Abstract Acute infected necrotizing pancreatitis (ANP) is a very complex disease with a high risk of complications and death. ANP is difficult to treat and is often associated with poor outcomes. Despite the increasing data on the technical details required to perform a mini-invasive necrosectomy for walled-off necrosis (WON), relatively few studies have focused on the presence and consequences of pancreatic duct disruption in the context of APN. Moreover, the long-term prognosis of patients with acute pancreatitis (AP) is scant. The aim of this study was to examine the diagnosis, treatment and prophylaxis of pancreatic fistulas (PFs) associated with APN. In addition, the long-term prognosis of AP was evaluated. The study population consists of the patients with AP treated at Oulu University Hospital, Finland (Studies I–IV) and Copenhagen University Hospital, Denmark (Study II) during 1995–2015. In the first part of the study, all consecutive patients following open necrosectomy for infected ANP were demonstrated to have PF. Endoscopic transpapillary pancreatic stenting (ETPS) was attempted and proven to be an effective and safe treatment for patients with PF. In Study II, prophylactic pancreatic stenting in the early stage of the disease was tested in a randomized controlled trial to the patients with ANP to prevent PFs associated with the disease. However, the study showed that the patients with ANP did not benefit from early prophylactic pancreatic ductal stenting (PPDS); instead, it seemed to be harmful for the patients. The results of Study III showed that single drain amylase level measurement after surgical necrosectomy is unreliable. According to this study, serial measurements are recommended to diagnose PFs after necrosectomy. Study IV including 1644 patients showed that AP, especially alcohol AP, was associated with a high long-term mortality. On the other hand, AP without an alcohol aetiology had a minimal impact on survival. In conclusion, in patients with infected ANP, a PF has to be considered in treatment, but the prevention of ductal leak with PPDS is not recommended. In addition, the poor long-term outcome among alcohol AP patients was due to alcohol-related diseases. / Tiivistelmä Akuutti nekrotisoiva haimatulehdus ja erityisesti siihen liittyvä bakteeri-infektio on sairaus, johon liittyy korkea komplikaatio- ja kuolleisuusriski. Tautia usein komplisoi infektion lisäksi nekroosiin liittyvä haimafisteli, joka tekee hoidosta entistä haasteellisemman. Viime aikaisissa tutkimuksissa on käsitelty runsaasti mini-invasiivista nekrosektomiaa, mutta suhteellisen vähän on tutkimuksia nekrotisoivaan haimatulehdukseen liittyvästä fisteliongelmasta. Haimatulehdus-potilaiden pitkäaikaisennuste on myös epäselvä. Tämän väitöskirjatutkimuksen tavoitteena oli selvittää nekrotisoivaan haimatulehdukseen liittyvän haimafistelin yleisyyttä, diagnostiikkaa, ehkäisyä ja hoitoa. Lisäksi tarkasteltiin akuuttiin haimatulehdukseen sairastuneiden potilaiden pitkäaikaisennustetta. Ensimmäisessä osatyössä ilmeni, että kaikille potilaille, joille suoritettiin haiman nekrosektomia kehittyi fisteli ja endoskooppinen transpapillaarinen haimateiden stenttaus (ETPS) osoittautui hyväksi ja turvalliseksi hoidoksi fistelin hoidossa. Toisessa prospektiivisessa randomoidussa kontrolloidussa osatyössä tutkittiin profylaktista haimateiden stenttausta nekrotisoivassa haimatulehduksessa. Tutkimus osoitti, etteivät potilaat hyötyneet stenttauksesta: toimenpiteestä oli enemmän haittaa kuin hyötyä. Tämän tutkimuksen mukaan protetisointia ei suositella tehtäväksi taudin alkuvaiheessa. Kolmannessa osatyössä selvitettiin haiman nekrosektomian jälkeisen haimafistelin diagnosointia. Tutkimustuloksen mukaan haimafistelin osoittamiseksi dreenieritteen amylaasitasoa mittaamalla tarvitaan useita mittauskertoja, koska yksittäisen mittauksen sensitiivisyys on matala. Neljännessä osatyössä analysoitiin Oulun yliopistollisessa sairaalassa 1995–2012 akuutin haimatulehduksen sairastaneiden työikäisten potilaiden pitkäaikaisennustetta ja kuolinsyitä. Noin kymmenen vuoden seurannassa tutkimusryhmän (n = 1 644) kuolleisuus oli yli nelinkertainen verrattuna ikä- ja sukupuolivakioituihin verrokeihin (n = 8 220). Merkittävin kuolleisuutta lisäävä tekijä oli alkoholi. Tutkimuksemme osoitti, että infektoituneen haimanekroosiin liittyvä haimafisteli on huomioitava hoidossa. Varhaisesta profylaktisesta haimateiden protetisoinnista ei tutkimuksessa osoitettu olevan hyötyä. Alkoholin aiheuttaman haimatulehduksen pitkäaikaisennusteen mortaliteetti on korkea johtuen alkoholin käytöstä ja siihen liittyvistä sairauksista.

No description available.

Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.

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.

S. aureus est un pathogène humain qui produit une grande variété de facteurs de virulence, dont la leucocidine de Panton Valentine. Cette toxine est à l’origine de symptomatologies potentiellement sévères telles que la pneumonie nécrosante et l’ostéomyélite compliquée récidivante. Malgré la faible prévalence des gènes de la PVL au sein de souches de S. aureus, nous assistons à une émergence et à une diffusion de souches S. aureus résistantes à la méticilline d’origine communautaire (SARM-C) produisant la PVL. Bien que hautement conservée au sein de différents génomes, la PVL produite par les souches SARM-C américaines USA300 porte une substitution His176Arg avec un impact potentiel sur la fonctionnalité de la PVL. Nous avons étudié la distribution de ce polymorphisme au sein d’une sélection de souches SARM-C isolées de divers continents et nous avons confirmé le rôle leucotoxique de la PVL envers les neutrophiles humains quel que soit le fond génétique de la souche d’origine. Nous avons mis au point un modèle expérimental de lapin afin d’étudier le rôle de la PVL secrétée par la souche USA300 dans la pathogenèse de l’ostéomyélite. La PVL s’est révélée être un déterminant de la gravité de la maladie en termes de persistance et extension de l’infection, ainsi que d’amplitude de la réaction inflammatoire. Dès lors que nous avons conforté le rôle pathogène de la PVL, nous avons exploré l’effet des antibiotiques sur l’expression de la leucocidine. Nous avons observé après traitement avec l’oxacilline et l’imipénème une augmentation de la production de PVL. Cette augmentation est le résultat d’une activation transcriptionnelle des gènes de la PVL, activation médiée par les protéines liant la pénicilline et des régulateurs de la virulence staphylococcique, sarA et rot. D’autres antibiotiques la clindamycine, le linézolide et la rifampicine exercent un effet inhibiteur sur la production de PVL et bloquent l’induction de toxine par les bêta-lactamines. Sur la base de ces observations, nous recommandons pour le traitement des infections sévères à S. aureus producteur de PVL l’utilisation de l’oxacilline en association avec la clindamycine ou la rifampicine pour S. aureus sensible à la méticilline et du linézolide pour le SARM. / S. aureus is a human pathogen producing a large variety of virulence factors such as the Panton Valentine leukocidin (PVL). This toxin has been epidemiologically related to severe diseases such as necrotising pneumonia and recurrent osteomyelitis. The world wide spreading of PVL producing community acquired methicillin resistant S. aureus (CA-MRSA) has been reported. Although highly preserved within various genomes, the sequence of PVL produced by the american CA-MRSA strain USA300 carries a His176Arg substitution with a potential impact on the functionality of the leukocidin. We studied the distribution of this polymorphism within a selection of CA-MRSA strains isolated from various continents and we confirmed PVL toxicity towards human neutrophiles regardless to the genetic background of the harboring strain. We used an experimental rabbit model in order to study the relevancy of USA300 secreted PVL in the pathogenesis of osteomyelitis. We showed that PVL is a determinant of disease severity in terms of persistence and extension of the infection, as well as amplitude of the inflammatory reaction. We investigated the effect of antibiotics on the expression of the leukocidin. We observed increase PVL production after oxacilline and imipenem treatment. This was the consequence of trascriptional activation of PVL genes, mediated by penicillin binding proteins and S. aureus virulence regulators sarA and rot. Other antibiotics such as clindamycin, linezolid and rifampicin decreased PVL production and suppressed the inductor effect of beta-lactams. Based on these observations, we recommend for the treatment of the severe PVL induced diseases the use of oxacillin associated with either clindamycin or rifampicin for methicillin susceptible S. aureus or linezolid for MRSA.

Les virus sont les causes principales d’encéphalites et plus d’une centaine d’entre eux sont impliquées dans ces pathologies. Cependant, plus de 50% des encéphalites restent sans étiologie, dû en partie à des techniques de diagnostic mal adaptées ou à une méconnaissance de l’agent infectieux impliqué. Nous rapportons les cas de deux enfants d’une même famille qui ont développé une encéphalite nécrosante aiguë (ANE) conduisant à une hospitalisation de plusieurs semaines en soin intensif. Les virus communément recherchés dans les cas d’encéphalites n’ont pas été retrouvés chez ces patients. Par contre, la culture réalisée à partir des prélèvements d’urine des enfants et d’un prélèvement de gorge pour l’un des deux, a permis d’isoler un nouveau réovirus nommé MRV2Tou05. L’analyse moléculaire des 10 segments du génome viral a indiqué qu’un nouveau réovirus de sérotype 2 avait été isolé chez ces patients et qu’il s’agissait d’un virus réassortant entre une souche isolée de porc et une souche humaine. Le virus se réplique dans des cellules neuronales humaines et a été retrouvé par amplification de gène dans les prélèvements d’urine et dans certains prélèvements de sérum des deux enfants. De plus, une réponse anticorps spécifique dirigée contre MRV2Tou05 a été détectée dans les sérums des patients de l’étude alors qu’aucune réponse anticorps n’a été observée dans 40 sérums de donneurs sains. Toutes ces données sont en faveur de l’implication du nouveau réovirus isolé dans l’encéphalopathie observée. Des études supplémentaires réalisées in vivo dans un modèle animal permettront de mieux comprendre le rôle étiologique de ce nouveau réovirus dans les cas d’encéphalites / Viruses remain the main cause of Acute Encephalitis and more hundred of them are implicated in these pathologies. However, the etiologic agent is not determined in approximately 50% of cases, owed partially to not well adapted techniques of diagnosis or to misunderstanding of the involved infectious agent. We report the cases of two children in a same family who developed an acute necrotizing encephalopathy (ANE) leading to hospitalization in intensive care unit. Viruses commonly found in encephalitis were not detected in cerebrospinal fluid of these patients. However, a new reovirus strain of serotype 2, MRV2Tou05 was isolated from urine and throat swab samples on epithelium cells and was identified by electron microscopy and genome sequencing. Molecular characterization of its segmented genome indicates that MRV2Tou05 is a reassortant issued from swine and human strains. This new reovirus strain was directly detected by a specific sensitive molecular test in urine and serum samples from the two children. A specific antibody response directed against this new reovirus strain was detected in patient sera, whereas no response was observed in 38 healthy adult sera. Both its isolation and molecular detection from samples of the patients and the specific immune response toward this strain suggest an etiologic role of this reovirus in the ANE cases described herein. The reproduction of symptoms in an animal model will help understanding the exact role of this strain in ANE cases

Die heterogene Gruppe der Myopathien kann sowohl die Funktion des Muskels beeinflussen, als auch andere Organsysteme. Erworbenen Muskelerkrankungen sind theoretisch behandelbar, jedoch stehen zumeist nur sehr unspezifische Behandlungsoptionen zur Verfügung, während für vererbte Formen bisher keine kausalen Therapiemöglichkeiten bekannt sind. In dieser Arbeit wurden drei verschiedene Muskelerkrankungen untersucht. Gemeinsam ist ihnen ein jeweils charakteristischer Einstrom von Entzündungszellen, wobei die Zusammensetzung des Zellinfiltrates (z.B. Lymphozyten oder Makrophagen) bei den verschieden Erkrankungen unterschiedlich war. Weiterhin unterscheidet sich das zugrunde liegende Zytokinmilieu für die einzelnen untersuchten Entitäten. Daher war es Ziel der Arbeit, die genauen Interaktionen zwischen den Immunzellen zu untersuchen, sowie die charakteristischen Phänomene der Erkrankungen (Hypoxie, Entzündung und Fibrose). Nekrotisierende Myopathien können sowohl durch eine immun-vermittelte Genese, als auch durch Kontakt mit toxischen Substanzen ausgelöst werden und beide Subgruppen können klar durch morphologische Kriterien, als auch durch spezielle Immunaspekte unterschieden werden. Makrophagen waren hier die vorherrschende Zellpopulation und im gesamten Muskel verteilt. Patienten mit Dermatomyositis dagegen zeigten ein typisches perifaszikuläres Atrophiemuster und hypoxische Effekte, wobei beide Phänomene deutlich ausgeprägter bei juvenilen, als bei adulten Patienten vorkamen. Erbliche Myopathien (z.B. Muskeldystrophie Duchenne) können ebenfalls entzündliche Infiltrate aufweisen und die Entwicklung von Fibrose in der Skelettmuskulatur ist dabei ein Hauptkriterium der Muskelfaserdegeneration. Ein neu entwickelter computer-basierter Algorithmus wurde genutzt, um diese Entwicklung zu quantifizieren. Die Menge an Bindegewebe steigt mit dem Alter der Patienten, während bei älteren Patienten außerdem ein fettgewebiger Umbau ein wichtiger Aspekt der Pathologie war. / The heterogeneous group of myopathies can affect the skeletal muscle or other organ systems and comprise a huge number of different entities. Acquired myopathies are potentially treatable, but there are often only unspecific treatment options, while there is no causative cure for inherited forms of myopathies. In this work, three different entities were analyzed, which all share common aspects of the immune response, but also feature distinct immunological aspects as well. They have an inflammatory part in common, which is mainly regulated by influx of immune cells. However, the composition of these cellular infiltrates (e.g. lymphocytes or macrophages) was varying between the diseases. In addition, the respective cytokine milieu was highly specific in the examined entities. Thus, the aim of the study was to precisely examine interactions between immune cells, and analyze characteristic pathological phenomena (hypoxia, inflammation and fibrosis). Necrotizing myopathies have an immune-mediated background or showed a toxic aetiology and both sub-groups can be distinguished by their morphological characteristics and certain immune aspects. Here macrophages are the predominant cell population and are spread throughout the muscle. Analyses of patients suffering from dermatomyositis showed a typical perifascicular pattern of atrophy, as well as effects of hypoxia and the described features are in general more pronounced in juvenile dermatomyositis than in the adult form. Inherited myopathies (e.g. Duchenne muscular dystrophy) harbor significant inflammatory infiltrates as well and development of fibrosis was a major feature of skeletal muscle degeneration. A computer-based algorithm was used to quantify fibrosis. The amount of connective tissue increased with the age of patients, while at late stage of disease fatty transformation was an additional important issue.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-06-07T18:12:48Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Baixa diversidade e sucessão microbiana anormal estão associadas à enterocolite necrosante em recém-nascidos prematuros.pdf: 1587508 bytes, checksum: c407e4cf94f25b2272a7d25213f72873 (MD5) / Made available in DSpace on 2017-06-07T18:12:48Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Baixa diversidade e sucessão microbiana anormal estão associadas à enterocolite necrosante em recém-nascidos prematuros.pdf: 1587508 bytes, checksum: c407e4cf94f25b2272a7d25213f72873 (MD5) Previous issue date: 2017-04-07 / 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.

Introdução: A infusão parenteral de emulsão lipídica (EL) de óleo de peixe (OP), rica em ácidos graxos ômega-3 (AG n-3), está associada à diminuição do perfil de mediadores pró-inflamatórios em estudos experimentais e clínicos. AG n-3 se incorporam em poucas horas em membranas celulares e geralmente são administrados após a agressão inflamatória. A pancreatite aguda (PA) experimental é modelo de inflamação, local e sistêmico, bem estabelecido. Objetivo: O presente estudo avaliou o efeito da infusão parenteral de EL de OP por curto período, antes da indução de PA, sobre a modulação da resposta inflamatória sistêmica e sobrevida. Métodos: Após cateterização do sistema venoso central, ratos isogênicos Lewis receberam infusão parenteral de EL de óleo de peixe ou solução salina durante 48 horas, quando então foram submetidos à pancreatite aguda, pela injeção retrógrada de 0,5 mL de solução de taurocolato de sódio a 3% no duto pancreático. Após indução de PA, nos períodos de 2, 12 e 24 horas, os animais foram sacrificados para coleta de amostras de sangue e de tecidos para dosagem de marcadores inflamatórios e histopatológicos. Paralelamente, 20 animais de cada grupo foram observados até sete dias após indução de PA, para avaliação de sobrevida. Resultados: O tratamento com EL de óleo de peixe foi associado com diminuição de citocinas pró-inflamatórias IL-1beta (p=0,0006) e IL-6 (p=0,05), diminuição de IL-4 (p= 0,0019) e tendência no aumento de anti-inflamatória IL-10 (p= 0,06), após 24 horas de PA; e com aumento da expressão pulmonar e hepática de proteínas de choque térmico HSP 90, 2 e 12 horas após PA, respectivamente. Após infusão de EL de óleo de peixe, não foram encontrados efeitos sobre os níveis de malonaldeído no fígado e na histopatologia do pâncreas, no período de 2 e 12 horas pós pancreatite aguda; e na taxa de sobrevida, em relação aos demais grupos (p > 0,05). Conclusão: Nossos resultados sugerem que a infusão parenteral de EL de OP 48 horas antes da indução de pancreatite aguda experimental parece influenciar favoravelmente a produção de citocinas inflamatórias e HSP90 hepática e pulmonar, sem impactar sobre a histopatologia da lesão pancreática e a taxa de sobrevida / The parenteral infusion of fish oil (FO) lipid emulsion (LE), rich in omega-3 fatty acids (n-3 FA), is associated with the decrease of pro-inflammatory mediators profile in experimental and clinical studies. N-3 FA are incorporated in a few hours in cell membranes and are generally administered after the inflammatory injury. The experimental acute pancreatitis (AP) is an inflammation, local and systemic well-established model. This study evaluated the effect of parenteral infusion of fish oil LE for a short period before AP induction, on the modulation of systemic inflammatory response and survival. For this, after the central venous catheterization Lewis rats received parenteral infusion of fish oil LE or saline solution for 48 hours, when they were induced to acute pancreatitis by retrograde injection of 0.5 mL of sodium taurocholate at 3% in pancreatic duct. After AP induction, in periods of two, 12 and 24 hours, the animals were sacrificed to collect blood samples and tissues for measurement of inflammatory markers and histopathological. In parallel, 20 animals in each group were observed up to 7 days after induction of AP, for survival analysis. The treatment with fish oil LE was associated with decreased of pro-inflammatory cytokines IL-1beta (p = 0.0006) and IL-6 (p = 0.05), reduction of IL-4 (p = 0.0019) and upward trend of anti-inflammatory IL-10 (p = 0.06) after 24 hours of AP; and increased pulmonary and hepatic expression of heat shock proteins HSP 90 two and 12 hours after AP, respectively. After infusion of fish oil LE, there were no effects on malondialdehyde levels in the liver and the pancreas histopathology in the periods of 2 and 12 hours after acute pancreatitis; and survival rate, compared to the other groups (p > 0.05). Our results suggest that parenteral infusion of FOLE 48 hours before the induction of experimental acute pancreatitis appears to favorably influence the production of inflammatory cytokines; hepatic and pulmonary HSP90, without impacting on the histopathology of pancreatic injury and the survival rate

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.