Støy, Ann Cathrine Findal

Nekrotiserende enterocolitis (NEC), som primært ses hos præmature spædbørn, er forbundet med høj morbiditet og mortalitet. Patogenesen er ikke kendt fuldt ud, men risikofaktorer inkluderer præmaturitet, enteral ernæring (især modermælkserstatning) og tarmmicrobiotaen. Modermælk, rig på bioaktive faktorer, har en beskyttende effekt imod NEC, men ikke alle for tidligt fødte spædbørn har mulighed for at få modermælk. Formålet med denne afhandling var at undersøge om kocolostrum (BC), som også er rig på bioaktive faktorer, kunne tjene som et alternativ til modermælk. En præmatur grisemodel for NEC blev anvendt i studierne til at undersøge effekten af BC produkter på tarmens struktur, fordøjelses- og absorptionskapacitet, mikrobiota, og inflammatoriske markører målt på både vævs- og plasma- proteinniveauer og på vævs-genekspression niveau. Formålet med Studie I var at undersøge, om BC kunne korrigere tarm dysfunktion og inflammation induceret af total parenteral ernæring (TPN) kombineret med en brat overgang til modermælkserstatning, hvilket har vist at igangsætte skadelige reaktioner i tarmen. En gruppe præmature grise fik modermælkserstatning efterfulgt af BC og blev sammenlignet med grupper af præmature grise, som fik enten modermælkserstatning eller BC efter TPN. Dette studie viste, at BC ernæring kunne reversere tarmdysfunktion og reducere inflammation forårsaget af kortvarig (6 timer) ernæring med moderate mængder af modermælkserstatning til præmature TPN-ernærede grise. En forudsætning for brug af BC i klinikken er, at et standardiseret produkt er let tilgængeligt, og Studie II undersøgte om pasteuriseret og/eller spraytørret BC var lige så gavnligt som frisk BC. Præmature grise fik TPN i to dage efterfulgt af 6 timers modermælkserstatning før de fik enten modermælkserstatning, BC, spraytørret BC, eller pasteuriseret og spraytørret BC. Studiet viste, at selvom spraytørring og pasteurisering påvirker BC proteiner, så blev en lavere grad af NEC observeret i grise, som fik spraytørret BC, eller pasteuriseret og spraytørret BC, mens grise, som fik BC, havde en tendens til lavere grad af NEC en modermælksernærede grise. All BC produkterne bevarede trofiske og antiinflammatoriske virkninger på den umodne tarm. Et simpelt og standardiseret system var nødvendigt til at undersøge effekterne af modermælkserstatning versus BC på tarmepitelceller. I Studie III blev IPEC-J2 cellelinien vurderet som en mulig in vitro model for den præmature grisetarm. Diæt-inducerede forskelle på ekspressionenaf 48 epitel- og immunrespons-relaterede gener blev undersøgt i IPEC-J2 celler stimuleret med modermælkserstatning, BC eller vækstmedium. Vævsprøver fra den distale tyndtarm fra præmature grise ernæret med modermælkserstatning eller BC blev inkluderet til sammenligning. Studiet viste, at grundige overvejelser skal foretages inden genekspressionsanalyse af diæt-inducerede reaktioner i IPEC-J2 celler som et system for den præmature tarm, da diæt-inducerede effekter i IPEC-J2 cellerne ikke kunne observeres. C. perfringens er en patogen bakterie associeret med NEC hos præmature spædbørn og grise, og i Studie IV blev mængden af C. perfringens samt total bakterier i tarmprøver fra præmature grise kvantificeret med qPCR. Endvidere blev vært-patogen interaktioner undersøgt i IPEC-J2 celler ved at analysere ekspressionen af 48 epitel- og immunrespons-relaterede gener i IPEC-J2 celler stimuleret med C. perfringens. En association mellem C. perfringens eller total bakterier og NEC blev observeret, og in vitro studiet viste, at et øget antal C. perfringens er forbundet et ændret immunrespons. For at øge BCs aktivitet imod C. perfringens, blev det i Studie V undersøgt om hyperimmunt colostrum med øget aktivitet mod C. perfringens kunne produceres. Western blot blev anvendt til at bekræfte, at anti-C. perfringens hyperimmunt BC blev produceret ved immunisering af drægtige køer med en clostridia specifik vaccine. Sammenfattende kan det konkluderes, at BC produkter har en gavnlig effekt på tarmmiljøet hos præmature grise baseret på en forbedring i tarmens struktur, fordøjelses- og absorptions funktioner, mikrobiotaen og et reduceret niveau af inflammatoriske markører. BC produkter, modificeret så de opfylder de ernæringsmæssige krav fra det præmature spædbarn, kan være værdifulde alternativer til modermælk i de første kritiske dage efter fødslen. Imidlertid er veldesignede studier i præmature spædbørn nødvendige for at undersøge om BC er sikkert og effektivt at bruge som ernæring for disse sårbare spædbørn, når modermælk ikke er tilgængelig.Necrotizing enterocolitis (NEC), primarily seen in preterm infants, is associated with high morbidity and mortality. The pathogenesis is not fully understood but risk factors include prematurity, enteral feeding (especially with milk formula), and the intestinal microbiota. Mother’s milk, rich in bioactive factors, has a protective effect against NEC, but not all preterm infants are able to receive mother’s milk. The overall aim of this thesis was to investigate if bovine colostrum (BC), also rich in bioactive factors, could serve as an alternative to mother’s milk. A preterm pig model of NEC was used to investigate the effect of BC products on intestinal structure, digestive and absorptive functions, microbiota, plasma and tissue proteins and tissue gene expression levels of inflammatory markers.In Study I, the aim was to investigate if BC could correct intestinal dysfunction and reduce inflammation induced by total parenteral nutrition (TPN) combined with an abrupt transition to milk formula, which has shown to initiate detrimental intestinal responses. A group of preterm pigs fed milk formula followed by BC was compared with groups of preterm pigs fed either milk formula or BC alone after the TPN period. This study showed that BC feeding restores intestinal dysfunction and reduces a proinflammatory response induced by short term (6 hours) formula feeding to preterm TPN-fed pigs.A prerequisite for the use of BC in clinical settings is that a standardized product is readily available, and in Study II it was investigated if pasteurized and/or spray dried BC had similar beneficial effects on intestinal dysfunction and inflammation as fresh BC. Preterm pigs were given TPN for two days followed by 6 hours of formula feeding before given either formula, BC, spray dried BC, or pasteurized and spray dried BC. The study showed that even though spray drying and pasteurization affected BC proteins, pasteurized and/or spray dried BC decreased the severity of NEC in pigs compared with milk formula, while a tendency towards lower NEC severity was observed in pig fed raw BC compared with milk formula. All three BC products maintained trophic and anti-inflammatory effects on the immature pig intestine. A simple and standardized system was required to investigate the effects of milk formula versus BC on intestinal epithelial cells. In Study III, the IPEC-J2 cell line was evaluated as an in vitro model for the premature pig intestine. It was investigated if diet-induced effects could be observed on the expression of 48 epithelial- and immune response-related genes in IPEC-J2 cells stimulated with milk formula, BC or growth medium. Distal small intestinal samples from preterm pigs fed milk formula or colostrum were included for comparison. This study showed that careful considerations must be made prior to gene expression analysis of diet-induced responses in IPEC-J2 cells as a system for the premature intestine, since no dietinduced effects in the IPEC-J2 cells were detected.C. perfringens is a pathogen associated with NEC in preterm infants and pigs, and in Study IV, the association between NEC and the abundance of C. perfringens and total bacteria in intestinal samples from preterm pigs was investigated using quantitative polymerase chain reaction. Furthermore, host-pathogen interactions were investigated in IPEC-J2 cells by analyzing the expression of 48 epithelial- and immune response-related genes after stimulation with C. perfringens. An association between the abundance of total bacteria and C. perfringens and NEC was observed, and an in vitro study in IPEC-J2 cells showed that increased numbers of C. perfringens were associated with changes in gene expression response.To increase the effect of BC against C. perfringens, Study V aimed to investigate if BC with increased activity toward C. perfringens could be produced. Western blot confirmed that anti-C. perfringens hyperimmune BC could be produced by immunization of pregnant cows with a clostridial specific vaccine.In conclusion, BC products have a beneficial effect on the intestinal environment in preterm pigs based on improvements in intestinal structure, digestive and absorptive functions, the microbiota, and by reduced level of inflammatory markers. BC products, modified to meet the nutritional requirements of preterm infants, may serve as valuable alternatives to mother’s milk during the first critical days after birth. However, well-designed studies in preterm infants are required to investigate if BC is a safe and effective alternative for these vulnerable infants when mother’s milk is not available

Online Research Database In Technology

The effect of bovine colostrum products on intestinal dysfunction and inflammation in a preterm pig model of necrotizing enterocolitis

http://orbit.dtu.dk/ws/files/54900213/Ann_Cathrine_F_St_y_Final_B5_thesis_2013.pdf

The effect of bovine colostrum products on intestinal dysfunction and inflammation in a preterm pig model of necrotizing enterocolitis

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