Increased intestinal permeability in rats with graft

Transcription

1 Gu 1996; 39: Increased inesinal permeabiliy in ras wih graf versus hos disease 291 Deparmen of Surgery, Pennsylvania Sae Universiy, Coliege of Medicine, The Milon S Hershey Medical Cener, Hershey, Pennsylvania, USA W A Kolun M M Bloomer P Colony G L Kauffman Correspondence o: Dr W A Kolun, Division of General Surgery, C484, Pennsylvania Sae Universiy, College of Medicine, 5 Universiy Drive, Hershey, PA , USA. Acceped for publicaion 11 March 1996 W A Kolun, M M Bloomer, P Colony, G L Kauffman Absrac Background/Aims-The sudy of graf versus hos disease ofhe inesine has significan clinical relevance and may also be a model for oher immune mediaed inesinal diseases. There presenly is no simple non-invasive es ha can be used o evaluae graf versus hos disease induced inesinal injury in humans or animal models. This sudy esed he hypohesis ha graf versus hos disease leads o an increase in hos bowel permeabiliy as assessed by he relaive urinary excreion of orally adminisered laculose and rhamnose. Mehods-The urinary excreion raio of orally adminisered laculose and rhamnose was deermined daily for wo weeks in (Lewis x Brown-Norway) Fl ras wih graf versus hos disease caused by eiher he ransplanaion of parenal (Lewis) small bowel or he inraperioneal injecion of parenal (Lewis) splenic lymphocyes. Resuls-Significan wofold o fourfold increases in he laculose o rhamnose raio were seen in boh small bowel ransplan and splenic lymphocye ransfer animals suffering from graf versus hos disease during he second posoperaive week. This effec occurred sooner in small bowel ransplan han in splenic lymphocye ransfer animals (posoperaive day 7 versus 11, respecively). The signs of graf versus hos disease, including splenomegaly and alered inesinal mucosal archiecure, as well as he increased laculose o rhamnose raio were significanly aenuaed in small bowel ransplan animals reaed wih cyclosporine A (1 mg(kg/day). Conclusions-Graf versus hos disease is associaed wih an increase in he laculose o rhamnose clearance raio reflecing an increase in hos bowel permeabiliy. This increase, along wih he signs of sysemic graf versus hos disease, can be significanly amelioraed by cyclosporine A. The laculose o rhamnose clearance raio is a non-invasive echnique ha can be used o assess he inesinal effecs of graf versus hos disease and he associaed increase in inesinal permeabiliy. (Gu 1996; 39: ) Keywords: graf versus hos disease, inesinal permeabiliy, small bowel ransplanaion, cyclosporine A. Graf versus hos disease (GVHD) occurs in up o 75 per cen of paiens afer bone marrow ransplanaion and recenly has also been described in small bowel ransplan recipiens.l1 This condiion resuls from he large number of lymphocyes presen in boh bone marrow and small bowel grafs ha recognise he hos issue as foreign.5 In addiion o he cuaneous effecs such as alopecia and rash, GVHD commonly affecs he inesine. An accurae diagnosis of GVHD can be difficul o esablish when i affecs he gasroinesinal rac because of is relaively non-specific sympomaology. Upper inesinal or recal endoscopy and biopsy can aid in he diagnosis, bu pose significan risks especially when performed repeiively o monior disease progression. Similar difficulies are encounered in he experimenal laboraory, where assessmen of inesinal GVHD in animal models usually requires deah of he animal. A minimally invasive es could be very useful in he appropriae clinical or experimenal seing o diagnose inesinal GVHD or monior response o reamen. A relaively non-invasive mehod of assessing small bowel injury is he measuremen of inesinal permeabiliy using orally adminisered, non-meabolised, and minimally absorbed sugars, such as laculose and rhamnose. Increases in he raio of inesinal laculose o rhamnose permeaion and subsequen urinary clearance (LIR) have been described in oher immune mediaed inesinal illnesses, such as Crohn's disease.68 I was hypohesised ha a similar increase in LIR would be seen in GVHD induced injury of he inesine. Such increases in inesinal permeabiliy in GVHD afer bone marrow ransplanaion have already been suggesed using radioacive racer echniques, bu hese sudies have eiher been anecdoal or complicaed by concurren chemoherapy, which iself is known o increase bowel permeabiliy.9 1 Small bowel ransplanaion has also been associaed wih increased inesinal permeabiliy, bu his has been in paiens or animal models where he surgical manipulaion of he inesine iself, malnuriion or simulaneous rejecion phenomenon could have been responsible. 1" 12 We herefore underook o sudy he IJR clearance raios in wo differen ra models of GVHD and in GVHD animals reaed wih cyclosporine A, o es he hypohesis ha an increase in he IIR urinary clearance raio is associaed wih GVHD injury of he inesine. The presen series of experimens used inbred ras in a sibling o parenal cross o eliminae

2 292 Kolun, Bloomer, Colony, Kauffman Inferior vena cava Naive bowel oma Aora ~~~~Poral vein Tansplaned bowel! 'W - ~Aoric cuff Figure 1: Small bowel ransplan induced graf versus hos disease animal model. Transplaned bowel (wih passenger lymphocyes) is anasomosed in an auxiliary fashion o he recipien aora and vena cava. The animal's naive bowel is unmanipulaed and has is permeabiliy subsequenly measured as graf versus hos disease progresses. he possibiliy of rejecion and he naive, nonoperaed inesine was evaluaed, so minimising he effecs of surgical injury o he inesine. To characerise and confirm he presence and progression of GVHD in hese models, food consumpion, animal and splenic weighs, and inesinal hisology a he ime of deah was performed. Mehods Animal models and groups Male Lewis (Lew) and Lewis x Brown Norway (LBN) Fl virus anibody free ras (25-3 g) were obained from a commercial breeder (Harlan Sprague Dawley, Indianapolis, IN) and handled in accordance wih he guidelines of he American Associaion for Accrediaion of Laboraory Animal Care. Animals were quaranined upon arrival from he supplier and moniored for one week for illness prior o use. Animals were housed in meabolic cages (65-1, Nalgene Co, Rocheser, NY) during experimenal proocol o permi easy collecion of urine for bowel permeabiliy assessmen. Animals had free access o waer and Purina Sandard Roden Chow 51, alhough food was removed from cages during he hours of permeabiliy measuremens o avoid conaminaion of urine collecions wih food debris. GVHD was produced by eiher he auxiliary ransplanaion of small bowel (Fig 1) or inraperioneal injecion of splenic lymphocyes from Lew (RT-1 1,1) donors ino LBN F1 (RT-1 1,n) hoss. Such a cross from inbred paren o Fl progeny produces GVHD wihou rejecion because he major hisocompaibiliy (MHC) anigens presen in he homozygous grafed issue are also presen in he Fl recipien. The heerozygous Fl hos however, expresses MHC anigens ha are recognised as foreign by he grafed parenal lymphocyes, resuling in GVHD. All animals, wheher receiving small bowel grafs or inraperioneal splenic lymphocyes, had heir naive inesines lef inac for subsequen permeabiliy measuremens. Five groups of animals were sudied: (1) SBTx-GVHD (n= 14): LBNF 1 animals wih auxiliary Lew small bowel ransplans (SBTx) resuling in GVHD; (2) Sham-op conrols (n= 13): LBNF1 animals wih he same operaive dissecion as SBTx-GVHD animals bu wihou small bowel grafs; (3) Spl-GVHD (n=9): LBNF1 animals wih an average of 3*9X18 (range X 18) Lew splenocyes injeced inraperioneally, resuling in GVHD; (4) Un-op conrols (n=8): LBNF1 animals wihou operaive manipulaion; (5) SBTx-CsA (n=8): LBNF1 animals wih Lew small bowel ransplans (SBTx) as in group 1 above who also received 1 mg/kg/day cyclosporine A (CsA) subcuaneously. All animals had he permeabiliy of heir naive (in siu) bowel assessed daily for wo weeks afer ransplanaion using he laculose/ rhamnose (LIR) clearance echnique (see below). Food cups were weighed daily o assess food consumpion. Animals were weighed and inspeced daily for signs of GVHD (cuaneous eryhema, alopecia, weigh loss, and dermaiis). Foureen days afer grafing or sham operaion, animals were killed by exsanguinaion under eher anaeshesia. Spleens were weighed and segmens of bowel harvesed for hisology o confirm he presence of inesinal GVHD. Technique of small bowel ransplanaion The echnique of small bowel ransplanaion has been previously described.13 Briefly, small bowel from proximal jejunum o erminal ileum was harvesed from he donor and subsequenly anasomosed in an auxiliary fashion o he recipien's aora and vena cava. The proximal and disal ends of he graf were brough ou as somas, creaing an auxiliary Thiry-Villa loop. The animal's naive bowel was lef inac for subsequen bowel permeabiliy assessmen (Fig 1). Splenic lymphocye ransplanaion Spleens were harvesed from Lew ras using asepic echnique and placed ino serile Dulbecco's phosphae buffered saline (D-PBS, GIBCO BRL, Life Technologies, Grand Island, NY). Tissue was genly minced using forceps and he resulan suspension passed hrough a 7,um nylon mesh filer (Falcon, Becon Dickinson, Franklin Lakes, NJ). Red cells were lysed by he addiion of 15 ml of red cell lysing buffer (Sigma, S Louis, MO). Spleen cells were suspended wih D-PBS in a 5 cc conical ube and cenrifuged for eigh minues a 2 g in a refrigeraed cenrifuge. The supemaan was discarded, he pelle washed wo more imes, and finally brough up o 1 cc wih D-PBS. A cell coun was done using a haemocyomeer and rypan blue exclusion o deermine viabiliy. An average of 3.9X 18 viable cells (range 3-6.2X 18) cells were injeced inraperioneally ino recipien LBN F1 animals using an 18 gauge needle. I has been shown ha a minimum of 1. 5 X 18 splenocyes are necessary o cause GVHD in his ra model.14

3 Increased inesinal permeabiliy in ras wih graf versus hos disease Laculose/rhamnose clearance Animals were gavaged daily each evening wih a soluion conaining 4 mg laculose and 1 mg rhamnose (Pfansiehl Laboraories, Waukegan, IL) in 5 cc of disilled waer. The urinary clearance of orally adminisered laculose and rhamnose has been shown o be largely complee in ras by eigh hours.'5 Urine was colleced for a leas eigh hours overnigh in recepacles conaining 2,ul of chlorhexidine (Sigma, S Louis, MO) as a preservaive. Urine samples were cenrifuged a 2 g for 1 minues and 1P ml aliquos of he supernaan were frozen a - 7 C for laer analysis by high pressure liquid chromaography (HPLC). Urine samples before gasric gavage were confirmed as conaining unmeasurable amouns of boh he es sugars and inernal sandard sugars (see below). Sored urine specimens were defrosed on ice and cenrifuged a 2 g for five minues. The supernaan was dilued 1-fold o 2- fold wih deionised waer o yield final es sugar concenraions ha fell wihin he linear range of sandard curves for each sugar. Inernal sandards, arabinose, and cellobiose (Pfansiehl Labs, Waukegan, IL) were added o obain a final concenraion of 2 mgll. Samples were filered hrough.2 pl pore size syringe ip filers (Supelco, Bellefone, PA) and 2,ul aliquos analysed by HPLC using an anion exchange column (Carbopac PAl, Dionex, Marlon, NJ) and pulsed amperomeric elecrochemical deecion (Coulochem II, ESA, New Bedford, MA).6 17 Sandard curves were run for es and inernal sandard sugars and confirmed o be linear by leas squares analysis (r2> 95). Peak heigh analysis of he chromaograms (Specra Physics SP 427) was used o deermine concenraions of he sugars. Tes sugar resuls were expressed as he raio: IJR=percen urinary recovery of orally adminisered laculose/percen urinary recovery of orally adminisered rhamnose. Absolue and relaive spleen weighs Boh absolue and relaive splenomegaly have been used as quanifiable indicaors of GVHD. A he ime of deah (14 days posoperaion) he absolue we weigh of each animal's spleen was deermined o he neares O1 mg. One animal in he Spl-GVHD group died on day 13 so only eigh animals in his group had spleen weighs deermined. Relaive spleen weigh was expressed as absolue spleen weigh (g)/animal body weigh a he ime of deah (kg). Inesinal hisology To confirm he effecs of GVHD on he hos inesine, samples of small bowel were harvesed a he ime of deah from eigh Sham-op, eigh SBTx-CsA animals, six SBTx- GVHD animals (and hree addiional animals done subsequenly for a oal of nine SBTx- GVHD animals), and six Spl-GVHD animals and evaluaed hisologically for cryp hyperplasia and villus arophy. These wo feaures have been shown o be indicaive of inesinal GVHD.18-2 Full hickness segmens of proximal jejunum, mid (5 cm) and disal (9-1 cm) small bowel were harvesed from each animal and sained in sandard fashion wih haemaoxylin and eosin. An average villus and cryp lengh per segmen of bowel sudied was generaed by a blinded hisologis by measuring 2 well oriened villi and cryps wih an eyepiece micromeer. Saisics Numerical daa are expressed as mean (SEM). Animal weigh, food consumpion, spleen and hisological daa were saisically evaluaed using paired Suden's es for comparisons wihin groups and unpaired Suden's es for comparisons beween groups. All ess were wo ailed. Significance was considered achieved when p@o.o5. The LR values from all he animals in each experimenal group for each day were combined yielding a group mean and sandard error for each day. I was noed ha all animals who underwen operaive manipulaion (Sham-op, SBTX-GVHD, SBTx-CsA) had a ransien increase in he LR raio hree o six days afer surgery (see Fig 7). This was fel o be a side effec of he operaion because i was absen in unoperaed animals (Un-op, Spl-GVHD) and a similar effec has been noed in human sudies.2' 22 To eliminae his variable, a six day posoperaive washou period was arbirarily se and LR daa were saisically compared only during he posoperaive day 7-13 period (inclusive). This ime period also coincided wih he clinical manifesaions of GVHD in he animals (see below). The LIR daa for his enire 7-13 day period for each group were hen saisically compared using repeaed measures analysis of variance as. -*-Un-op (n = 8) -'-SBTx-GVHD (n = 14) -*nsham-op (n = 13) -'Spi-GVHD (n = 9) --SBTx-CsA (n = 8) E95 c cc * 9.F_ o ' 8 ) -7r Time (days pos-op) Figure 2: Mean weighfor each animal group, expressed as per cen of iniial weigh. All experimenal animal groups had los a significan amoun ofweigh by he ime of deah (14 days pos-op) relaive o sham or un-operaed conrols (*p<5, p<-1 v sham-op; p<1 v un-op by unpaired Suden's es). Sandard error bars omiedfor clariy.

4 294 Kolun, Bloomer, Colony, Kauffman SBTx-CsA Sham-op SBTx-GVHD 1( 2 SBTx-CsA 15 SBTx-GVHD 1 SpI-GVHD E a- 6 F g/animal/day Days -6 Days 7-13 Figure 3: Average daily food consumpion (grams/animal/day) for all animal groups for he firs versus he second posoperaive seven day periods. All animals undergoing surgery (sham-op, SBTx-GVHD, SBTx-CsA) had significanly (p< 1) lowerfood consumpion han un-operaed animals (un-op, Spl-GVHD) during hefirs posoperaive week. All animal groups had near idenicalfood consumpion during he second week, excep for he Spl-GVHD group, which had a significan decrease (p< 1 v all oher groups and v day -6, by unpaired and paired es, respecively). Error bars eliminaedfor clariy. -C 4- f CD 3._ c U) Q) _ 2 1 Sham-op (n = 13) M* SBTx-GVHD (n = 14) SpI-GVHD (n = 8) 6 M SBTx-CsA (n = 8) I~~~~~~~ Relaive * ADsolue CO spleen weigh spleen weigh Figure 4: Mean relaive spleen weighs (lef) and mean absolue spleen weighs (righ) in animal groups. Graf versus hos disease was confirmed a deah (14 days posop) in animals wih small bowel ransplans (SBTx- GVHD) and splenocye ransfer (Spl-GVHD) by an increase in boh relaive spleen weigh (absolue spleen weigh (g)/body weigh a deah (kg)) and absolue spleen weigh (g). (*p<.1, p<o5 v sham-op by unpaired Suden's es). There was no saisical difference in eiher absolue or relaive spleen weighs beween cyclosporine A reaed animals (SBTx-CsA) and sham-op conrols. implemened in SAS PROC GLM (SAS Insiue, Cary, NC) and p values repored. Resuls Clinical appearance Boh he SBTx-GVHD and Spl-GVHD animals recovered and appeared normal unil he sevenh or eighh posoperaive day when he firs signs of GVHD disease became manifes as mild cuaneous eryhema of he paws, ears and snou, wih some hair loss. These feaures gradually progressed o dramaic cuaneous hyperemia and dermaiis, alopecia, nasal and ocular discharge, and phimoic appearing so m * 4 H 3 H 2 1-1K 5 4 V 3 _- 2 H 1 o 3r 25K 2K 15 H- 1 5 o -n-1 VIllUS E Sham-op (n = 8) M SBTx-GVHD (n = 9) M SpI-GVHD (n = 6) SBTx-CsA (n =8) * * * Cryp Figure 5: Villus heigh and cryp deph in proximal, mid, and disal small bowel in experimenal groups of animals. Animals afer SBTx (SBTx-GVHD group) had very significan decreases in villus heigh and increases in cryp deph in all regions of bowel sudied compared wih shamop animals, confirming he effecs of GVHD on hos inesine. All hese changes improved significanly when animals were reaed wih cyclosporine A (SBTx-CsA group) hough hey did no compleely reurn o sham-op values. GVHD afer splenic ransplanaion (Spl-GVHD group) also caused significan decreases in villus heigh bu appeared o cause less severe cryp lengh changes when compared wih ha seen in he SBTx-GVHD animals (*p < 1 versus respecive sham-op group; p<.1 versus respecive SBTx-GVHD group). genialia. There was no diarrhoea. Sools became viscid, bu were always in pelle form, a foruious siuaion as urine collecions were no conaminaed by liquid faecal maerial. Animals became less acive and assumed a hunched posure wih apparen difficuly in fully exending heir limbs by he 13h o 14h day. Sham operaed animals recuperaed and reurned o normal aciviy and appearance promply wihou clinical deerioraion during he experimenal period. Unoperaed animals exhibied normal aciviy and behaviour. SBTx-CsA animals appeared generally healhy even during he second posoperaive week. Mild cuaneous eryhema was -observed in some,

5 Increased inesinal permeabiliy in ras wih graf versus hos disease 295 Bi- EE D Figure 6: Proximal small bowel micrographs from sham-op(a) and SBTx-GVHD(B) animals (original magnificaion xl25). Higher power micrographs (original magnificaion X312) of disal small bowelfrom sham-op(c) and SBTx-GVHD(D) animals. Noe villus bluning and cryp elongaion in GVHD specimens compared wih sham-op. Inesinal epihelial inegriy is mainained wihou evidence of ulceraion or increased apoposis in he GVHD animals. however, here were no hair or mucous membrane changes. Food consumpion/weigh changes Figure 2 shows he mean weigh change of he animal groups over he course of he experin insignif week pi 4._ Co W-._ co.5 r.4 h if Z:r:.3 V E.2 m..1 P emporary period of posoperaive weigh loss before reurning o a weigh comparable o he preoperaive level. Animals wih eiher form of GVHD, as well as he CsA reaed animals all had a similar paern of weigh loss over he experimenal period ha resuled in hese hree groups having a final weigh a deah significanly less han Sham-op or Un-op conrols. Figure 3 shows he mean daily food consumpion for each of he groups during he firs and second 7 day posoperaive periods of he experimen. Food consumpion during he firs week was very similar, bu significanly depressed in hose animal groups ha underwen surgery compared wih hose animal groups ha did no have surgery. Food consumpion during he second week reurned o conrol levels in all animal groups, excep he Spl-GVHD group in which here was a significan decline. This decrease in food inake may have accouned, in par, for he concurren rapid weigh loss seen in his animal group. The weigh loss seen in he SBTx-GVHD and SBTX-CsA groups during he second week, however, could no be aribued o decreased dieary inake. Spleen weighs Relaive and absolue spleen weighs confirmed he presence of GVHD in he experimenal groups and he efficacy of CsA reamen in reaing GVHD in animals wih small bowel ransplans (Fig 4). nen. The Un-op group gained an Inesinal hisology can amoun of weigh during he wo There were significan reducions in villus eriod, while he Sham-op group had a heigh and increases in cryp deph in SBTx- GVHD animals compared wih Sham-op conrols in he proximal, mid, and disal regions of SpB-GVHD (n = 9) he small bowel confirming he effecs of GVHD Un-op (n = 8) on he hos inesine (Fig 5). In SBTx-GVHD -o- Sham-op (n=13) animals, villus bluning appeared o be mos severe in he disal bowel where mean villus heigh was 38% less han Sham-op conrols whereas i was only 12% and 2% less in he proximal and mid small bowel areas, respecively. Though here was also significan villus bluning diffusely in Spl-GVHD animals, cryp elongaion was less prominen, suggesing less severe inesinal injury compared wih SBTx- GVHD animals. I is noeworhy ha epihelial //-/, \ inegriy was mainained in all segmens of bowel sudied in boh GVHD animal groups wih no loss of surface epihelium or ulceraions noed (Fig 6). When SBTx animals were reaed wih CyA (SBTx-CyA group), here were I i I I I I significan improvemens in villus and cryp lenghs compared wih SBTX-GVHD animals, o Time (days pos-op) approaching hough no compleely reurning Figure 7: Bowel permeabiliy as defined by IR raios in o Sham-op conrol values in all segmens of rnnrnlc I (snham-nnand un-on) and animals wih zraf inesine sudied. ;un,rug, ju"-uf Unu F-VF - -- W*S* -j versus hos disease afer small bowel ransplanaion (SBTx-GVHD) or afer splenic lymphocye ransfer (Spl- GVHD). For days 7-13, LIR was significanly increased in SBTx-GVHD animals v sham-op conrols (p<1) and in Spl-GVHD animals v un-op conrols (p<2) by repeaed measures analysis of variance. The overall increase in LIR in he SBTx-GVHD group for he 7-13 day period was saisically greaer han ha in he Spl-GVHD group a he p= 5 level. Laculose/rhamnose bowel permeabiliy Unoperaed conrol animals (Un-op group) showed very consisen mean L/R raios from day o day during he wo week experimenal periods (Fig 7). Sham-op L/R raios were

6 296 Kolun, Bloomer, Colony, Kauffman.7 n.6.5h.4.3 H-.2.1 (1 2 4 Time Figure 8: Effec of cyclosporirne A (CsA, 1 mg/kg/day) on migh augmen local injury or promoe LIR in ras wih graf versus hos disease afer small bowel sysemic sepsis by allowing he enry of ransplanaion. The LIR curves of he SBTx-GVHD and noxious luminal microflora or oxins. The sham-op animals are reprodc uced from Figure 7for increase in he UR raio found in his comparison wih he CsA re,aed animals (SBTx-CsA). sudy The LIR raios of he SBTx--Csperiod was significanly less Ihan ha in he SBTx-GVHD inesinal barrier in GVHD injury of he groupfor he 7-13 day suppors his concep of a compromised group (p< 25), bu sill ix 4creased relaive o he inesine. I is imporan o realise, however, sham-op conrol group (p< 4) by repeaed measures analysis of variance. ha his increase in bowel permeabiliy as idenified by he raised L/R raio need no saisically no differen han hose of he necessarily correlae wih increased bowel per- 7-13, alhough here meabiliy o oher compounds, including Un-op group during dlays appeared o be a ranisien increase in IhR a eneric organisms. Differen mechanisms for days 5 and 6 as previoiusly menioned. upake of various maerials exis in he Compared wih hie Sham-op group, he inesine. Translocaion of baceria and fungi SBTx-GVHD group exhibied a saisically has been shown o occur ranscellularly, for significan increase in L/R raios for he 7-13 example.29 The presenly idenified increase in day period (p<1) i, when he clinical signs LIR herefore, canno be freely exrapolaed o of GVHD were manife.s. Similarly, here was a oher maerials, including much larger saisically significan increase in he LJR raio subsances such as endooxin or baceria bu in he GVHD-Spl aniimals versus Un-op ani- does reinforce he noion ha he inesinal mals during he saime 7-13 day period defensive barrier is compromised in GVHD. found o be significan v GVHD-Spl). Figure 8 shows he CsA group compared IR raios of he SBTx- Increased serum concenraions of TNF in wih he SBTx-GVHD GVHD afer SBTx have been described and and Sham-op groups. When animals receiving anibodies o boh IFN y and TNF have been small bowel ransplanis were reaed wih CsA shown o ameliorae some of he hisopaho- (SBTx-CsA group), ] LJR raios for he 7-13 logical changes of GVHD injury in mice day period were signiificanly lower han ha Of significan ineres, IFN y has also been seen in he unreae:d SBTx-GVHD group shown o play a par in inesinal barrier (p<25). Cyclosporine A did no compleely funcion. Ussing chamber sudies using culhir raios seen in he ured monolayers of T-84 human inesinal ameliorae he raised SBTx-GVHD group however, because raios cells have shown a decrease in ransepihelial in he SBTx-CsA groiup were sill significanly resisance a he level of he inercellular igh increased relaive c (p<4). Discussion The concep of he baceria or oxins ha leads o sysemic illni eneric permeabiliy b one ha has been illnesses, including C or selecive deconan aenuaed GVHD ir.{i Sham-GVHD Sham-op (n= 13) = 13) ransplan models and passive immunisaion using ani-escherichia coli aniserum has been shown o improve animal survival.2>27 - SBTx-CsA (n = 8) Similarly, human sudies in bone marrow ransplan recipiens have suggesed ha gu deconaminaion wih oral anibioics may decrease he clinical signs and infecious complicaions of GVHD.28 A wo sage model of inesinal injury in GVHD includes an immunological phase, in which grafed lymphocyes recognise alloanigen on he hos issues, and an effecor or issue damage phase, where he release of cyokines or oher inflammaory mediaors incur or aggravae I I injury o he bowel.25 The resuling increased permeabiliy of he injured inesinal epi- (days pos-op) helium may represen a hird phase, which (p< 2). Qualiaivefly, he increase in he This sudy suggess increases in LJR are a IJR raio in he SBTx-GVHD group occurred consequence of immune mediaed inflammain he Spl-GVHD group ion direced owards he inesine during earlier han ha seen: (posoperaive day 7 nversus posoperaive day GVHD. The exac mechanism is unclear bu 1 1, respecively). When hese wo groups were may enail he local release of poen saisically comparecd, his difference was cyokines.3 Two cyokines ha have been (p=5, GVHD-SBTx implicaed in GVHD injury include inerferon y (IFN y) and umour necrosis facor (TNF). he Sham-op group juncion afer IFN y adminisraion As he LIR echnique of bowel permeabiliy measuremen is fel o assess paracellular inegriy, he aleraion defined in his sudy may represen he in vivo correlae of his in viro increase in gu as a reservoir of epihelial permeabiliy ha occurs secondary aggravaes local injury or o he local release of his cyokine during ess when he proecive GVHD inesinal injury. arrier is compromised, is Weigh loss during GVHD has been proposed in numerous described before and may resul from a NVHD Serilisaion number of facors, including inesinal dysninaion of he gu has funcion, compromised abiliy o uilise he i murine bone marrow absorbed nuriens or a hypermeabolic sae.34

7 Increased inesinal permeabiliy in ras wih graf versus hos disease 297 I is noeworhy ha here was a similar paern of weigh loss in he SBTx-CsA animals who also had conrol levels of food inake bu did no manifes over signs of GVHD. This suggess ha eiher CsA iself had cachexia inducing effecs, or ha CsA imperfecly reaed he GVHD. The laer is probably he case as firsly, he LR raios did no reurn compleely o conrol values in he SBTx-CsA group and secondly, a subsequen group of six animals injeced daily wih CsA (1 mg/kg) had a mean weigh afer 14 days virually idenical o heir mean saring weigh (13 (3.5)%). The weigh loss seen in he SBTx- CsA animals also dispues he possibiliy ha he significan increases in he IR raios seen in he GVHD groups of animals were simply due o generalised nuriional deerioraion causing inesinal arophy and injury, because he SBTx-CsA group had a relaively low L/R raio associaed wih significan weigh loss. The increased L/R raio seen in his sudy was significan and presens he sugar clearance echnique as a comparaively noninvasive mehod by which o follow GVHD injury in he conrolled laboraory or clinical seing. Assessmen of reamen direced a amelioraing inesinal GVHD injury could be accomplished, in par, by repeiive measuremen of he IhR clearance raio. Such a sudy in Crohn's disease paiens showed an increased laculose/rhamnose raio in hose wih acive disease, which decreased when paiens were reaed and became clinically improved.6 I would be emping o sugges ha he laculose/rhamnose measuremen could provide a non-invasive mehod by which o diagnose inesinal GVHD in paiens undergoing bone marrow or small bowel ransplanaion. Limiaions on he applicabiliy of he echnique may exis, however, as many oher facors have been associaed wih increased bowel permeabiliy. These include surgery, chemoherapy, neoplasia, sepsis, and malnuriion, many of which could also be presen in he paien wih GVHD Repeiive IJR measuremens may none he less be clinically useful in he individual GVHD paien by providing an improving or deerioraing rend ha could minimise he need for more morbid endoscopic biopsy procedures. In his regard, he hr echnique has significan advanages over oher echniques of bowel permeabiliy measuremen, especially hose using radioacive racers, as i can be repeiively applied in he clinical arena wih minimal risk.1 In he conrolled laboraory seing, he [R raio could similarly be effecive. Therapeuic inervenions aimed a decreasing inesinal GVHD injury, such as was done here wih CsA, could be assessed in animals on a daily basis wihou he need for deah. The clinical manifesaions of GVHD in he SBTx-GVHD and he Spl-GVHD groups were similar, bu boh he IhR and inesinal hisology daa sugges ha a more severe inesinal injury occurred in he SBTx-GVHD animals. Cryp elongaion occurred in proximal, mid, and disal small bowel in he SBTx-GVHD animals, bu was presen only in he proximal porions of he bowel in Spl- GVHD animals. The IIR increase appeared sooner in SBTx-GVHD animals compared wih Spl-GVHD animals (day 7 v 11, respecively). The dispariy in food consumpion beween he Spl-GVHD and SBTx-GVHD animals during he second week also sugges ha GVHD afer SBTx may be differen han ha seen wih splenocye ransfer. Such differences may be relaed o he finding ha lymphocyes from he inesine end o 'home' back o he gu.38 GVHD inesinal injury afer SBTx may be greaer han ha caused by peripheral or splenic lymphocye ransfer because of his rafficking phenomenon, direcing and preferenially localising he donor eneric lymphocyes and injury o he hos bowel. This suggess ha daa regarding GVHD mediaed inesinal injury obained from clinical bone marrow ransplanaion paiens may no be direcly applicable o he increasing number of paiens receiving small bowel ransplans. Furher clinical and animal sudies are warraned, evaluaing his concep of lymphocye homing and hos inesinal injury afer bowel ransplanaion. In summary, using boh splenocye and small bowel ransplan models of GVHD, increases in hos bowel permeabiliy, as measured by he laculose/rhamnose urinary clearance echnique were observed. This increase in he laculose/rhamnose raio was aenuaed by reamen of small bowel ransplan animals wih cyclosporine A. These daa sugges ha he laculose/rhamnose urinary clearance assay may provide a non-invasive mehod by which o assess inesinal injury and he effec of reamen in GVHD mediaed injury of he bowel. 1 Brubaker DB. Immunopahogenic mechanisms of posransfusion graf-vs-hos disease. Proc Soc Exp Biol Med 1993; 22: Gran D, Garcia B, Wall W, Oheme-Fianko D, Zhong R, Mimeaul R, e al. Graf-versus-hos disease afer clinical small bowel/liver ransplanaion. Transplan Proc 199; 22: Cohen Z, Silverman RE, Wassef R, Levy GA, Burnsein M, Cullen J, e al. 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