset of NEC so that changes can be made to prevent the progression of the disease.

This study tested a method of overexpressing the apical sodium-dependent bile acid transporter (ASBT) in IEC-6 cells to replicate necrotizing enterocolitis (NEC), a gastrointestinal disease prevalent in premature infants. The expression vector pcDNA4/TO was altered to pDR1019, containing the Rattus norvegicus ASBT coding sequence. Sequencing confirmed the ASBT sequence in pDR1019 using forward, reverse, and midsequence primers (respective E-values: 0.0, 0.0, and 2e ⁻¹⁷⁴). IEC-6 cells were transfected with varying ratios of pcDNA6/TR (tetracycline-controlled repression vector) and pDR1019: 6:1, 15:1, and 30:1 pcDNA6/TR:pDR1019. Using relative quantitative real-time PCR (qrt-PCR), the 30:1 transfection had the greatest fold-change difference of ASBT mRNA expression relative to non-transfected IEC-6 cells (overexpression-induced and noninduced trials: 3120.0-fold and 1445.6-fold, respectively). To test the effects of bile acids and cytokines on ASBT expression in IEC-6 cells with overexpressed ASBT, a 30:1 pcDNA6/TR:pDR1019 transfection was performed, followed by treatments of chenodeoxycholic acid (CDCA), tumor necrosis factor-alpha (TNF-α), and interleukin 18 (IL18). According to a qrt-PCR, the ASBT mRNA expression fold-change of non-transfected trials were: CDCA (2.09x10⁹-fold), TNF-α (0.39-fold), IL18 (2.12x10⁹-fold); insufficient cells survived the transfection followed by treatments to yield usable RNA. Using this cell-based model to replicate NEC will aid future molecularly-based investigations of the disease.

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As múltiplas causas de Enterocolite Necrosante (NEC) e seus indicativos clínicos utilizados para o diagnóstico ainda se mantêm elusivos. Biomarcadores alternativos para o diagnóstico precoce de NEC em recém-nascidos prematuros e um melhor entendimento dos fatores de risco para o desenvolvimento de NEC são desafios emergentes. Em uma tentativa de contribuir para a solução deste problema, neste trabalho nós rastreamos as mudanças no microbioma dos recém-nascidos (diversidade microbiana, abundância e estrutura) com NEC, iniciando com a primeira evacuação (mecônio) e continuando até a liberação, e comparamos essas mudanças com os prematuros sem o diagnóstico de NEC. Um estudo metataxonomico foi conduzido usando 88 amostras fecais, a partir da primeira evacuação até a 5ª semana de vida, obtidas de 25 recém-nascidos prematuros (14 controles e 11 casos de NEC) selecionados de um grupo de 52 prematuros. Nossos dados revelaram que casos de NEC apresentaram baixa diversidade e uma transição anormal da comunidade microbiana até o diagnóstico de NEC. Um microrganismo pertencendo a família Enterobacteriaceae foi consistentemente mais abundante em prematuros com NEC do que nos controles, mesmo nas amostras de mecônio, e foi considerado um constituinte chave da comunidade microbiana correlacionada com a doença. Finalmente, nos também detectamos uma distorção na associação micróbio-micróbio nas amostras de mecônio dos casos de NEC. Portanto, nossos dados sugerem que a detecção precoce de elevada dominância de Enterobacteriaceae, baixa diversidade e associações micróbio-micróbio nos primeiros dias de vida poderiam ser utilizados como indicativo de risco de desenvolvimento de Enterocolite Necrosante nas UTIs neonatais brasileiras.
The multiple causes of Necrotizing Enterocolitis (NEC) as well as the clinical predictors used for diagnosis have remained elusive to date. Alternative biomarkers for early diagnosis of NEC in premature infants and a better understanding of risk factors for NEC development are emergent challenges. In attempt to contribute to solve this problem, in this work we tracked the changes in the newborn’s microbiome (microbial diversity, abundance and structure) with Necrotizing Enterocolitis beginning with the first stool (meconium) continuing until discharge and compare those changes with preterns without NEC diagnosis. A metataxonomy study was conducted using 88 fecal samples from the first stool (meconium) until the 5th week of life obtained from 25 preterm babies (14 controls and 11 NEC cases) selected from a cohort of 52 premature infants. Our data revealed low microbial diversity in NEC cases and an abnormal transition of the microbial community until NEC diagnosis. A microbial phylotype belonging to the Enterobacteriaceae family were consistently more abundant in NEC than in the controls even in meconium samples and was considered a key constituent of the microbial community that correlated with the disease. Finally, we also detected a disruption of microbial-microbial associations in the meconium samples of NEC cases. Thus, our data suggests that early detection of high dominance of Enterobacteriaceae, low diversity and altered microbial-microbial associations at the first days of life could be used as an indicative of risk of preterm development of Necrotizing Enterocolitis in Brazilian NICU’s.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Jun. 4, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Nursing." Discipline: Nursing; Department/School: Nursing.

Orientador: Diana Rodrigues de Pina
Resumo: A enterocolite necrosante (NEC – do inglês necrotizing enterocolitis) é caracterizada pela necrose isquêmica da mucosa intestinal de recém-nascidos prematuros. Uma vez que existe a suspeita de NEC, é instituída uma rotina de radiografias abdominais de acordo com a gravidade da doença. Os principais achados radiológicos de pacientes com NEC são: distensão abdominal generalizada, pneumatose intestinal, pneumoperitônio. Entretanto, a interpretação dessas radiografias é um processo difícil devido à falta de especificidade da maioria dos achados radiológicos. O objetivo desse estudo foi desenvolver uma ferramenta computacional que auxilie o corpo clínico na análise de radiografias abdominais para a diferenciação de alças normais e alças inflamadas em recém-nascidos prematuros. Para o desenvolvimento desta pesquisa foi utilizado um banco de dados composto por 45 radiografias abdominais e algoritmos computacionais desenvolvidos em ambiente MatLab. As espessuras das alças intestinais foram quantificadas através da ferramenta computacional Largura a Meia Altura (FWHM – do inglês Full Width at Half Maximum), e classificadas como alças edemaciadas ou alças normais. Para a análise de textura e extração de características, a fim de diferenciar regiões de pneumatose, aplicamos a técnica de Transformada Wavelet. Com a utilização do algoritmo, as alças intestinais normais apresentaram mediana igual a 10,30 pixels, enquanto as alças edemaciadas, foram estatisticamente maiores (Mann Whitney, p... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Microarray experiments generate vast amounts of data that evidently reflect many aspects of the underlying biological processes. A major challenge in computational biology is to extract, from such data, significant information and knowledge about the complex interplay between genes/proteins. An analytical approach that has recently gained much interest is reverse engineering of genetic networks. This is a very challenging approach, primarily due to the dimensionality of the gene expression data (many genes, few time points) and the potentially low information content of the data. Bayesian networks (BNs) and its extension, dynamic Bayesian networks (DBNs) are statistical machine learning approaches that have become popular for reverse engineering. In the present study, a DBN learning algorithm was applied to gene expression data produced from experiments that aimed to study the etiology of necrotizing enterocolitis (NEC), a gastrointestinal inflammatory (GI) disease that is the most common GI emergency in neonates. The data sets were particularly challenging for the DBN learning algorithm in that they contain gene expression measurements for relatively few time points, between which the sampling intervals are long. The aim of this study was, therefore, to evaluate the applicability of DBNs when learning genetic networks for the NEC disease, i.e. from the above-mentioned data sets, and use biological knowledge to assess the hypothesized gene interactions. From the results, it was concluded that the NEC gene expression data sets were not informative enough for effective derivation of genetic networks for the NEC disease with DBNs and Bayesian learning.

Background. Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in neonates. No specific therapy is available and the treatment is only supportive. Amniotic Fluid Stem (AFS) cells represent a novel class of pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all embryonic germ layers and do not form tumors after implantation in vivo. These characteristics, together with the absence of ethical issues concerning their obtainment, make AFS cells good candidates for cell therapy of human diseases. Aim. The aim of this study was to explore the therapeutic potential of Amniotic Fluid Stem (AFS) cells in a rat model of NEC. Methods. AFS cells were obtained from green fluorescent (GFP+) transgenic pregnant rats at 16 days p.c. by c-kit selection. NEC was induced in newborn rats by hyperosmolar milk formula, oral lipopolysaccharide and hypoxia. Rats were divided into 2 groups receiving at 24 and 48 hours of life an intraperitoneal injection of: (i) phosphate buffered saline (PBS; n=120) or (ii) 2x106 AFS cells (n= 121). Additional groups of animals, either injected with bone marrow-derived mesenchymal stem cells (i.e. rat BM-MSCs) or committed cells (i.e. rat myoblasts), or non subjected to NEC induction (i.e. healthy breast fed newborn rats), were used as additional controls. All groups were blindly compared regarding survival, clinical status, radiological features (abdominal MRI), gut motility (carmine red transit time) and intestinal permeability (plasma lactulose/mannitol ratio). Intestines were blindly analyzed for macro- and microscopic appearance, transcriptional profile (microarray-based expression analysis), neutrophil infiltration (myeloperoxidase activity), enterocyte proliferation (EdU assay) and apoptosis (cleaved caspase 3 immunohistochemistry). AFS cell integration in the gut was evaluated by GFP amplification and immunostaining. Cyclooxygenase 2 (COX2+) cells in the lamina propria were evaluated by immunofluorescence. COX2 activity was inhibited in vivo using selective (celecoxib) and non-selective (ibuprofen) inhibitors; the effects of COX2 pharmacological inhibition on rat survival and clinical status were evaluated. Results. Compared to animals injected with PBS, rats receiving AFS cells survived longer (p<0.0001), and showed: improved clinical conditions (p<0.001), better abdominal appearance at MRI, restored intestinal transit (p<0.01), decreased intestinal permeability (p<0.05), reduced macroscopical (p<0.001) and histological gut damage (p<0.001). These beneficial effects were specific to AFS cells as neither BM-MSCs nor myoblasts were able to improve animal morbidity/mortality in comparison to PBS (p=n.s.). AFS cells integrated in the intestine with various degrees of spreading in all the animals. cDNA arrays comparing the intestinal transcriptional profile of PBS vs. AFS cell rats showed differences in the expression of genes involved in inflammation, apoptosis and cell proliferation which were respectively down-regulated (inflammation and apoptosis) and up-regulated (proliferation) in the AFS cell group. At a protein level, AFS cell rats had lower gut neutrophil infiltration (p<0.05), reduced enterocyte apoptosis (p<0.05) and increased enterocyte proliferation (p<0.0001) compared to PBS rats. In rats treated with AFS cells vs. rats injected with PBS, COX2+ cells in the lamina propria were increased (p<0.001) and repositioned under crypts (p<0.001). Moreover, both the total number of COX-2+ cells per villus unit and the number of cryptal COX-2+ cells inversely correlated with the degree of intestinal damage (p=0.014). The pharmacological inhibition of COX2 activity did not exert any effect in PBS rats, whereas it completely abolished AFS cell beneficial effects on animal survival and clinical behavior. Conclusions. In experimental NEC, AFS cell administration via the intraperitoneal route is associated with reduced animal morbidity/mortality and decreased incidence of NEC. AFS cell beneficial effects seems to be related to decreased intestinal neutrophil infiltration, enhanced enterocyte proliferation and reduced epithelial apoptosis. We hypothesize that is achieved through activation of COX2+ cells in the lamina propria. Stem cell therapy may represent a new therapeutic option for infants with NEC.
Premesse. L’enterocolite necrotizzante (NEC) rappresenta la causa più frequente di insufficienza intestinale in età pediatrica. Non esistono tuttora terapie specifiche per la NEC ed il suo trattamento si basa unicamente sulla terapia medica di supporto e sulla rimozione chirurgica delle porzioni di intestino affetto. Le cellule staminali derivanti da liquido amniotico (AFSC) sono una popolazione di cellule staminali di origine fetale descritta per la prima volta nel 2007. Esse possiedono delle caratteristiche intermedie fra le cellule staminali embrionali (i.e. pluripotenza) e le cellule staminali adulte (i.e. mancata tumorigenicità dopo iniezione in vivo) che le rendono candidati ideali per la terapia cellulare. Scopo dello studio. Valutare il potenziale terapeutico delle cellule staminali derivanti da liquido amniotico (AFSC) in un modello animale di NEC. Materiali e metodi. AFSC sono state derivate da ratti Sprague-Dawley GFP+ (i.e. esprimenti in modo costitutivo la proteina reporter “Green Fluorescent Protein”) al 16^ giorno p.c. tramite immunoselezione per il loro caratterstico marcatore di superficie (i.e. c-kit/CD117). Le cellule ottenute sono state caratterizzate per morfologia e immunofenotipo. La NEC e’ stata indotta in ratti neonati tramite l’utilizzo di elementi simili ai fattori patogenetici implicati nell’insorgenza della NEC umana: alimentazione con latte formulato iperosmolare, eventi ipossici, somministrazione di lipopolisaccaride. I ratti, suddivisi in due gruppi principali, hanno ricevuto a 24 e 48 ore di vita, per via intraperitoneale: i. 50 ul di soluzione salina (PBS; n=120) o ii. 2x106 AFSC (n= 121). Altri gruppi di animali, trattati con cellule staminali mesenchimali di ratto derivanti da midollo osseo o con mioblasti, oppure non sottoposti all’induzione di NEC (i.e. neonati sani allattati al seno), sono stati utilizzati come gruppi aggiuntivi di controllo. I diversi gruppi di animali sono stati valutati in cieco per i seguenti parametri: sopravvivenza, stato clinico, aspetto radiologico intestinale (RM ad alta risoluzione), motilita’ intestinale (studio del tempo di transito con coloranti vitali), permeabilita’ intestinale (rapporto lattulosio/mannitolo plasmatici). L’intestino e’ stato valutato in cieco per: aspetto macroscopico ed istologico, profilo di espressione genica (tramite tecnologia cDNA-microarray), infiltrazione neutrofila (saggio di attivita’ della mieloperossidasi), proliferazione (EdU) e apoptosi degli enterociti (immunoistochimica per caspasi 3 attivata). L’integrazione di AFSC nell’intestino e’ stata analizzata sia tramite PCR (amplificazione del gene gfp) che tramite immunoistochimica (immunofluorescenza per GFP). Il numero e la localizzazione delle cellule stromali esprimenti COX2 nella mucosa sono stati valutati con immunofluorescenza. L’attivita’ di COX2, in vivo, e’ stata inibita farmacologicamente con inibitori selettivi (celecoxib) e non selettivi di COX2 (ibuprofene); gli effetti di tale inibizione sulla sopravvivenza e sulla morbidita’ degli animali trattai con AFSC o PBS sono stati analizzati in cieco. Risultati. La somministrazione di AFSC, per via intraperitoneale a ratti neonati affetti da NEC: migliora significativamente la sopravvivenza degli animali sia rispetto alla somministrazione di PBS (p<0.0001) che di linee cellulari di controllo (i.e. cellule staminali mesenchimali di ratto derivanti da midollo osseo [p=0.024] e mioblasti di ratto [p<0.0001]). Rispetto alla somministrazione di PBS, inoltre, il trattamento con AFSC: i. riduce la morbidita’ degli animali migliorandone l’aspetto clinico (p<0.001); ii. riduce significativamente il danno intestinale sia alla valutazione dell’addome con RM ad alta risoluzione che all’esame macroscopico (p<0.001) ed istologico dell’intestino (p<0.001); iii. migliora significativamente la funzionalità dell’intestino sia per quanto concerne la motilità (p<0.01) che l’assorbimento di nutrienti (p<0.05). AFSC somministrate per via intraperitoneale migrano preferenzialmente verso l’intestino dove, seppur con un basso tasso di integrazione tissutale, sono in grado di localizzarsi in tutti gli strati della parete e talora di differenziarsi in cellule con fenotipo mesenchimale (i.e. cellule muscolari lisce). La somministrazione di AFSC in ratti neonati affetti da NEC è in grado di modificare il profilo di espressione genica dell’intestino incrementando l’espressione di geni coinvolti nella proliferazione e riducendo l’espressione di geni coinvolti in apoptosi e infiammazione. Tali dati di espressione sono stati confermati a livello proteico dimostrando che nell’intestino dei ratti affetti da NEC trattati con AFSC v.s. PBS è maggiore la proliferazione delle cellule epiteliali (p<0.0001), minore l’apoptosi degli enterociti (p<0.05) e ridotta l’infiltrazione neutrofila tissutale (p<0.05). La somministrazione di AFSC, inoltre, determina l’attivazione di una popolazione di cellule stromali esprimenti la ciclossigenasi 2 (COX2) nella lamina propria della mucosa intestinale. Più in dettaglio la somministrazione di AFSC v.s. PBS causa un significativo aumento del numero delle cellule COX2+ nella lamina propria (p<0.001) e un loro spostamento dall’asse del villo alla niche delle cripte intestinali (p<0.001). Tale effetto costituisce il meccanismo d’azione di AFSC poiché la somministrazione in vivo di inibitori selettivi e non selettivi di COX2 (ma non di COX1) a ratti affetti da NEC abolisce gli effetti positivi di AFSC su morbidità e mortalità degli animali ma non ha alcun effetto sugli animali trattati con PBS. Conclusioni. In un modello animale di NEC, AFSC sono in grado di migliorare in modo significativo la mortalita’ e la morbidita’ degli animali e il danno intestinale. AFSC non determinano direttamente tali effetti rigenerando di per sé l’intestino ma indirettamente attivando le cellule stromali esprimenti COX2 presenti nella lamina propria le quali a loro volta stimolano la proliferazione e riducono l’apoptosi delle cellule epiteliali intestinali residenti. Sebbene ulteriori studi siano necessari (e.g. per identificare i fattori/meccanismi molecolari responsabili dell’attivazione delle cellule COX2+), riteniamo che la terapia con cellule staminali derivanti da liquido amniotico possa rappresentare una nuova prospettiva terapeutica per i pazienti affetti da NEC.

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The Necrotizing enterocolitis (ECN) is a severe gastrointestinal illness caused by multiple factors and is among the leading causes of neonatal mortality in the Neonatal Intensive Care Unit of the Environment (NICU). The incidence of NEC is inversely proportional to gestational age and birth weight, reaching 12% of children weighing less than 1500g and triggering death in 30% of casos.Tem up to analyze the occurrence of ECN and associated factors its development in newborns (NB) with low weight. It is an epidemiological case-control study, conducted from March 1, 2014 to June 30, 2015, in two NICUs in Sao Luis, MA. The sample size was calculated considering a case to three controls (1:3), establishing confidence level of 95% and power of 80% study, sufficient to detect an OR = 2.5, making 236 newborns (RN) underweight, and 59 infants with NEC diagnoses (case group) and 177 infants without NEC (control group). In analyzing the data maternal variables (gestational period and the type of delivery) and newborn (birth and hospitalization) were organized in six blocks, arranged in a hierarchical structure, and analyzed in STATA 11.0 program. The differences between means were assessed by Student's t-test, whereas the differences between the medians by Man Whitney test. It was considered as the dependent variable, the ECN, and as independent, maternal and neonatal variables. Univariate analysis was performed between independent variables of two groups: case and control, estimated the OR values, with reference category OR = 1, built the confidence intervals of 95% and certain values of p. In the hierarchical analysis was carried out by the group of variables in levels according to influence the outcome. Of the 59 cases of NEC, 61.02% were female, with a median of 45 days hospital stay; and 177 controls without ECN, 54.55% were male, with a median hospital stay of 19.5 days. As for the clinical outcome of cases of NEC, 40.68% progressed to death. At the end of hierarchical analysis remained statistically significant association, the use of antenatal corticosteroids (OR = 2.90; p <0.001), reduced amniotic fluid (OR = 2.03;p<0.001), resuscitation at birth (OR = 1, 35, p = 0.010), birth weight ≤1500g (OR = 3.32, p <0.001), transfusions (OR = 2.11, p = 0.040) and the use of surfactant (OR = 2.41, p = 0.020). It concludes that maternal aspects related to pregnancy and neonatal concerning the birth and hospitalization may be influencing the appearance of NEC.
A Enterocolite Necrosante(ECN) é uma grave enfermidade gastrintestinal, de causa multifatorial e está entre as principais causas de mortalidade neonatal no ambiente da Unidade de Terapia Intensiva Neonatal (UTIN). A incidência da ECN é inversamente proporcional à idade gestacional e o peso de nascimento, atingindo 12% das crianças com peso inferior a 1500g e desencadeando o óbito em 30% dos casos.Tem-se como objetivo analisar a ocorrência de ECN e os fatores associados ao seu desenvolvimento em recém-nascidos (RN)de baixo peso. Trata-se de um estudo epidemiológico tipo caso controle, realizado no período de 01 de março de 2014 a 30 de junho de 2015, em duas UTINs de São Luís-MA. O tamanho da amostra foi calculado considerando um caso para três controles (1:3), estabelecendo nível de confiança de 95% e poder do estudo de 80%,suficiente para detectar um OR=2,5, perfazendo 236 recém-nascidos (RN) de baixo peso, sendo 59 RN com diagnósticos de ECN (grupo caso) e 177 RN sem ECN (grupo controle). Na análise dos dados as variáveis maternas (período gestacional e o tipo de parto) e neonatais (nascimento e hospitalização) foram organizadas em seis blocos e dispostas em uma estrutura hierarquizada, e analisadas no programa STATA 11.0. As diferenças entre as médias foram avaliadas pelo teste T-Student, enquanto que as diferenças entre as medianas pelo teste de Man Whitney. Considerou-se como variável resposta, a ECN, e como independentes, as variáveis maternas e neonatais. Foi realizada análise univariada entre as variáveis independentes dos doisgrupos: caso e controle, estimados os valores das OR, tendo como categoria de referência OR=1, construídos os intervalos de confiança de 95% e determinados os valores de p. Na análise hierarquizada foi realizadoo agrupamento das variáveis em níveis segundo a influência no desfecho. Dos 59 casos de ECN, 61,02% eram do sexo feminino,com mediana do tempo de hospitalização de 45 dias; edos 177 controles sem ECN, 54,55% eram do sexo masculino, com mediana do tempo de hospitalização de 19,5 dias. Quanto ao desfecho clínico dos casos de ECN, 40,68% evoluíram a óbito. Ao final da análise hierarquizada mantiveram associação estatisticamente significante, o uso de corticóide antenatal (OR=2,90; p<0,001), líquido amniótico reduzido (OR=2,03;p<0,001), reanimação ao nascimento (OR=1,35; p=0,010), peso ao nascimento ≤1500g (OR=3,32;p<0,001), transfusão (OR=2,11;p=0,040) e uso de surfactante (OR=2,41;p=0,020). Conclui-se que os aspectos maternos relacionados ao período gestacional e os neonatais referentes ao nascimento e hospitalização podem estarinfluenciando no aparecimento da ECN.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Introduction: An association between maternal human immunodeficiency virus (HIV) infection and Necrotizing Enterocolitis (NEC) in preterm infants has been reported. The impact of probiotics in an HIV-exposed very low birth weight (VLBW) infant on the occurrence of NEC is uncertain at present; however it is known that probiotics have protective effects against inflammation and prevent NEC. Postnatal growth restriction is a major issue in preterm, especially extremely-low-birth-weight (ELBW) infants and probiotics have been found to improve feeding tolerance in preterm infants. Human milk oligosaccharides (HMO) also known as the prebiotics of human milk, are known to have bifidogenic and anti-adhesive effects. Infants that receive human milk show a reduced incidence of NEC compared to those who receive infant formula. Very little is known about the composition of breast milk in the HIV-infected mother. Objective: The primary objective of the study was to assess the effect of probiotics on the incidence and severity of NEC in high-risk infants born to HIV-positive and HIV-negative women. The secondary objectives were to assess the effect of probiotic administration on feeding tolerance and growth outcomes of HIV-exposed but uninfected preterm infants, to describe the HMO composition of HIV-infected mothers breast milk and lastly to determine if HMO composition affects the incidence of NEC in HIV-exposed preterm very low birth weight infants. Patients and Methods: A randomized, double blind, placebo controlled trial was conducted for the period July 2011 to August 2012. HIV-exposed and HIV-unexposed premature (<34 weeks gestation) infants with a birth weight of ≥500g and ≤1250g were randomized to receive either a probiotic or a placebo. The probiotic consisted of 1x109 CFU, L. rhamnosus GG and B. infantis per day and was administered for 28 days. NEC was graded according to Bell’s criteria. Anthropometrical parameters and daily intakes were monitored. Breats milk samples were analysed for oligosaccharide content. Results: 74 HIV-exposed and 110 HIV-unexposed infants were enrolled and randomized (mean birth-weight, 987g; mean gestational 28.7 weeks). The incidence of death and NEC did not differ significantly between the HIV-exposed and unexposed groups but a significantly higher NEC incidence was found in the control group. There was no difference in the average daily weight gain for treatment groups or HIV exposure. The HIV-exposed group achieved significantly higher z-scores for length and head circumference at day 28 than the unexposed group (p<0.01 and p=0.03, respectively). There were no differences in the incidence of any signs of feeding intolerance and abdominal distension between the groups. Our results show significantly higher absolute concentrations of 2’-fucosyllactose, laco-N-tetraose and lacto-N-fucopentaose 1 and higher relative abundance of 3’-sialyllactose, difucosyl-lacto-N-tetraose and fucosyl-disialyllacto-N-hexaose in HIV-infected compared to -uninfected Secretor women. DSLNT concentrations were significantly lower in the breast milk of mothers whose infants developed NEC compared to infants without NEC. Conclusion: Probiotic supplementation reduced the incidence of NEC in the premature infants; however results failed to show a lower incidence of NEC in HIV-exposed premature infants. Probiotic supplementation did not affect growth outcomes or the incidence of any signs of feeding intolerance in HIV-exposure. The data confirms previous reports that HIV-infected mothers have higher 3’sialyllactose milk concentrations. Most intriguing though, the data also indicates that low levels of DSLNT in the mother’s milk increase the infant’s risk for NEC, which is in accordance with results from previously published animal studies and warrants further investigation.
AFRIKAANSE OPSOMMING: Inleiding: ʼn Verwantskap tussen moederlike menslike immuniteitsgebreksvirus (MIV) en nekrotiserende enterokolitis (NEK) in premature babas is aangemeld. Die impak van probiotika in ʼn MIV-blootgestelde baie lae geboortemassa (BLGM) baba op die voorkoms van NEK is tans nog onseker, maar dit is wel bekend dat probiotika ʼn beskermende effek het teen inflammasie en die voorkoms van NEK. Nageboortelike groei beperkings is ʼn groot probleem in premature, veral ekstreme lae geboortemassa (ELGM) babas. Daar is gevind dat probiotika voeding toleransie in premature babas kan verbeter. Menslike melk oligosakkariede (MMO), ook bekend as die prebiotika van menslike melk, is bekend om bifidogeniese en anti-kleef effekte te hê. Babas wat moedersmelk ontvang toon ʼn verlaagde voorkoms van NEK in vergelyking met diegene wat baba formule melk ontvang. Baie min inligting is bekend oor die samestelling van borsmelk in die MIV-positiewe moeder. Doel: Die primêre doel van die studie was om die effek van probiotika op die voorkoms en die graad van NEK in hoë risiko babas van MIV-positiewe en MIV-negatiewe vroue te bepaal. Die sekondêre doelwitte was om die effek van probiotika op voeding verdraagsaamheid en groei uitkomste van MIV-blootgestelde, maar nie- geinfekteerde premature babas te evalueer sowel as die MMO samestelling van MIV-positiewe moeders se borsmelk te beskryf en laastens om die invloed van die MMO samestelling op die voorkoms van NEK in baie lae geboortegewig MIV-blootgestelde premature babas te beskryf. Pasiënte en Metodes: ʼn Gerandomiseerde, dubbelblinde, plasebo-beheerde studie is vir die tydperk Julie 2011 tot Augustus 2012 onderneem. MIV-blootgestelde en nie-blootgestelde premature (<34 weke) babas met 'n geboorte gewig van ≥500g en ≤1250g was ewekansig verdeel om probiotika of plasebo te ontvang. Die probiotika het bestaan uit 1x109 kolonie vormende eenhede, L. rhamnosus GG en B. infantis per dag en is toegedien vir 28 dae. NEK is gegradeer volgens Bell se kriteria. Antropometriese parameters en daaglikse inname is gemonitor. Borsmelk monsters is geanaliseer vir oligosakkaried inhoud. Resultate: 74 MIV-blootgestelde en 110 MIV-nie-blootgestelde babas is ingesluit en ewekansig ingedeel (gemiddelde geboorte gewig, 987g, gemiddelde gestasie 28,7 weke). Die voorkoms van die sterftes en NEK het nie beduidend verskil tussen die MIV-blootgestelde en nie-blootgestelde groepe nie, maar 'n beduidende verskil is gevind vir NEK voorkoms tussen die studie en die kontrole groep. Daar was geen verskil in die gemiddelde daaglikse gewigstoename tussen die behandelings groepe of MIV-blootstelling nie. Die MIV-blootgestelde groep het beduidend hoër z-tellings vir lengte en kopomtrek op dag 28 getoon teenoor die nie-blootgestelde groep (p <0.01 en p = 0,03, onderskeidelik). Daar was geen verskille in die voorkoms van voeding onverdraagsaamheid en abdominale distensie tussen die twee groepe nie. Ons resultate dui op aansienlik hoër absolute konsentrasies van 2'-fucosyllactose, laco-N-tetraose en lakto-N-fucopentaose 1 en hoër relatiewe voorkoms van 3'-sialyllactose, difucosyl-lakto-N-tetraose en fucosyl-disialyllacto-N-hexaose in MIV-positiewe vroue in vergelyking met-negatiewe Sekretor vroue. DSLNT konsentrasies was aansienlik laer in die melk van moeders wie se babas NEK ontwikkel het in vergelyking met babas sonder NEK. Gevolgtrekking: Probiotika aanvullings verminder die voorkoms van NEK in premature babas, maar die resultate kon nie ʼn laer voorkoms van NEK in MIV-blootgestelde premature babas bewys nie. Probiotiese aanvulling het geen invloed op groei uitkomste of die voorkoms van voeding onverdraagsaamheid in MIV-blootstelling getoon nie. Die data bevestig vorige verslae wat aandui dat MIV-besmette moeders hoër 3'sialyllactose borsmelk konsentrasies het. ʼn Interessante aspek is dat lae vlakke van DSLNT in die moeder se melk beduidend is van ʼn verhoogde risiko vir NEK, wat in ooreenstemming is met die resultate uit voorheen gepubliseerde dier studies en regverdig verdere ondersoeke.

INTRODUÇÃO: O conhecimento de valores de normalidade do fluxo sanguíneo da artéria mesentérica superior (AMS) em recém-nascidos prematuros (RNPT) saudáveis pode prevenir quadros de intolerância alimentar e a ocorrência da enterocolite necrosante. MÉTODOS: Com o objetivo de descrever a evolução dos índices de avaliação da dopplerfluxometria da AMS em RNPT saudáveis de idade gestacional entre 27 e 34 semanas completas, no primeiro, no terceiro, no sétimo e semanalmente (14, 21, 28, 35 e 42 dias de vida), foi realizado este estudo coorte prospectivo em RNPT de idade gestacional ao nascimento entre 27 e 34 semanas completas. O exame dopplerfluxométrico foi realizado, após o consentimento livre e esclarecido dos responsáveis pelos RNPT, através do aparelho Logic Book 8C-RS (General Eletric EUA); obtendo-se as seguintes medidas: pico de velocidade sistólica (PVS), pico de velocidade diastólica final (PVDF) e média de velocidade de fluxo; sendo, após, calculadas o Índice de Pourcelot, sendo: [pico de velocidade sistólico pico diastólico final] / pico de velocidade sistólico, que representa um índice de resistência (IR); e índice de pulsatilidade (IP). Foram excluídos: recém-nascidos com instabilidade hemodinâmica; em ventilação assistida com altos parâmetros; síndromes mal-formativas; intolerância alimentar ou enterocolite necrosante; fototerapia; presença de cateteres umbilicais, persistência de canal arterial e pequenos para a idade gestacional. O exame pré-prandial foi realizado antes da alimentação (até 30 minutos) e pós-prandial entre 15 e 60 minutos após a alimentação. Foram realizados no primeiro dia (entre 6 a 24 horas de vida), no terceiro, no sétimo, e após, semanalmente até 42 dias de vida. Os resultados foram expressos em médias e desvios-padrão e descritos de maneira evolutiva. RESULTADOS: Ao total, foram estudados 77 RNPT e realizados 125 exames. Os valores em média±desvio-padrão são descritos na seqüência do primeiro, terceiro, sétimo e, consecutivamente a cada semana, até 42 dias de vida; sendo: IR pré-prandial de 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 e IR pós-prandial de 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. Os resultados de IP pré-prandial foram: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 e IP pós-prandial: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. Obtivemos PVS pré-prandial: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 e pós-prandial: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. Quanto a PVDF pré-prandial, obtivemos: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 e PVDF pós-prandial: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. A partir dos resultados acima, demonstra-se que o fluxo sanguíneo da AMS em RNPT saudáveis apresenta uma evolução peculiar a partir do nascimento tanto dos valores basais quanto após a estimulação com a dieta, representados por uma evolução característica dos índices de resistência, melhora dos picos de velocidades sistólica e diastólica e melhora da resposta vasodilatadora após a alimentação enteral. CONCLUSÕES: RNPT saudáveis de idade gestacional ao nascimento de 27 a 34 semanas completas apresentam uma evolução do fluxo sanguíneo da artéria mesentérica superior de maneira peculiar, do nascimento até 42 dias de vida, tanto dos valores basais quanto em resposta à alimentação. O conhecimento destes valores pode indicar a dopplerfluxometria como um método preventivo de avaliação específico de cada RNPT para a introdução e progressão mais segura da alimentação, reduzindo a ocorrência de quadros gastrintestinais, melhorando os índices de morbi-mortalidade neonatal.
INTRODUCTION: The knowledge of the normal values of indices of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates may help to prevent feeding intolerance situations and necrotizing enterocolitis. METHODS: In order to describe the indices for evaluation of Doppler velocimetry of the superior mesenteric artery in healthy premature neonates with gestational age between 27 and 34 weeks, on the first, third, seventh days, and then weekly, until six weeks of life; this is a prospective cohort study. The Doppler velocimetric examination was done by means of the Logic Book 8C-RS (General Electric USA), using a 8 MHz imaging transducer, with the pulsed color Doppler readings being obtained by sonographic waves at 4 MHz. The neonate was kept in a supine position, with the transducer positioned in the epigastric region, immediately below the xyphoid appendix, obtaining two-dimensional images of the celiac trunk and of the superior mesenteric artery, a few millimeters after its emergence from the aorta in the sagittal plane. The flux measurements were obtained in the longitudinal direction of the vessel and at an angle of insonation between 0 and 20 degrees. The blood flow curves were recorded after a sequence of five stable measurements, with respect to the quality of the waves, and with respect to their audible characteristics; thus obtaining the following measurements: peak systolic velocity (PSV), end diastolic velocity (EDV) and average flow velocity; with the Pourcelot Index being calculated subsequently, that is: [peak of systolic velocity end diastolic velocity / peak of systolic velocity, which represents a resitance index (RI); and pulsatility index (PI). The values obtained were expressed as averages and standard deviations. The results were stored in an Excel database, with blind analysis after the conclusion of data gathering. Uncomplicated and appropriate for gestational age premature neonates with gestational age between 27 and 34 weeks at birth were included in the study. We adopted as criteria for exclusion from the study: neonates in unstable hemodynamic conditions; needing assisted ventilation with high parameters; large deformations or clinical syndromes; feeding intolerance or diagnosis of necrotizing enterocolitis; conditions that alter the mesenteric flow, such as: phototherapy, presence of umbilical catheters, patent ductus arteriosus and sepsis. The exams were done prior to feeding (up to 30 minutes) and after feeding (between 15 and 60 minutes). If the neonate was fasting, only one of the above parameters was measured, in order to establish behavior of the basal mesenteric flow at that moment. The exams were done on the first day (between the 6th and 24th hours of life), third, seventh days, and then weekly, until six weeks of life. Data are shown as the mean ± standard deviation and described for each postnatal age group. RESULTS: A total of 77 neonates were studied and realized 125 exams. The values of the resistance and pulsatility indices (RI and PI); peaks of systolic (PSV) and final diastolic velocity (EDV) on the first, third, seventh days, and then, on sequentially for each week until six weeks of postnatal life; as mean and standard deviations, was described: RI prior to feeding were 0,69±0,09; 0,67±0,15; 0,75±0,07; 0,74±0,07; 0,75±0,07; 0,76±0,07; 0,79±0,03; 0,78±0,05 and RI after feeding were 0,66±0,10; 0,70±0,21; 0,74±0,07; 0,73±0,08; 0,75±0,06; 0,76±0,06; 0,77±0,04; 0,77±0,03. The results of PI prior to feeding: 1,45±0,30; 1,35±0,28; 1,68±0,29; 1,50±0,23; 1,47±0,22; 1,52±0,20; 1,62±0,09; 1,68±0,06 and PI after feeding: 1,38±0,39; 1,40±0,29; 1,58±0,26; 1,46±0,26; 1,45±0,24; 1,50±0,27; 1,58±0,10; 1,64±0,04. The values of PSV prior to feeding were: 60,51±22,24; 55,24±26,04; 90,61±12,74; 95,33±18,11; 92,89±15,40; 96,96±12,18; 63,18±14,08; 58,12±9,78 and after feeding: 59,60±24,14; 110,82±32,45; 118,10±20,15; 121,95±24,18; 124,15±25,16; 126,07±18,17; 96,68±11,12; 96,12±8,98. And the results of EDV prior to feeding: 18,85±6,09; 18,66±10,01; 20,99±8,12; 22,02±8,50; 23,04±7,89; 22,24±8,02; 11,99±6,15; 12,05±5,12 and EDV after feeding: 20,63±6,89; 30,15±12,78; 27,98±9,72; 29,02±10,05; 34,56±9,00; 32,02±8,45; 19,02±4,95; 21,15±3,43. These results shows that healthy premature neonates with gestational age between 27 and 34 weeks presents a peculiar evolution in blood flow in the superior mesenteric artery after birth, represented by the resistance patterns caracteristics, improvement in peaks of systolic and diastolic velocity, and improvement in vasodilation in response to feeding. CONCLUSION: These results suggest for the Doppler velocimetry as specific and preventive evaluation method for each premature neonate, as a way to a safer introduction and progression of feeding, reducing the prevalence of gastrointestinal inflammatory diseases in neonates, and improving the indices of neonatal morbidity and mortality. Knowledge of blood-flow velocity in the superior mesenteric artery in uncomplicated preterm infants might provide a clue in investigating the maturation of intestinal circulation and the pathogenesis or pathophysiology of gastrointestinal diseases in newborn infants.

La dysbiose est une cause importante dans la survenue de maladies, favorisant la prolifération de pathogènes ou induisant l’inflammation. L’étude de ce phénomène est devenue possible grâce aux approches d’analyse génomique (AG) associé avec d’autres techniques. L’entérocolite nécrosante (ECN) et l’infection du pied diabétique (IPD) demeurent deux maladies associées à la dysbiose dans lesquels différents bactéries ont été décrites, notamment C. butyricum dans l’ECN, E. coli et S. aureus dans l’IPD. Dans le cadre de l’ECN, C. butyricum demeure l’espèce la plus fréquente chez les ECN. L’identification clusters liés géographiquement et en fonction du temps. Le portage asymptomatique est suggéré par une similarité génomique des souches patients et contrôles. La prédiction d’un gène de β-hémolysine ainsi leur effet cytotoxique sur les cellules Jurkat avait été observé. De même, sur les cellules Caco-2 malgré le KO du gène de β-hémolysine. En se basant sur l’analyse physico-chimique du surnageant bactérien, nous avons suggéré que la fraction cytotoxique est protéique. La purification de la fraction cytotoxique a permis de trouver une protéine codant pour PspC family possédant un domain conservé commun avec celui de la toxine A/B. L’inactivation du gène codant pour cette protéine n’a pas supprimé l’effet cytotoxique, suggérant la présence d’une combinaison gènes. En parallèle, nous avons ciblé l’impact de C. neonatale par qRPC spécifique rpoB. Cette espèce était plus fréquente chez les patients, ainsi de clones géographiques ont été identifiées. Enfin, des SNPs ont été observés dans des gènes de virulence dans le cas des E. coli et S. aureus isolés de l’IPD
Dysbiosis remains a main cause during the establishment of several diseases, by promoting bacterial translocation, leading to inflammation process. Specific microorganisms were involved in the pathogenesis of dysbiosis-associated diseases, notably necrotizing enterocolitis (NEC) and diabetic foot (DF). This was possible by the implication of whole-genome analysis (WGA) in association with other techniques. In case of NEC, C. butyricum was significantly associated with in NEC; tested on a South-East French cohort. Geographical and/or temporal clusters were identified, thus genomic relationship between NEC-associated isolates and controls, suggesting the presence of asymptomatic carriage. Genes encoding for β-hemolysin was detected and C. butyricum supernatant exhibited cytotoxic effect on Jurkat cells. Cytotoxic effect was also presented on Caco-2 cells. Supernatant of β-hemolysin-mutant C. butyricum showed enterotoxic effect. Basing on physico-chemical data, we assumed that the evaluated fraction was a protein. Proteomics analysis revealed that PspC family was the cytotoxic protein. This protein owned a glucan-binding domain, shared by C. difficile toxin A/B. The KO of PspC gene was enterotoxic, suggesting by this the existence of a combination of genes. In parallel, a specific rpoB-based qPCR was developed to identify C. neonatale. We found that, C. neonatale was more prevalent in NEC than in controls. Although co-identified in association with C. butyricum. C. neonatale clones were distinguished especially in strains isolated from the same hospital. Regarding to DF infection, SNPs were identified within S. aureus and E. coli genomes, especially in virulent genes

Le microbiote digestif humain joue un rôle essentiel et bénéfique pour son hôte mais il est également impliqué dans un nombre croissant de pathologies. Les connaissances sur la composition de cet écosystème ont récemment été révolutionnées grâce à l’utilisation de techniques moléculaires. Cependant, ces techniques comportent des limites importantes. C’est ainsi que le concept de « culturomique » a été introduit ; il consiste en la multiplication de milieux et conditions de culture et l’identification rapide de colonies bactériennes par spectrométrie de masse (MALDITOF) ou par amplification et séquençage du gène de l’ARN ribosomal 16S. Dans la première partie de ce travail, nous avons mis en évidence une association entre la présence de Clostridium butyricum dans les selles et la survenue d’entérocolite ulcéro-nécrosante que ce soit par méthodes de pyroséquençage et culture ou par PCR quantitative en temps réel spécifique de C. butyricum; identifié après séquençage du génome complet de toutes nos souches de C. butyricum, la présence du gène de la β-hémolysine (toxine). Dans la deuxième partie de ce travail, nous avons montré par cuturomique que les bactéries à Gram-négatif (BGN) étaient fréquemment disséminées au sein du microbiote cutané transitoire des patients hospitalisés en réanimation ; le réservoir serait essentiellement digestif. En conclusion, le microbiote digestif constitue un réservoir sousestimé de bactéries pathogènes. La microbiologie moderne incluant les nouvelles méthodes de culture permet d’étendre de manière considérable les connaissances sur la composition de cet écosystème et son implication en pathologie humaine
He human gut microbiota plays an important and beneficial role in its host but it is also involved in a growing number of diseases. Knowledge of the composition of this ecosystem have recently been revolutionized by the use of molecular techniques. However, these techniques have significant limitations. Thus, the concept of "culturomics" has been introduced; it consists of the multiplication of culture conditions and the rapid identification of bacterial colonies by mass spectrometry (MALDI-TOF) or by PCR 16S RNA gene sequencing. In the first part of this work, we have demonstrated an association between the presence of Clostridium butyricum in the stool and the occurrence of necrotizing enterocolitis whether by pyrosequencing methods and Culture or by quantitative PCR specific real time C. butyricum; identified after sequencing the complete genome of all our strains of C. butyricum, the presence of the gene of β-hemolysin (toxin). In the second part of this work, we showed by cuturomics that Gram-negative bacteria (BGN) were frequently spread out over the transitional skin microbiota of patients hospitalized in intensive care; the reservoir would essentially digestive. In conclusion, the gut microbiota is an underestimated reservoir of pathogenic bacteria. Modern microbiology including new culture-based methods is currently extending exponentially our knowledge on gut microbiota giving rise to new insights into the pathogenesis or the transmission of infectious diseases

La recherche en bactériologie et virologie est à la fois de nature cognitive et appliquée. Elle consiste à fédérer et mettre en place une capacité de recherche multidisciplinaire et pouvoir l'intégrer sur un champ très vaste de microorganismes et de maladies. Les nouvelles avancées conceptuelles et technologiques dans le domaine de la génomique, notamment les avancées dans les techniques à haut débit (séquençage, PCR...) permettent actuellement d’avoir rapidement des génomes bactériens et viraux entiers, ou seulement sur quelques gènes d’une grande population. Les progrès dans ce domaine permettent l’accès à ces informations en évitant une combinaison de plusieurs méthodologies, et à moindre coûts. Dans notre travail de thèse, nous avons été porté à analyser et traiter les données de deux études genomiques et métagenomiques, mettant en évidence avantages, limites et attentes liés à ces techniques. La première étude porte sur l'analyse génomique de nouveaux virus géants et chlamydia infectant Vermamoeba vermiformis. La deuxième étude concerne le pyroséquençage 16S de microbiote intestinal de nouveau-nés atteint de l'entérocolite nécrosante. Pour le premier projet du travail de thèse, nous avons analysé les génomes de trois nouvelles espèces de Chlamydiae et onze virus giants (premiers membres de deux probables nouvelles familles) qui se multiplient naturellement dans Vermamoeba vermiformis. L'objectif étant de mettre en évidence les caractéristiques génétiques spécifiques à ces micro-organismes. La deuxième partie a été consacrée à l'analyse des données de pyroséquençage 16S des selles de nouveau-nés atteints de l'entérocolite nécrosante
Research in bacteriology and virology is both cognitive and applied. It involves federating and developing a multidisciplinary research capacity and being able to integrate it into a very broad field of microorganisms and diseases. New genomic and conceptual advances in genomics, including advances in high-throughput techniques, now permit rapid bacterial and viral genomes, or only a few genes of a large population. Progress in this area allows access to this information by avoiding a combination of several methodologies and at lower costs. In our thesis work, we were led to analyze and process the data of two genomic and metagenomic studies, highlighting advantages, limitations and expectations related to these techniques. The first study focuses on the genomic analysis of new giant viruses and chlamydia infecting Vermamoeba vermiformis. The second study concerns the 16S pyrosequencing of intestinal microbiota of neonates with necrotizing enterocolitis. The first project of the thesis work analyzed the genomes of three new species of Chlamydiae and eleven giant viruses (first members of two probable new families) which naturally multiply in Vermamoeba vermiformis. The objective is to highlight the genetic characteristics specific to these microorganisms. The second part was devoted to the analysis of 16S pyrosequencing data from neonatal enterocolitis neonatal stools. The goal was to identify an agent responsible for this disease

L'enterocolite necrotizzante è un emergenza sia neonatale che chirurgica; lo scopo di questo studio prospettico è di valutare il ruolo del drenaggio addominale preventivo in stadio 2 di nec con lo scopo di ridurre il numero di pazienti che progrediscono verso lo stadio 3 (perforazione intestinale). Materiali e metodi. Prospettivamente abbiamo osservato 56 pazienti trattati con l'apposzione del drenaggio o con il classico wait and see. sono stati creati criteri di inclusione ed esclusione. Abbiamo trattato con drenaggio solo i pazienti con consenso dei genitori. L'apposizone di drenaggio è stata eseguita con anestesia locale risultati: dopo aver studiato i dati è emerso che il drenaggio preventivo è un metodo efficace e sicuro per non far progredire clinicamente la nec. Ulteriori importanti dati vengono discussi. Conclusioni. Il drenaggio peritoneale è sicuro e potrebbe essere utilizzato come primo step per il trattamento della nec
Necrotizing enterocolitis is an emergency in neonatology and pediatric surgery. the aim of this prospective study was to evaluate the role of preventive abdominal drain in stage II nec in order to avoid clinical progression to stage III (intestinal perforation) Materials and methods: we prospectively treated 56 patients for NEC at stage II with two different management: peritoneal drain or wait and see, patients were selecteted with inclusion and exclusion criteria, we treat patients after parents consent. each patients underwent peritoneal drain under local anesthesia. data were collected for statistical analysis. results: after reviewing the medical charts our study showed that peritoneal drain in safe and effective to avoid the clinical progression to stage III.other important clinical aspect were described. Conclusions: peritoneal drain is safe and could be used as first treatment for stage II NEC to avoid clinical progression to stage III

Leung, Wan Lun Fiona.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 179-204).
Abstracts in English and Chinese.
Abstract --- p.i
中文摘要 --- p.v
Acknowledgement --- p.viii
List of Abbreviations and Symbols x --- p.vi
List of Tables --- p.xx
List of Figures --- p.xxi
Chapter CHAPTER ONE --- Introduction --- p.1
Chapter 1.1 --- General Overview --- p.1
Chapter 1.2 --- Necrotizing Enterocolitis (NEC) --- p.3
Chapter 1.2.1 --- Epidemiology of NEC --- p.3
Chapter 1.2.2 --- "Clinical Presentation, Diagnosis and Management of NEC" --- p.5
Chapter 1.2.3 --- Pathophysiology of NEC --- p.9
Chapter 1.2.3.1 --- Prematurity --- p.9
Chapter 1.2.3.2 --- Bacterial Colonization --- p.12
Chapter 1.2.3.3 --- Enteral Feeding --- p.15
Chapter 1.2.3.4 --- Hypoxia and Ischemia --- p.16
Chapter 1.2.3.5 --- Genetic Polymorphism --- p.17
Chapter 1.2.3.6 --- Inflammatory Mediators --- p.20
Chapter 1.3 --- Spontaneous Intestinal Perforation (SIP) --- p.24
Chapter 1.3.1 --- Epidemiology of SIP --- p.24
Chapter 1.3.2 --- "Clinical Presentation, Diagnosis and Management of SIP" --- p.26
Chapter 1.3.3 --- Risk Factors of SIP --- p.28
Chapter 1.3.3.1 --- Prematurity --- p.29
Chapter 1.3.3.2 --- Use of Drugs --- p.30
Chapter 1.4 --- Comparison between NEC and SIP --- p.32
Chapter 1.5 --- Role of Cytokines in Pathogenesis of NEC and SIP --- p.38
Chapter 1.6 --- Immunoregulatory Molecules of Interest in This Study --- p.46
Chapter 1.6.1 --- Angiopoietin-2 (Ang-2) --- p.46
Chapter 1.6.2 --- v-erb-b2 Erythroblastic Leukemia Viral Oncogene Homolog 2 (avian) (ErbB3) --- p.48
Chapter 1.6.3 --- Type II Interleukin-1 Receptor (IL-1RII) --- p.52
Chapter 1.6.4 --- Urokinase Plasminogen Activator Receptor (uPAR) --- p.54
Chapter CHAPTER TWO --- Objectives --- p.57
Chapter CHAPTER THREE --- Materials and Methodology --- p.58
Chapter 3.1 --- Overview of the Experimental Procedures --- p.58
Chapter 3.1.1 --- Investigation on the Profile of Circulatory Immunoregulatory Proteins in Plasma of NEC and SIP High Risk Neonates --- p.58
Chapter 3.1.2 --- Investigation on the mRNA Expression Level of Targeted Immunoregulatory Molecules on Resected Intestinal Tissues in NEC and SIP Neonates --- p.58
Chapter 3.1.3 --- Investigation on the mRNA and Protein Expression Levels of Targeted Immunoregulatory Molecules in Human Intestinal Cell Lines --- p.60
Chapter 3.2 --- Reagents and Lab-wares with Their Sources --- p.61
Chapter 3.3 --- Study Population --- p.63
Chapter 3.4 --- Collection of Neonatal Whole Blood Samples --- p.65
Chapter 3.5 --- Cytokine Antibody Array Analyses --- p.67
Chapter 3.6 --- Enzyme-linked Immunosorbant Assays (ELISA) --- p.69
Chapter 3.6.1 --- Angiopoietin-2 --- p.69
Chapter 3.6.2 --- sErbB3 --- p.71
Chapter 3.6.3 --- sIL-lRII --- p.72
Chapter 3.6.4 --- suPAR --- p.74
Chapter 3.7 --- Collection of Neonatal Resected Intestinal Tissues --- p.76
Chapter 3.8 --- Resected Intestinal Tissue RNA Isolation --- p.78
Chapter 3.9 --- Purity Assessment of the Purified Tissue RNA Samples --- p.80
Chapter 3.10 --- Integrity Assessment of the Purified Tissue RNA Samples --- p.81
Chapter 3.11 --- In vitro Stimulation of Human Enterocytes by Lipopolysaccharides (LPS) and/or Platelet Activating Factor (PAF) --- p.84
Chapter 3.12 --- mRNA Expression Level Assessment of Selected Target Genes in Resected Intestinal Tissues and Human Intestinal Cell Lines --- p.86
Chapter 3.12.1 --- Synthesis of First Strand cDNA --- p.86
Chapter 3.12.2 --- Quantitative Polymerase Chain Reaction (qPCR) --- p.87
Chapter 3.13 --- Statistical Analysis --- p.89
Chapter CHAPTER FOUR --- Screening of Immunoregulatory Target Protein Molecules in Plasma of NEC and SIP Patients by Cytokine Array Analyses --- p.104
Chapter 4.1 --- Results --- p.104
Chapter 4.1.1 --- Screening of Detectable Immunoregulatory Target Molecules --- p.104
Chapter 4.1.2 --- Selection of Target Molecules Based on the Fold Change in NEC or SIP Compared with Control Samples --- p.105
Chapter 4.1.2.1 --- Similar Regulation of Target Molecules in Both NEC and SIP patients --- p.105
Chapter 4.1.2.2 --- Differential regulation of Target Molecules in NEC and SIP Patients --- p.106
Chapter 4.1.2.3 --- "Relative Normalized Expressions of Selected Circulatory Immunoregulatory Protein Molecules in NEC, SIP and Control Neonates" --- p.108
Chapter 4.1.2.3.1 --- Anti-inflammation --- p.108
Chapter 4.1.2.3.2 --- Pro-inflammation --- p.109
Chapter 4.1.2.3.3 --- Cell Growth --- p.110
Chapter 4.1.2.3.4 --- Wound Healing --- p.110
Chapter 4.1.2.3.5 --- Angiogenesis --- p.111
Chapter 4.1.2.3.6 --- "Anti-apoptosis, Cell Adhesion and Extracellular Matrix Organization" --- p.112
Chapter 4.1.3 --- Further Selection of Novel Target Molecules Based on Statistical Significance and Fold Change of NEC versus SIP --- p.113
Chapter 4.2 --- Discussion --- p.115
Chapter CHAPTER FIVE --- Validation of Target Proteins in Plasma of NEC and SIP Patients by Enzyme-linked Immunosorbant Assay --- p.132
Chapter 5.1 --- Results --- p.133
Chapter 5.1.1 --- Demographic Data of the Study Group --- p.133
Chapter 5.1.2 --- "Comparison of Plasma Levels of Target Proteins between NEC, SIP and Respective Controls" --- p.134
Chapter 5.1.3 --- Longitudinal Study of the Pre- and Post-operative Target Proteins Levels in Plasma --- p.136
Chapter 5.2 --- Discussion --- p.138
Chapter CHAPTER SIX --- Investigation on mRNA Expression Levels of Target Immunoregulatory Protein Molecules in Intestinal Tissue and Intestinal Cell Lines --- p.151
Chapter 6.1 --- Results --- p.152
Chapter 6.1.1 --- mRNA Expression Levels of Target Molecules in the Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.152
Chapter 6.1.2 --- mRNA Expression Levels of Target Molecules in the Macroscopically Normal and Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.154
Chapter 6.1.3 --- mRNA Expression Levels of Target Molecules in Human Intestinal Cell Lines upon LPS and PAF Challenge --- p.156
Chapter 6.1.3.1 --- FHs-74 Int Cell Line --- p.156
Chapter 6.1.3.2 --- Caco-2 Cell Line --- p.157
Chapter 6.2 --- Discussion --- p.158
Chapter CHAPTER SEVEN --- General Discussion --- p.171
Chapter 7.1 --- Overall Findings --- p.171
Chapter 7.2 --- Limitations of Study --- p.174
Chapter 7.3 --- Future Investigations --- p.177
References --- p.179

INTRODUCTION: Infants who have dysphagia (difficulty swallowing) are often recommended thickened oral liquids, which can be easier to swallow and allow infants to continue feeding orally. In the last decade, a xanthan gum thickener, SimplyThick®, was commonly used in preterm infants with dysphagia because of its ability to thicken breast milk. In 2011, the FDA cautioned against the use of SimplyThick in preterm infants, because of case reports of necrotizing enterocolitis (NEC), a condition where the bowel becomes inflamed and can lead to intestinal perforation or necrosis, systemic infection, the failure of multiple organs and death (Moore, 2016; Press Announcements, 2011). However, since the FDA warning, there have been no studies examining the prevalence of necrotizing enterocolitis in infants who consume SimplyThick. AIMS: Among infants at BCH who used SimplyThick and other thickeners at <1-year old between October 1st, 2012- December 31st, 2015 to 1) describe the patients’ clinical characteristics, including indications for SimplyThick and other thickeners and 2) determine the prevalence of necrotizing enterocolitis and adverse effects. METHODS: We performed a retrospective chart review in infants who had been seen at Boston Children’s Hospital, and prescribed or recommended SimplyThick thickener under the age of 1 (defined as from 0 up to and including 12 months) from October 1, 2012 to December 31, 2015. We collected information from electronic medical records and an existing quality improvement database of infants who had an abnormal modified barium swallow study. We collected information regarding clinical variables (e.g. patient age, patient sex, patient weight, gestational age at birth, clinical indications), nutritional information, and outcomes (presence of NEC or other adverse effects). These data were entered into a REDCap database and analyzed using SAS statistical software. RESULTS: We identified 20 cases of infants meeting our inclusion criteria. The duration of follow-up ranged from 6 months to 9.3 months. This follow up was either until the case turned 12 months of age or 6 months after the use of SimplyThick if the age started SimplyThick was greater than 6 months old. Mean corrected age at the time that SimplyThick was started was 6.2 months (range, 2.7 to 10.6 months), and 6 (30%) were born preterm at a gestational age ranging from 24.7 to 36.5 weeks. In cases that eventually stopped using SimplyThick (14 cases, 70%), SimplyThick was continued for a mean duration of 42.1 weeks (range 1.1 to 117.1 weeks). The most common indications for SimplyThick were aspiration documented on a modified barium swallow test, dysphagia and GERD. The most common reasons for discontinuation of SimplyThick were no longer requiring thickened feeds, or needing to stop oral feeding. No cases of necrotizing enterocolitis were reported among the 20 subjects. No adverse effects of SimplyThick were reported. CONCLUSION: Among 20 infants started on SimplyThick at 6.2 months and followed for up to 6 to 9.3 months, there were no cases of necrotizing enterocolitis. Further data collection is required to confirm these findings.
2019-07-11T00:00:00Z

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: A enterocolite necrosante é a doença adquirida do trato gastrointestinal mais frequente no período neonatal que afeta principalmente os recém-nascidos pré-termo e apresenta uma taxa de mortalidade elevada. Este estudo tem como objetivo principal averiguar o efeito protetor que o leite materno tem per si no desenvolvimento desta patologia devastadora. Materiais e Métodos: Realizou-se uma pesquisa da literatura entre 2007 e 2017 nas bases de dados PubMed/Medline, B-on e Cochrane Library utilizando as palavras-chave definidas. Adicionalmente, foram consultadas plataformas digitais nacionais e internacionais de referência. Os artigos encontrados foram selecionados tendo em conta a relevância científica para o estudo. Resultados: O leite materno é o melhor fator protetor contra o desenvolvimento da enterocolite necrosante, apresentando eficácia e segurança comprovadas. Este apresenta propriedades imunológicas e anti-inflamatórias únicas com ação protetora sobre a mucosa gastrointestinal, minimizando o dano aos enterócitos que resulta da imaturidade inerente ao parto pré-termo. Embora seja unânime a existência de efeitos benéficos associados ao aleitamento, ainda persistem algumas questões fundamentais relativas ao protocolo ideal de alimentação entérica que carecem de consenso científico. Discussão e Conclusão: O leite materno assume um papel determinante na prevenção da enterocolite necrosante e constitui a forma ideal de nutrição e alimentação para todos os recém-nascidos. Contudo, são necessários mais estudos controlados e randomizados que permitam retirar conclusões mais robustas e inequívocas relativamente ao protocolo de alimentação entérica que apresenta maior eficácia na prevenção desta patologia.
Introduction: Necrotizing enterocolitis is the most commonly acquired gastrointestinal disease in the neonatal period, affecting mainly preterm newborns and having a high mortality rate. The main purpose of this study is to determine the protective effect of breast milk against the development of this devastating disease. Materials and Methods: A literature search was performed in the Pubmed/Medline, B-on and Cochrane Library databases using the chosen keywords and focusing on articles published between 2007 and 2017. Additionally, we also used nationally and internationally renowned websites. The articles were selected taking into consideration their scientific relevance to the study. Results: Breast milk is the best protective factor against the development of necrotizing enterocolitis, having proven effectiveness and safety. It has unique anti-inflammatory and immunological properties that protect the gastrointestinal mucosa, minimizing the enterocyte damage inherent to preterm immaturity. Despite the unanimous recognition of its beneficial effects, there is still no scientific consensus regarding the ideal feeding protocol. Discussion and Conclusion: Breast milk has a crucial role in preventing against necrotizing enterocolitis and constitutes the ideal form of nutrition and feeding for all newborns. Nevertheless, a higher number of randomized controlled studies is necessary to draw more solid conclusions regarding the feeding protocol which provides the biggest benefit on the prevention of the disease.

introduzione l'enterocolite necrotizzante è la principale emergenza chirurgica neonatale. lo scopo di questo studio è di valutare il ruolo del drenaggio addominale preventivo sull'evoluzione clinica materiali e metodi sono stati arruolati con randomizzazione i pazienti con NEC allo stadio 2 e trattati con drenaggio addominale o classicamente riultati i pazienti trattati con drenaggio preventivo hanno sviluppato minor complicanze rispetto al trattamento classico discussione il drenaggio preventivo riesce a detendere l'addome e creare quindi una miglior complicance che garantisce una miglior vascolarizzazione delle anse intestinali
After the birth and development of Neonatal Intensive Care, necrotizing enterocolitis (NEC) has become the most common perinatal gastrointestinal emergency.The aim of this trial is to find out risk and predictive factors of NEC development, to identify prognostic factors on patients’ survival as well as, and most of all, to identify the preventive role of the peritoneal drain. materials and methods There are TWO study groups: Group A: placement of abdominal drainage in stage 2; Group B: surgical treatment only with perforation (plus retrospective data for each study centre)(patients treated with PD on stage III are included). results: When comparing NEC with non-NEC group, there was a significant difference between Apgar score at 1 minute (2±1 vs. 4±1 respectively, p<0.05) but not for Apgar at 5 minutes (p>0.05). The use of prenatal steroids was associated with lower mortality rate and lower NEC stage (p<0.05). Patients who received bowel enemas starting from day 2 after birth did not developed NEC or advanced NEC (p<0.05). If compared to our previous data, only 13 infants out of 58 (22%) developed advanced NEC(stage III)(p<0.05): Nine infants from the NEC group and seven infants from the non NEC group died. Only 20% of patients treated with PPD underwent laparotomy for bowel perforation (p<0.05). discussion abdominal drain is safe and could be considred an alternative treatment

For most of human history, breastfeeding has been the optimal source of nutrition for infants. Human milk contains a variety of important nutritional sources including vitamins, fats, proteins, and immunological components. With the rise of artificial infant formulas, however, breastfeeding as a whole has decreased around the world. Preterm infants are especially susceptible to diseases such as necrotizing enterocolitis in the first few weeks of life. Therefore, they have the most to gain from the extra immunological and nutritional support that is present in human milk. Within the last few decades, donor human milk has been viewed as the next best option if mothers own milk is not available. Donor human milk contains many of the same beneficial milk properties as regular human milk including immunoglobulins and human milk oligosaccharides. Studies have shown decreases in preterm cases of NEC and fewer deaths in infants who received DHM. One argument against the use of DHM is that pasteurization can reduce the beneficial enzymes and immunoglobulins present in samples. However, the increased use of human milk fortifiers has been able to significantly decrease the nutrient gap between regular human milk and donor milk. Overall, DHM along with proper fortification serves as the best and most cost effective way to feed preterm infants if mother’s milk is unavailable.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine in the Department of Paediatrics and Child Health, Faculty of Health Sciences, University of the Witwatersrand. Johannesburg, 2015
Introduction Little is known about the association between maternal HIV status and the development of necrotizing enterocolitis (NEC) in premature infants. The few studies that have been published give no clear picture. In the light of the high maternal HIV infection in South Africa, it is important to explore the association between maternal HIV infection and all aspects of the paediatric population. Objectives The primary objective of this study was to determine if maternal HIV positive status was associated with the development of NEC in preterm infants who were born in one of our academic hospitals. The second objective was to determine the severity, need for surgery and mortality of preterm infants with NEC according to maternal HIV status. Finally, the third objective was to determine risk factors associated with NEC. Method The study population included preterm newborns less than 1500 grams born at the CMJAH and admitted to the neonatal unit within 24 hours of birth. Data on maternal and infant characteristics were collected from the computerized neonatal database from January 2006 to December 2013. Results A total of 2355 infants <1500g constituted the study population. Of these 126 met the inclusion criteria for NEC and 2229 did not. Therefore, large proportions were not entered for a multivariate analysis. Univariate analysis did not demonstrate an association between maternal HIV positive status and NEC (OR: 1.3, 95% CI: 0.8-1.9, p= 0.2). Therefore it was not entered into the multivariate analysis. On multivariate analysis antenatal corticosteroids showed a protective association with NEC (OR: 0.2, 95% CI: 0.1-0.4, p< 0.05). Multiple pregnancy and the need for resuscitation at birth was associated with NEC (OR: 1.6, 95% CI: 1-2.5, p= 0.03), (OR: 8, 95% CI: 4.5-15, p< 0.05), respectively. The analysis also found that severity of NEC, the need for surgery and mortality among infants with NEC did not differ according to maternal HIV status (p= 0.9, p=0.7 and p= 0.4), respectively. Conclusions The analysis was not able to demonstrate an association between maternal HIV positivity and the risk of NEC. Risk factors for NEC that were identified were multiple pregnancies and the need for resuscitation at birth. Antenatal corticosteroids were found to have a protective association with NEC. Finally, severity, need for surgery and mortality did not also differ according to maternal HIV status among the NEC group.
MT2017