Milk diets influence doxorubicin-induced intestinal toxicity in piglets

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1 Downloded from orit.dtu.dk on: Dec 18, 217 Milk diets influence doxoruicin-induced intestinl toxicity in piglets Shen, R. L.; Pontoppidn, P. E.; Rthe, M.; Jing, P.; Hnsen, C. F.; Buddington, R. K.; Heegrd, Peter Mikel Helweg; Müller, K.; Sngild, P. T. Pulished in: Americn Journl of Physiology: Gstrointestinl nd Liver Physiology Link to rticle, DOI: /jpgi Puliction dte: 216 Document Version Pulisher's PDF, lso known s Version of record Link ck to DTU Orit Cittion (APA): Shen, R. L., Pontoppidn, P. E., Rthe, M., Jing, P., Hnsen, C. F., Buddington, R. K.,... Sngild, P. T. (216). Milk diets influence doxoruicin-induced intestinl toxicity in piglets. Americn Journl of Physiology: Gstrointestinl nd Liver Physiology, 311(2), G324-G333. DOI: /jpgi Generl rights Copyright nd morl rights for the pulictions mde ccessile in the pulic portl re retined y the uthors nd/or other copyright owners nd it is condition of ccessing pulictions tht users recognise nd ide y the legl requirements ssocited with these rights. Users my downlod nd print one copy of ny puliction from the pulic portl for the purpose of privte study or reserch. You my not further distriute the mteril or use it for ny profit-mking ctivity or commercil gin You my freely distriute the URL identifying the puliction in the pulic portl If you elieve tht this document reches copyright plese contct us providing detils, nd we will remove ccess to the work immeditely nd investigte your clim.

2 Am J Physiol Gstrointest Liver Physiol 311: G324 G333, 216. First pulished July 21, 216; doi:1.1152/jpgi Milk diets influence doxoruicin-induced intestinl toxicity in piglets Rene L. Shen, 1 Peter E. L. Pontoppidn, 1 Mthis Rthe, 2 Pingping Jing, 1 Crl Frederik Hnsen, 1 Rndl K. Buddington, 3 Peter M. H. Heegrd, 4 Klus Müller, 5,6 nd Per T. Sngild 1,5 1 Section of Comprtive Peditrics nd Nutrition, Deprtment of Clinicl Veterinry nd Animl Science/Nutrition Exercise nd Sports, University of Copenhgen, Copenhgen, Denmrk; 2 Hns Christin Andersen Children s Hospitl, Odense University Hospitl, Odense, Denmrk; 3 Deprtment of Helth nd Sport Sciences, University of Memphis, Memphis, Tennessee; 4 Ntionl Veterinry Institute, Technicl University of Denmrk, Lyngy, Denmrk; 5 Deprtment of Peditrics nd Adolescent Medicine, Copenhgen University Hospitl, Rigshospitlet, Copenhgen, Denmrk; nd 6 Institute of Inflmmtion Reserch, Deprtment of Rheumtology, Copenhgen University Hospitl, Rigshospitlet, Copenhgen, Denmrk Sumitted 26 Octoer 215; ccepted in finl form 1 July 216 Shen RL, Pontoppidn PE, Rthe M, Jing P, Hnsen CF, Buddington RK, Heegrd PM, Müller K, Sngild PT. Milk diets influence doxoruicin-induced intestinl toxicity in piglets. Am J Physiol Gstrointest Liver Physiol 311: G324 G333, 216. First pulished July 21, 216; doi:1.1152/jpgi Chemotherpy-induced gstrointestinl (GI) toxicity is common dverse effect of cncer tretment. We used prewened piglets s models to test our hypothesis tht the immunomodultory nd GI trophic effects of ovine colostrum would reduce the severity of GI complictions ssocited with doxoruicin (DOX) tretment. Five-dy-old pigs were dministered DOX (1 1 mg/m 2 ) or n equivlent volume of sline (SAL) nd either fed formul (DOX-Form, n 9, or SAL- Form, n 7) or ovine colostrum (DOX-Colos, n 9, or SAL-Colos, n 7). Pigs were euthnized 5 dys fter initition of chemotherpy to ssess mrkers of smll intestinl function nd inflmmtion. All DOX-treted nimls developed dirrhe, growth deficits, nd leukopeni. However, the intestines of DOX-Colos pigs hd lower intestinl permeility, longer intestinl villi with higher ctivities of rush order enzymes, nd lower tissue IL-8 levels compred with DOX- Form (ll P.5). DOX-Form pigs, ut not DOX-Colos pigs, hd significntly higher plsm C-rective protein, compred with SAL- Form. Plsm citrulline ws not ffected y DOX tretment or diet. Thus single dose of DOX induces intestinl toxicity in prewened pigs nd my led to systemic inflmmtory response. The toxicity is ffected y type of enterl nutrition with more pronounced GI toxicity when formul is fed compred with ovine colostrum. The results indicte tht ovine colostrum my e eneficil supplementry diet for children sujected to chemotherpy nd susequent intestinl toxicity. chemotherpy-induced mucositis; intestinl toxicity; enterl nutrition; milk; ovine colostrum NEW & NOTEWORTHY In this study, prewened pigs were used s models to study intestinl complictions fter doxoruicin s models for peditric oncology ptients receiving chemotherpy. This study shows tht the type of enterl nutrition is n importnt fctor tht cn influence the toxic responses relted to doxoruicin tretment. Compred with formul, ovine colostrum nutrition provided eneficil effects ginst the intestinl injury, which occurred erly fter doxoruicin dministrtion. Address for reprint requests nd other correspondence: P. T. Sngild, Comprtive Peditrics nd Nutrition, Dept. of Clinicl Veterinry nd Animl Science, Univ. of Copenhgen, Denmrk, 68 Dyrlegevej, DK-187 Frederikserg C, Denmrk (e-mil: pts@sund.ku.dk). GASTROINTESTINAL (GI) TOXICITY is mjor dose-limiting dverse effect of chemotherpy with implictions for oth moridity nd mortlity (12, 48). The ssocited symptoms include pin, nuse, vomiting, nd dirrhe, which ffect nutritionl sttus nd qulity of life. These complictions re relted to dverse clinicl outcomes, prolonged hospitliztion, nd need for prenterl nutrition. Due to mucosl rrier injury, there re incresed risks of fever nd infections, potentilly leding to incresing mortlity (12, 18, 48, 53). Emerging clinicl strtegies for GI toxicity, including drugs, proiotics, nd nutritionl interventions (3, 28, 65), hve not een effective, nd GI dysfunction often hinders dequte enterl nutrition support, which provides energy, mcronutrients, nd dietry ioctive fctors (37, 63). Enterl nutrition my, therefore, help to reduce inflmmtory conditions in the GI trct (1), nd this could e importnt in ssocition with chemotherpy. While the survivl of childhood cncer hs improved over the lst decdes, it is still the leding cuse of disese-relted mortlity in children (62). The improved survivl is prtly relted to intensified nd risk-strtified chemotherpy regimens, which hve led to incresed toxicity, underlining the need for etter understnding nd mngement of tretmentrelted complictions in these ptients. At the forefront is chemotherpy-induced intestinl dysfunction. The ssocited mlnutrition nd toxic complictions my led to dose reduction or tretment delys, compromising tretment efficcy. Nutrition could e prticulrly importnt for outcome in peditric cncer ptients tht re still in stte of growth nd orgn mturtion (4, 8). Studies lso suggest tht cncer nd chemotherpy in infncy nd erly childhood cn e more chllenging to mnge nd my hve worse outcomes compred with tht in older children (4, 42, 62), potentilly due to immturity of orgns such s the GI trct. Knowledge out this ptient group is limited, nd comprle peditric niml models for chemotherpy-induced mucositis re relevnt to provide new sic knowledge. Until now, such models hve focused minly on rodents (33), ut their physiology differs mrkedly from humns, nd their smll size nd immture stte t irth limit their use s trnsltionl model for infnts nd young children, compred with species like the pig (39, 51). The lrger size of prewened nd juvenile pigs lso mkes it esier to use stndrd clinicl nd medicl cre procedures when inducing nd evluting chemotherpy toxicity (35, 36, 45, 46, 57). Bovine colostrum (Colos) contins mny ioctive compounds, such s immunogloulin (Ig), trnsforming growth Downloded from y on August 29, 216 G /16 Copyright 216 the Americn Physiologicl Society

3 fctor-, nd insulin-like growth fctor I tht my stimulte gut growth nd function nd provide mucosl protection vi immunomodultory effects nd chnges to the gut microiot (44). Thus Colos my promote GI mucosl protection vi ntimicroil nd endotoxin-neutrlizing effects, suppression of gut inflmmtion, nd y fcilitting mucosl tissue repir. In preterm pigs, enterl feeding with ovine Colos protects ginst necrotizing enterocolitis, severe condition with inflmmtory nd ulcertive lesions in the GI trct (24). Thus ovine Colos might provide GI nd systemic enefits in severl disese sttes involving dmge to the GI mucos (44). Recent studies hve used prewened nd wened pigs s models for chemotherpy-induced intestinl toxicity (36, 45), nd these my provide niml models to study liquid diet interventions during period of rpid growth nd development, similr to peditric ptients. Liquid milk-sed diets re highly digestile nd esy to djust in nutrient composition nd intke for chemotherpy ptients requiring tue feeding. In this study, we hypothesized tht GI toxicity fter tretment of prewened pigs with doxoruicin (DOX) would e reduced y feeding ovine colostrum compred with stndrd formul diet. DOX is n nthrcycline, which cuses DNA intercltion nd is commonly used in mny nticncer regimens, with multiple orgn toxicities, including myelosuppression, crdiotoxicity, nd GI toxicity (5, 7, 64). MATERIAL AND METHODS Animls, surgicl preprtion, clinicl procedures, nd chemotherpy. All niml procedures were pproved y the Dnish Ntionl Committee on Animl Experimenttion (no. 21/ ). An overview of the experimentl pproch is shown in Fig. 1. A totl of 32 pigs (Lrge White Dnish Lndrce) of oth sexes were otined on the 3rd dy of life fter hving suckled their mother from irth. The pigs were rought to the reserch fcilities where they were housed in individul cges in fcility kept t constnt temperture ( 3 C) with 12:12-h light-drk cycle. Ech cge contined ehviorl enrichments (toy-cloths) nd llowed visul contct mong individul pigs. Piglets Dy: Ctheteriztion DOX or SAL Blood smples Lctose test Lc/mn test Tissue collection DOX Form SAL DOX Colos SAL Fig. 1. Study design with enterl nutrition intervention using formul (Form) or ovine colostrum (Colos). Ctheters nd feeding tues were fitted surgiclly 2 dys efore chemotherpy. Doxoruicin (DOX) or sline (SAL) ws dministered on dy. Blood smples were collected efore the tretment, dys 3 nd 5, nd nlyzed for hemtologicl prmeters nd C-rective protein. Digestive cpcity of lctose ws tested on dy 4, nd intestinl permeility ws ssessed with the lctulose/mnnitol test (lc/mn test) on dy 5 when nimls were killed. Results for doxoruicin-treted pigs fed formul (DOX- Form, n 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicin-treted pigs fed ovine colostrum fed (DOX-Colos, n 9), nd slinetreted pigs fed ovine colostrum (SAL-Colos, n 7) re shown. x G325 Shortly fter rrivl, the pigs were nesthetized using zoletil mix (intrmusculr, tiletmine,.28 mg/kg; zolzepm,.28 mg/kg; xylzine,.56 mg/kg; ketmine,.56 mg/kg; utorphnol,.11 mg/kg) for plcement of n orogstric feeding tue (6F Portex, Kent, UK) tht ws inserted through the cheek nd secured with sutures. A ctheter (Tygon OD.7 ID.4 Sint-Goin Performnce Plstics, Frnce) ws surgiclly inserted into the externl jugulr vein, tunneled under the skin, nd exteriorized through the dorsl prt of the neck nd used for lood smpling nd dministrtion of DOX. The pigs were fed either ovine colostrum (Colos) or formul (Form), nd within ech diet group pigs received DOX chemotherpy or sline (SAL), with rndomized lock design resulting in four groups with similr distriution of initil ody weight, sex, nd litter of origin: DOXtreted nimls fed ovine Colos (DOX-Colos, n 9) or Form (DOX-Form, n 9), nd SAL groups fed ovine Colos (SAL-Colos, n 7) or Form (SAL-Form, n 7). The pigs were fed every 3hy plcing 15 ml/kg of their respective diet in feeding trough. Voluntry food intke ws llowed, ut, if ppetite ws reduced, ny remining feed ws fed through the orogstric tue. The composition of the Form diet ws sed on commercilly ville clinicl nutrition products, i.e., Pepdite (6 g/l; powdered enterl nutrition with nonmilk-derived low-moleculr-weight peptides, essentil mino cids, crohydrte, ft, vitmins, nd minerls; Nutrici, Allerød, Denmrk), Lcprodn (5 g/l; whey protein; Arl Foods Ingredients, Arhus, Denmrk), Miprodn (5 g/l; csein; Arl Foods Ingredients), Clogen (5 g/l; long-chin triglyceride ft emulsion; Nutrici), Liquigen (8 g/l; medium-chin triglyceride from frctionted coconut oil; Nutrici), nd Servit-SHS (12 g/l; vitmins nd trce elements; Nutrici). The mcronutrient composition in the Form diet ws 5,291 kj/l energy, 96 g/l protein, 4 g/l crohydrte, with 36 g/l mltodextrins, nd 79 g/l ft. The Colos ws collected from Dnish diry cows within 24 h of prturition, pooled, nd spry-dried to powder nd -irrdited to eliminte microorgnisms (Biofier-Dmino, Gesten, Denmrk). The Colos powder ws reconstituted in wter (2 g/l) nd yielded mcronutrient vlues of 3,986 kj/l energy, 94 g/l protein, with 28 g/l IgG, 36 g/l crohydrte, nd 48 g/l ft. Chemotherpy. Two dys fter the surgery nd the strt of the respective feeding regimens, the pigs were dministered single intrvenous dose of DOX over 3 min, corresponding to 1 mg/m 2 ody surfce re or n equivlent volume of SAL. This dose of DOX is comprle to doses used in tretment of peditric cncers, such s cute lympholstic leukemi, nd hs een shown to induce intestinl toxicity in wened pigs (36). Body surfce re ws clculted s 7 kg ody wt.75 /1, (1), providing mg/kg DOX for the 2.5- to 3.5-kg pigs. Clinicl ssessment. Animls were monitored for clinicl sttus, including dily mesurements of ody weight, temperture, nd dirrhe scoring. Dirrhe ws ssessed y visully ctegorizing wste collection trys elow the cges nd niml stining, providing dily score of (no stools or norml stools), 1 (mild dirrhe), or 2 (severe dirrhe). Humne endpoints for euthnsi of nimls efore dy 5 were s descried elsewhere (46). Hemtology, CRP, nd in vivo mrkers of intestinl function. Blood smples were collected t seline, immeditely efore dministrtion of DOX, on dy 3 nd on dy 5 (the time of euthnsi) for hemtology nd nlyses of C-rective protein (CRP) nd citrulline. Hemtologicl prmeters included red lood cell (RBC), white lood cells (WBC), neutrophils (NEU), lymphocytes (LYM), nd pltelet (PLT) count (Advi 212 Hemtology System, Siemens, Erlngen, Germny). These prmeters hve previously een shown to correlte with toxicity in pigs (46). Circulting CRP is mrker of systemic inflmmtory response commonly used to indicte inflmmtion nd infection in oth humns nd pigs nd ws mesured with n enzymelinked immunosorent ssy (ELISA) using vlidted method for pigs (21). Plsm citrulline is synthesized y enterocytes nd is considered mrker for intestinl mss nd function, s well s mrker of dmge fter chemotherpy (58). For detection of citrulline Downloded from y on August 29, 216

4 G326 (17), nlytes extrcted from serum smples were seprted nd detected y ultr-performnce liquid chromtogrphy- triple qudrupole mss spectrometry (Wters, Milford, MA). Quntifiction of citrulline ws crried out using n nlyzing softwre, QunLynx (Wters). At seline nd gin on dy 4, the pigs were fed one olus (15 ml/kg) of 1% lctose solution vi the feeding tues to ssess the cpcity for lctose digestion s mrker for intestinl function. The olus ws given 3 h fter the lst feed insted of the norml diet. Blood smples were collected 2 min fter the olus, nd plsm glctose levels were mesured spectrophotometriclly (Pentr 4, Hori ABX, Montpellier, Frnce) using commercil kit (55). Before deth on dy 5, nimls received olus (15 ml/kg) of 5% lctulose nd 5% mnnitol solution vi the feeding tue 3 h fter the previous mel to ssess intestinl permeility. A urine smple ws collected y cystocentesis during necropsy 3 5 h fter the lctulose/ mnnitol (lc/mn) olus. Urinry concentrtions of lctulose nd mnnitol were determined spectrophotometriclly (Pentr 4), nd intestinl permeility ws ssessed y the rtio of lctulose to mnnitol (lc/mn rtio). Necropsy nd tissue collection. On dy 5, the pigs were nesthetized using the zoletil mix efore intrcrdil collection of lood smple fter which the pigs were euthnized y intrcrdil injection of sodium pentoritl, 2 mg/kg. After deth ws confirmed, the domen ws opened, nd the smll intestine (from the pyloric sphincter to the ileo-cecl junction) ws removed quickly, plced on cooled surfce, nd divided into three segments of equl length, designted proximl (Prox), middle (Mid), nd distl (Dist) intestine. For ech segment, two pieces of.5 cm were collected nd fixed in 4% neutrl uffered formlin. After fixtion, smples were dehydrted with ethnol, emedded in prffin nd cross-sectioned (5 m), mounted on glss slides, nd stined with hemtoxylin nd eosin. Additionl tissue smples from ech intestinl region were snpfrozen in liquid nitrogen for susequent nlyses. Finlly, 1-cm piece of ech region ws collected for determining the proportionl weight of mucos, reltive to whole intestine. The remining orgns were collected, nd their weights recorded. Intestinl tissue structure, rush order enzymes, nd proinflmmtory cytokines. Villus height nd crypt depth were determined s previously descried (46). The proportion of the smll intestine represented y mucos ws determined y scrping off the mucosl lyer nd drying (49). The frozen intestinl smples were processed for nlyses of rush order enzymes nd cytokine levels, s descried elsewhere (46, 5). Briefly, the tissues were homogenized in 1% Triton X-1, nd the ctivities of three disschridses (lctse, mltse, sucrse) nd three peptidses (minopeptidse A, minopeptidse N, dipeptidylpeptidse IV) were expressed s hydrolytic rte of 1 mol sustrte relesed per minute t 37 C per grm wet whole intestinl tissue. Intestinl inflmmtory responses were sed on cytokine levels in the Dist region nd mesured using the DuoSet ELISA Development kit (R&D Systems, Aingdon, UK) trgeted for porcine interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin 8 (IL-8), nd tumor necrosis fctor- (TNF- ), ccording to the mnufcturer s instructions. Intestinl prolifertion, poptosis, nd structurl proteins. Enterocyte prolifertion ws ssessed y immunohistochemistry, stining for Ki-67 on sections from the Mid intestine, s descried previously (6). Quntifiction ws performed y imge nlysis using Visiomorph 4.6 (Visiophrm). The totl re of Ki-67-positive stined nuclei were clculted nd presented s frction of ll nuclei. Expression of PCNA ws used s mrker of prolifertion nd tht of cspse-3 s mrker of poptosis. -Actin nd GAPDH proteins were quntified y Western lot nd used s indictors of sic cytostructurl nd metolic conditions, s previously descried (25). Briefly, 25-mg protein extrcted from the Mid intestine segments ws resolved y electrophoresis nd trnsferred onto polyvinylidene difluoride memrnes. The expression of PCNA, cspse-3, nd -ctin ws shown with specific ntiodies (Snt Cruz, CA; nd Acm, Cmridge, UK). The protein nds on the memrnes were visulized, nd the nd densities were detected y Imge J (Ntionl Institutes of Helth, Bethesd, MD). Protein loding ws controlled y direct stining of the electrophoresis gels run prllel to the ones for Western lot, using methods descried previously (2). Sttisticl nlyses. Sttisticl nlyses were performed with the sttisticl softwre R (version 2.15.). Weight gin ws expressed s the percent chnge in weight, reltive to the strt of DOX dministrtion. Continuous dt were nlyzed using mixed models (lmer function) with the groups s independent vriles nd with djustments for sex, seline ody weight, nd study round (3 rounds of pig studies with evenly distriuted groups nd sexes) s explntory vriles. For repeted-mesures nlyses, the individul pig identifier ws included in the model s rndom effect. Normlity ws ssessed y mens of stndrdized residuls nd log-trnsformed to ccount for vrince heterogeneity, when necessry. Adjustments for seline vlues were included in the model for longitudinl dt. Group comprisons were done with the lsmens pckge with multiplicity djustments of P vlues with the single-step method. For smple vlues lower thn ssy detection limit, these were ssigned the lowest vlue of the ssy rnge. Dirrhe score ws nlyzed s nonprmetric dt with the nprcomp pckge. P vlues.5 were considered significnt. Grphs were mde with GrphPd Prism (version 5.1; GrphPd Softwre, L Joll, CA). Dt re presented s rithmetic mens nd SE of rw dt, unless otherwise specified. Fig. 2. Weight gin indicted s percent chnge reltive to initil ody weight efore tretment on dy (A) nd dirrhe score (B) re presented s mens SE. Results for doxoruicin-treted pigs fed formul (DOX-Form, n 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicin-treted pigs fed ovine colostrum (DOX-Colos, n 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 7) re shown., Different superscript letters indicte significnt differences mong groups within the sme time point (P.5). Downloded from y on August 29, 216

5 G327 RESULTS Clinicl sttus nd growth. All pigs continued to et nd remined ctive nd cliniclly stle during the 5-dy study period. No pigs required euthnsi ccording to humne endpoints efore the end of the protocol. All of the pigs gined weight etween dy, when DOX (or SAL) ws dministered, nd dy 2 (Fig. 2A). After dy 3, weight gin decresed for oth groups of DOX pigs, resulting in stle ody weight of DOX-Colos pigs from dys 3 5 nd weight reduction for DOX-Form pigs. The two groups of SAL pigs continued to gin weight until dy 5. Growth on dy 5 ws significntly lower in DOX-Form pigs, compred with oth groups of SAL pigs (P.5, Fig. 2A), wheres weight for DOX-Colos pigs ws not different from tht in SAL pigs. Initil weight gin (dys 2) tended to e greter for oth groups of Form pigs, compred with Colos pigs (Fig. 2A). Dirrhe ws commonly oserved from dy 3 fter DOX dministrtion with incresing severity therefter (Fig. 2B). Dirrhe scores were similr in DOX-Form nd DOX-Colos pigs. However, DOX-Form pigs hd higher dirrhe score thn SAL groups on dys 3 nd 4, wheres scores in DOX-Colos were not significntly elevted until dy 4, reltive to SAL pigs (Fig. 2B). Dirrhe in SAL pigs ws restricted to one mild trnsient cse t the strt of the study period. Vomiting ws oserved in one DOX-Form pig on dys 4 nd 5. No effects on ppetite or food intke were oserved. Blood hemtology nd systemic inflmmtory response. Administrtion of DOX reduced WBC, compred with SAL on dys 3 nd 5 (P.5, Fig. 3A). DOX lso reduced LYM vlues on oth dys, wheres NEU were not ffected, nd no diet effect ws detected for WBC, LYM, or NEU (Fig. 3, A C). On dy 5, PLT vlues were lower in DOX vs. SAL pigs, s nlyzed for ech of the diet regimens (P.5, Fig. 3D), with no differences etween DOX-Colos nd DOX-Form. RBC did not differ mong groups (Fig. 3E). Longitudinl Fig. 3. Circulting levels of white lood cells (WBC; A), neutrophils (NEU; B), lymphocytes (LYM; C), pltelets (PLT; D), red lood cells (RBC; E), nd C-rective protein (CRP; F) re presented s mens SE. Results for doxoruicin-treted pigs fed formul (DOX- Form, n 4 9), sline-treted pigs fed formul (SAL-Form, n 4 7), doxoruicintreted pigs fed ovine colostrum (DOX-Colos, n 4 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 2 7) re shown.,,c Different superscript letters indicte significnt differences mong groups within the sme time point (P.5). mesurements of CRP did not differ significntly mong groups on dys nd 3. However, on dy 5, levels were higher for DOX-Form pigs, compred with DOX-Colos nd SAL- Form pigs (P.5, Fig. 3F). Gut function mrkers in vivo. After 2 dys of Form or Colos feeding, nd efore the DOX (or SAL) ws dministered, plsm glctose mesured 2 min fter lctose olus ws lower for Form thn for Colos pigs (92 12 vs mol/l, P.1). On dy 4, glctose vlues were similr for oth groups of Colos pigs, nd oth were higher thn for the two groups of Form pigs, regrdless of SAL or DOX dministrtion (pooled mens SE: vs mol/l, P.5), indicting tht diet, ut not DOX tretment, ffected glctose uptke cpcity. Among the groups, men gut per- Fig. 4. Intestinl permeility mesured on dy 5 s the urinry rtio of lctulose to mnnitol 3 5 h fter n enterl olus of lctulose (A), nd mnnitol solution nd proportion of mucosl mss reltive to totl intestinl mss (B) re presented s mens SE. Results for doxoruicin-treted pigs fed formul (DOX-Form, n 4 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicin-treted pigs fed ovine colostrum (DOX-Colos, n 8 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 7) re shown., Different superscript letters indicte significnt differences mong groups within the sme time point (P.5). Downloded from y on August 29, 216

6 G328 Tle 1. Orgn weights t necropsy Form Colos SAL DOX SAL DOX Stomch Intestine , Colon , , Liver Lungs Hert Spleen Kidneys Vlues re mens SE. Orgn weights re expressed reltive to ody weight (g/kg). Groups re doxoruicin-treted pigs fed formul (DOX-Form, n 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicintreted pigs fed ovine colostrum (DOX-Colos, n 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 7)., Different superscript letters indicte significnt differences mong the four groups (P.5). meility, s ssessed y the lc/mn rtio, ws lowest for DOX-Colos pigs (P.5 compred with DOX Form, Fig. 4A). Plsm citrulline did not differ significntly mong groups, regrdless of diet or whether DOX ws dministered. Intestinl nd inflmmtory responses. Mcroscopic signs of GI mucosl dmge were not oserved in ny of the groups, nd only one pig from the DOX-Form group hd signs of edem in the colon. Only mong the Form pigs did DOX tretment reduce smll intestinl weight ( 38%, P.5), with less (nonsignificnt) effects of DOX within the Colos pigs (Tle 1). The percentge of intestinl weight represented y mucos ws significntly reduced in DOX-Form pigs, ut not in DOX-Colos pigs, oth compred with their respective SAL controls (P.5, Fig. 4B). Anlyzed cross the two diets, the DOX tretment decresed intestinl weight nd colon weight compred with SAL (P.5), ut, when nlyzed for ech diet seprtely, only the DOX-induced reduction in intestinl weight in Form-fed piglets ws significnt (3% reduction, P.5, Tle 1). For the weight of other internl orgns, the spleen ws most ffected y DOX with reduction to 3 5%, compred with SAL (P.5), with no effect of diet (Tle 1). Across SAL nd DOX tretments, liver weight ws significntly lower in Colos pigs compred with Form (P.5, Tle 1). DOX-Colos pigs hd the longest villi in the Mid nd Dist intestinl regions compred with DOX-Form nd SAL-Colos (P.5, Fig. 5A). The DOX tretment reduced crypt depths Downloded from y on August 29, 216 Fig. 5. Villus height (A), crypt depth (B), villus-crypt rtio (VC-rtio; C), nd ctivity of lctse (D), sucrse (E), mltse (F), dipeptidylpeptidse IV (DPP-IV; G), minopeptidse A (ApA; H), nd minopeptidse N (ApN; I) mesured in the proximl (Prox), middle (Mid), nd distl (Dist) regions of the smll intestine, respectively (mens SE) re shown. Results for doxoruicin-treted pigs fed formul (DOX-Form, n 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicin-treted pigs fed ovine colostrum (DOX-Colos, n 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 7) re shown., Different superscript letters indicte significnt differences mong the four groups within the sme region (P.5).

7 cross ll three regions nd cross diets, indicting lowered crypt cell prolifertion (P.5 for Colos nd trend for Form, Fig. 5B). Consequently, the villus-to-crypt rtios in the Mid nd Dist regions were incresed in the DOX-Colos group, compred with ll the other groups (P.5, Fig. 5C). Discchridse ctivities were highest in Prox nd Mid intestinl regions, while peptidse ctivities were highest in Mid nd Dist regions (Fig. 5, D I). Only for Colos pigs were the lctse, sucrose, nd mltse ctivities higher for DOX vs. SAL pigs (P.5, Fig. 5, D F). No significnt DOX effect ws detected for Form pigs. Similrly, it ws only for Colos pigs tht dipeptidylpeptidse IV ctivity ws higher for DOX vs. SAL pigs (Fig. 5G). Concentrtions of the proinflmmtory cytokines, IL-8 nd IL-6 (Fig. 6) were lower in the Dist intestine of pigs fed Colos (P.5 cross tretments), ut only for IL-8 were vlues for DOX-Colos lower thn for DOX-Form pigs (P.5, Fig. 6A). Tissue levels of IL-1 nd TNF- did not differ etween diets nd tretments. Prolifertion, poptosis, nd intrcellulr structure. Immunohistochemistry stining of Ki-67-positive cells in the intestine showed lower numer of cells in DOX-Colos pigs compred with SAL-Colos pigs (P.5), nd numers were similr to those in the DOX-Form nd SAL-Form pigs, which showed no differences (Fig. 7, A nd G). Yet there ws significnt negtive effect of chemotherpy on Ki-67-positive cells when nlyzed cross diets (P.1). Western lot of PCNA reveled similr results in tht vlues were lower in DOX-Colos thn in SAL-Colos pigs (P.5), while DOX- Form tended to show lower vlues thn SAL-Form pigs (P.19, Fig. 7D), with no significnt effects of diet. Cleved cspse-3 vlues were significntly lower in DOX-Form thn in the other three groups, which hd similr levels (P.5 Fig. 7B). Uncleved cspse-3 did not differ significntly mong groups (Fig. 7E), lthough DOX incresed the vlues, reltive to SAL, s nlyzed cross diets (P.1). Overll, DOX tretment decresed the -ctin nd GAPDH levels, with limited effects of diet (Fig. 7, C nd F). DISCUSSION Fig. 6. Tissue levels of proinflmmtory cytokines interleukin-8 (IL-8; A) nd interleukin-6 (IL-6; B) in the distl region of the smll intestine (mens SE) re shown. Results for doxoruicin-treted pigs fed formul (DOX-Form, n 9), sline-treted pigs fed formul (SAL-Form, n 7), doxoruicin-treted pigs fed ovine colostrum (DOX-Colos, n 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 7) re shown., Different superscript letters indicte significnt differences mong the four groups (P.5). G329 Nutritionl support is criticl during tretment of peditric cncer ptients to prevent mlnutrition, which decreses chemotherpy tolernce, increses toxicity nd infection rtes, nd compromises outcome (4). Enterl nutrition formuls re preferred over totl prenterl nutrition ecuse of ssumed enefits for the gut (37) nd re recommended in ptients with functionl GI trct. Yet there is no consensus regrding the type of enterl nutrition support for peditric ptients. Our findings indicte tht the type of liquid diet support influences the response to the chemotherpeutic gent, DOX, nd tht diet rich in milk ioctive fctors, such s ovine colostrum, might e eneficil. In humns, gut toxicity develops erly fter chemotherpy (27), nd the onset of dirrhe nd weight loss 3 4 dys fter tretment in the current study coincides with the onset of GI toxicity in older, wenling piglets fter single dose of DOX (36). Likewise, the intestinl structurl response to DOX (e.g., decresed intestinl weight nd mount of mucos) is consistent with dt from studies in juvenile pigs (36). On the other hnd, the dministrtion of DOX did not significntly chnge lctose digestive cpcity, gut permeility, nd tissue proinflmmtory cytokines, nd possily some intestinl responses to DOX re less pronounced in prewened vs. wened juvenile pigs, t lest on dy 5 fter tretment. This my e explined y more immture inflmmtory response during erly life, compred with dult ptients, despite tht gut inflmmtory response is thought to e centrl element in the complictions of chemotherpy cross ll ptients (19, 26). Regrdless, it is importnt to note tht the present results of DOX re documented in stte of erly intestinl development, nd effects on more mture intestine could e different. CRP is systemic mrker of inflmmtory nd infectious complictions (38), nd the elevted CRP levels in this study re consistent with systemic inflmmtory response fter DOX tretment. Likewise, the mrkedly reduced spleen weight nd chnges in hemtology fter single dose of DOX were consistent with reduced hemtopoiesis. Growing children nd their GI trct re highly responsive to the type of nutrition support (6, 55). In ddition to mcronutrients, ovine colostrum contins numerous ioctive compounds tht influence oth gut mturtion nd systemic immunity. This includes GI growth, digestive function, mucosl immune function nd inflmmtion, nd the resident microiot. These my hve contriuted to the different responses of the SAL pigs fed colostrum, compred with those fed formul. Nevertheless, the colostrum diet fed to piglets not exposed to DOX ws ssocited with slower ody weight gin, compred with pigs fed formul, nd colostrum did not in this study increse the length nd weight of the smll intestine, villus height, crypt depth, or ctivity of digestive enzymes in the SAL pigs. Potentilly, the most eneficil effects of colostrl ioctive fctors re restricted to the immedite neontl period, consistent with the lcking effect of ovine colostrum on intestinl nutrient function in helthy dults or stle short owel syndrome ptients (13, 34, 47). Nevertheless, the oserved increses in lctose digestive cpcity nd reduced permeility in colostrum-fed SAL pigs my suggest some intestinl enefits, reltive to formul. These differences could lso e relted to potentil direct negtive effects of the chosen formul diet. In preterm pigs, detrimentl effects on gut structure nd function hve een induced y formuls very similr to the one used in this study (43, 55), nd the dmging effects were t lest prtly relted to replcing milk lctose with Downloded from y on August 29, 216

8 G33 % positive nuclei A 5 Ki Expression level B Cleved Cspse C c β-ctin c Expression level G D SAL-Form PCNA DOX-Form SAL-Colos SAL-Form DOX-Colos E SAL-Form Cspse-3 P=.11 DOX-Form SAL-Colos DOX-Colos DOX-Form SAL-Colos F SAL-Form GAPDH DOX-Form SAL-Colos DOX-Colos DOX-Colos Downloded from Fig. 7. Mrkers of prolifertion, poptosis, nd intrcellulr structure re shown. Immunohistochemistry stining nd nlysis of Ki-67 (A nd G) nd Western lot quntifiction of PCNA (D), cleved cspse-3 (B), cspse-3 (E), -ctin (C), nd GAPDH (F) in intestinl tissue smples from the middle region (mens SE) re shown. Results for doxoruicin-treted pigs fed formul (DOX-Form, n 9), sline-treted pigs fed formul (SAL-Form, n 6 7), doxoruicin-treted pigs fed ovine colostrum (DOX-Colos, n 8 9), nd sline-treted pigs fed ovine colostrum (SAL-Colos, n 6 7) re shown.,,c Different superscript letters indicte significnt differences mong the four groups (P.5). 3 µm y on August 29, 216 mltodextrins (56). Such formuls only cuse severe intestinl inflmmtion nd pthology in preterm pigs, not in norml term pigs, lthough some intestinl functions my e negtively ffected lso in term pigs (55). Bioctive fctors in milk hve previously een reported to meliorte mucosl toxicity fter dministering chemotherpeutic gents to rts nd hmsters, nd there is lso some evidence for eneficil effects in humn chemotherpy ptients (11, 14, 15, 22, 23, 44, 59). Such effects include reducing cteril trnsloction cross the gut mucos in ptients fter dominl surgery (9) nd reducing intestinl permeility cused y nonsteroidl nti-inflmmtory drugs tht fcilitte cteril trnsloction to the mesenteric lymph nodes, liver, spleen, nd peripherl lood (29, 3). The different intestinl nd systemic responses etween DOX pigs fed colostrum or formul suggests tht ovine colostrum my e prticulrly eneficil during inflmmtory processes. This is consistent with the eneficil colostrum effects oserved for inflmmtory conditions in humns (44, 47, 52) nd for the devstting GI inflmmtory disese in preterm infnts, necrotizing enterocolitis (24, 41, 54). The greter mucos proportion, villus height, lctose digestive cpcity, nd reduced permeility in the DOX pigs fed colostrum, reltive to formul, highlight the enefits of ovine colostrum on gut structure nd function during DOX chemotherpy. This is corroorted y lowered IL-8 in ilel tissue

9 nd lower circulting CRP levels nd is consistent with other results from pigs (36, 46). The higher ctivities of the mesured rush-order enzymes nd incresed villus length, prticulrly in the DOX-Colos pigs, my indicte tht colostrum stimulted intestinl regenertion fter chemotherpy. Regrdless, Ki-67 nd PCNA nlyses did not show signs of incresed intestinl prolifertion on dy 5 fter DOX tretment, suggesting tht most of the colostrum-stimulted dptive prolifertion hd lredy occurred y this time point. In line with this, other experimentl studies hve demonstrted very erly onset of cellulr toxicity fter DOX tretment, nd tht intestinl responses re highly dynmic with cellulr regenertion within few dys of DOX dministrtion (16, 61). Interestingly, we found very low levels of cleved cspse-3 in the DOX-Form group, the group with the worst clinicl outcome, while DOX- Colos pigs hd similr levels s SAL controls. This suggests tht the cspse-3 pthwy is not dominting the DOX-relted cell deth, t lest not 5 dys fter DOX tretment, similr to findings in other studies (16, 31). The reduced level of cleved cspse-3 in DOX-Form is even more evident when djusted reltive to the level of uncleved cspse-3. This indictes tht cspse-3 ctivtion is somehow ffected (66). The ccumultion of uncleved cspse-3 in the DOX groups could indicte ltered expression s response to chemotherpy. Conversely, there ws more consistent negtive effect of DOX on -ctin nd GAPDH protein undnce, reflecting tht chemotherpy ffected structurl nd metolic processes of the intestinl cells (2, 32), even 5 dys fter tretment. These two proteins re normlly considered high-undnce housekeeping proteins with fundmentl functions for most cells nd re often used for protein loding control when nlyzing Western lots (2, 32). Such cellulr protein controls re, therefore, negtively ffected nd, therefore, unrelile in studies of chemotherpyinduced mucositis, s lso indicted in other studies (2, 32). We hve demonstrted tht prewened pigs provide good model for studying the responses to chemotherpeutic gents during erly life nd for evluting liquid diets for enterl nutrition support to ddress the ssocited toxicity nd GI disturnce. Although the incidence of cncer is low mong infnts, the unique nture of the immture intestine nd the responses to chemotherpy need to e etter understood for more effective chemotherpy in this sensitive popultion. A limittion of our study is the use of only single dose of DOX. Chemotherpeutic regimens typiclly involve multiple rounds of tretment, which, comined with other drugs, increses the toxicities. Questions remin regrding the more long-term outcomes in oth nimls nd children (35, 57) nd whether colostrum would shorten the recovery period, llowing for more frequent chemotherpy. The findings indicte tht ovine colostrum or other ioctive milk products my e eneficil to include into the enterl nutrition support during chemotherpy. DOX hd more severe effects on GI prmeters in Form-fed compred with Colos-fed pigs, illustrting tht the intestinl response to chemotherpy is diet dependent. However, the colostrum diet ws ssocited with reltively low ody weight gins, even in control pigs, nd it might e suoptiml nd lso unfesile to supply ovine colostrum s the sole source of enterl nutrition for peditric cncer ptients for extended periods. Still, the present study highlights how nutritionl intervention my reduce toxic responses to DOX in piglets, nd, consequently, we hve initited clinicl study on ovine colostrum s diet supplement to children with leukemi receiving chemotherpy (Clinicl- Trils.gov Identifier: NCT176684). ACKNOWLEDGMENTS G331 We thnk Elin Skytte, Kristin Møller, nd Mndy Greig from Comprtive Peditrics nd Nutrition, Dept. of Clinicl Veterinry nd Animl Science, University of Copenhgen, for help with niml procedures nd lortory nlyses. We lso thnk Lrs Ove Drgsted from Dept. of Nutrition, Exercise nd Sports, University of Copenhgen, for generous help in providing nlytic resources to detect citrulline in plsm. Finlly, we thnk Christin Ritz from Dept. of Nutrition, Exercise nd Sports, University of Copenhgen, for sttisticl counseling. GRANTS R. L. Shen received the Erly Investigtors Exchnge Progrm Grnt y the Interntionl Peditric Reserch Foundtion. DISCLOSURES None of the uthors hve ny conflicts of interest. University of Copenhgen hs filed ptent ppliction on use of ovine colostrum for humn infnts. P. T. Sngild is listed s sole inventor, ut hs declined ny shre of potentil revenue rising from commercil exploittion of such ptent. AUTHOR CONTRIBUTIONS R.L.S., P.E.L.P., nd P.M.H. performed experiments; R.L.S., P.E.L.P., P.J., C.F.H., nd P.M.H. nlyzed dt; R.L.S., P.E.L.P., M.R., P.J., C.F.H., R.K.B., P.M.H., K.M., nd P.T.S. interpreted results of experiments; R.L.S. prepred figures; R.L.S. drfted mnuscript; R.L.S., P.E.L.P., M.R., P.J., C.F.H., R.K.B., P.M.H., K.M., nd P.T.S. edited nd revised mnuscript; R.L.S., P.E.L.P., M.R., P.J., C.F.H., R.K.B., P.M.H., K.M., nd P.T.S. pproved finl version of mnuscript; P.E.L.P., R.K.B., K.M., nd P.T.S. conception nd design of reserch. REFERENCES 1. Aldhous MC, Meister D, Ghosh S. Modifiction of enterl diets in inflmmtory owel disese. Proc Nutr Soc 6: , Aldridge GM, Podrerc DM, Greenough WT, Weiler IJ. The use of totl protein stins s loding controls: n lterntive to high-undnce single-protein controls in semi-quntittive immunolotting. J Neurosci Methods 172: , Brsch A, Eld S, Altmn A, Dmto K, Epstein J. 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