Tumor Necrosis Factor Alpha Inhibitors as Immunomodulatory Antirejection Agents after Intestinal Transplantation

American Journal of Transplantation 2011; 11: 1041–1050Wiley Periodicals Inc.

C© 2011 The AuthorsJournal compilation C© 2011 The American Society of

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2011.03497.x

Tumor Necrosis Factor Alpha Inhibitors asImmunomodulatory Antirejection Agents afterIntestinal Transplantation

U. A. Gerlacha, M. Kochb, H.-P. Mullerc,

W. Veltzke-Schliekerc, P. Neuhausa and

A. Paschera,∗aDepartment of General, Visceral and TransplantationSurgery, Charite-Universitaetsmedizin Berlin, CampusVirchow Klinikum, Berlin, GermanybInstitute of Pathology, Charite-UniversitaetsmedizinBerlin, Campus Mitte, Berlin, GermanycDepartment of Internal Medicine, Division ofGastroenterology and Hepatology,Charite-Universitaetsmedizin Berlin, Campus VirchowKlinikum, Berlin, Germany∗Corresponding author: A Pascher,[email protected]

We reported the successful administration of inflix-imab for late-onset OKT3-resistant rejection in twopatients, who presented persistent ulcerative inflam-mation of the ileal graft after intestinal transplanta-tion (ITX). Based on this experience, the present studydemonstrated our long-term experience with inflix-imab for different types of rejection-related and inflam-matory allograft alterations. Infliximab administration(5 mg/kg body weight (BW)) was initiated at a meanof 18.2 ± 14.1 months after transplantation. The num-ber of administrations per patient averaged 8.4 ± 6.7.Repeat dosing was timed according to clinical signsand graft histology in addition to serum-levels of tu-mor necrosis factor alpha (TNFa ), lipopolysaccharidebinding protein (LBP) and C-reactive protein (CRP).Infliximab was successful in the following patients:patients with late-onset OKT3- and steroid-refractoryrejection who presented persistent ulcerative alter-ations of the ileal graft (n = 5), patients with ul-cerative ileitis/anastomositis, who did not show typ-ical histological rejection signs (n = 2), and onepatient with early-onset OKT3-resistant rejection. In-fliximab was not successful in one patient with early-onset OKT3-resistant rejection that was accompaniedby treatment-refractory humoral rejection. In conclu-sion, infliximab can expand therapeutic options forlate-onset OKT3- and steroid-refractory rejection andchronic inflammatory graft alterations in intestinal al-lograft recipients.

Key words: Monoclonal antibodies, rejection, smallintestine, steroid refractory rejection, TNF-alpha

Abbreviations: ACR, acute cellular rejection; ATI,antibodies to infliximab; BW, body weight; CMV,cytomegalovirus; CNI, calcineurin inhibitors; CRP, C-reactive protein; EBV, Epstein–Barr Virus; HACA, hu-man antichimeric antibodies; HSV, herpes simplexvirus; HHV 6/7, human herpes virus type 6/7; IBD, in-flammatory bowel disease; ITX, intestinal transplan-tation; LBP, lipopolysaccharide binding protein; MMF,mycophenolate mofetil; MVTX, multivisceral trans-plantation; OKT3, Orthoklone (Muromonab); POD,postoperative day; PTLD, post-transplant lymphopro-liferative disease; SEM, standard error of mean; Th1,T-helper-1 cells; TNF a , tumor necrosis factor a ; VZV,varicella zoster virus.

Received 14 July 2010, revised 17 January 2011 andaccepted for publication 04 February 2011

Introduction

Acute cellular rejection (ACR), which is the major causeof graft loss, continues to be a severe complication afterintestinal transplantation (ITX) (1).

In experimental models of ITX, allograft rejection was ac-companied by a significant upregulation of TNFa (2), whichnot only correlated with the severity of rejection but alsopredicted ACR (3–5). Several clinical studies have demon-strated an increase in TNFa serum levels during rejec-tion episodes following solid organ transplantations (6)which suggests that TNFa is an important marker cytokine.Therefore, TNFa has become a target molecule for antire-jection treatment. Indeed, TNFa has been assessed in ex-perimental studies after lung (7,8), renal (9) and aortic (10)transplantation, and TNFa inhibition resulted in a reductionin inflammatory responses and prolonged graft survival. Ina standard rat model of ITX, short-term treatment witha combination of anti-CD4 and anti-TNFa antibody pro-longed graft survival in more than 50% of the recipients(11). Furhermore, clinical experience with TNFa inhibitorsafter solid organ transplantation has revealed good resultsin terms of safety, reduction in toxicity and allograft survival(12–14); however, there is limited use of TNFa inhibitors inITX patients (15,21).

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Table 1: Characteristics of patients, who received infliximab after intestinal or multivisceral transplanatation due to treatment refractoryacute cellular rejection or chronic ulcerations of the intestinal graft

Patient Age Gender Indication for ITX Graft type Indication for anti-TNFa therapy

1 27 f Congenital malrotation ITX Late-onset OKT 3 resistant ACR2 33 m Ileus 12 years after appendectomy ITX Late-onset OKT 3 resistant ACR3 39 m Mesenterial infarction ITX Early-onset OKT 3 resistant ACR and humoral

rejection4 31 m Volvulus ITX Steroid-resistant ACR5 31 f Chronic intestinal pseudo-obstruction ITX Chronic ulcerations of distal Tx-ileum and

anastomotic region6 39 m Mesenterial infarction ITX Steroid-resistant ACR7 26 m Volvulus ITX Steroid-resistant ACR8 25 m Volvulus MVTX Chronic ulcerations of distal Tx-ileum and

anastomotic region9 44 m Mesenterial infarction ITX Early-onset OKT3-resistant ACR

ITX, intestinal transplantation; MVTX, multivisceral transplantation; ACR, acute cellular rejection.

TNFa inhibitors are firmly established in the treatment ofsevere active forms of inflammatory bowel disease (IBD),because TNFa levels are elevated in all types of cells,tissues and secretory fluids of IBD patients and in ani-mal models of the disease (16). Additionally, TNFa mod-ulates different processes within the cascade of inflam-matory reactions by acting as a cofactor for mucosal Thelper 1 (Th1) responses (17) Therefore, TNFa has be-come a target molecule for interventional studies. Indeed,TNFa inhibitors are an effective option in patients who donot respond to conventional therapy with steroids or im-munomodulators (18). For example, infliximab (a chimericanti-TNFa monoclonal antibody that binds to soluble andtransmembrane TNFa (19) has demonstrated efficacy forthe induction and maintenance of mucosal healing and re-mission in severe inflammatory and fistulizing Crohn’s dis-ease (20).

Based on our experience with the successful administra-tion of infliximab for OKT3-resistant rejection and ulcerativeinflammation of the graft in two ITX patients (21), thepresent study examined infliximab administration in thesetting of early- and late-onset OKT3- and steroid-refractoryrejection as well as inflammatory graft alterations after ITX.

Patients and Methods

Patients

Nine patients (seven male, two female, median age 31.7 ± 6.3 years)were prospectively observed between June 2001 and November 2009.All patients received an intestinal graft because of irreversible intestinalfailure, which resulted from short bowel syndrome or motility disorder.One patient received a multivisceral transplantation (MVTX), the others anisolated ITX. The underlying indications and patient characteristics are de-scribed in Table 1. Operative and perioperative procedures and standardsincluding anti-infective prophylaxis and monitoring have been described(21–23).

Immunosuppressive and anti-inflammatory treatment

Baseline immunosuppression consisted of tacrolimus (Prograf R© Astel-las, Japan) and steroids, induction agents were daclizumab (Zenapax R©,

Hoffmann-LaRoche, Switzerland), alemtuzumab (Campath R©, Genzyme,USA) antihuman T-lymphocyte immunglobulin (ATG-Fresenius S R©,Fresenius-Biotech, Germany) or antithymocyte globulin (Thymoglobulin R©,Genzyme, USA). Some patients received a combination therapy withsirolimus (Rapamune R©, WYETH AYERST PHARMACEUTICALS, USA)(Table 2).

The identification of ACR and chronic inflammation was achieved via endo-scopies and graft biopsies, which were performed routinely and wheneverthey were necessary for diagnosis. Graft biopsies were assessed accord-ing to established histologic rejection criteria (24). Steroid therapy wasemployed for mild rejections (1000 mg methylprednisolone) on five con-secutive days. Muromonab (Orthoklone R©, OKT3, Janssen-Cilag, Germany)was considered for steroid resistant and moderate or severe rejections(5 mg/day, 5 or 10 days sequentially). Alternatively, antithymocyte globulinwas administered for 5 days with 1–1.5 mg/kg body weight (BW) to achievelymphocyte counts below 500 cells/nL.

Infliximab (Remicade R©, Centocor Inc., ESSEX PHARMA GmbH) was ad-ministered intravenously (5 mg/kg BW) for ACR and recurrent allograft alter-ations that had not responded to standard antirejection therapy.

The indication was guided by laboratory markers and clinical symptoms: (I)elevated serum TNFa levels (reference: 8.1 pg/mL; including relative TNFa

peaks upon already elevated TNFa levels), (II) elevated serum LBP levels(reference:<15 lg/mL), (III) elevated CRP levels (reference:<0.5 mg/dL),(IV) decreased citrulline levels (reference:12–55 lmol/L). Clinical symptomsincluded diarrhoea, abdominal pain, weight loss and intestinal wall thicken-ing/inflammation/hyperperfusion on intestinal power Doppler sonography.Alterations in graft histology that were indicative of nonspecific inflamma-tion other than viral/bacterial enteritis or rejection were also specific criteriafor repeat dosing.

Patient Selection and Indication

Indications for infliximab administration included therapy-refractory rejec-tions (steroid and/or OKT3 resistant), chronic ileal graft inflammation, andulcerations (Tables 1 and 3).

All patients underwent a thorough analysis of their response to standard im-munosuppression. Patients who developed late-onset allograft alterationswere treated by an escalation of conventional immunosuppression (doseescalation, steroid pulse therapy, addition of antiproliferative drugs, addi-tion of topic steroids) for 3 months before receiving infliximab. Because

1042 American Journal of Transplantation 2011; 11: 1041–1050

TNFa Inhibition after Intestinal Transplantation

Table 2: Initial immunosuppression after ITX or MVTX, consisting of a baseline immunosuppression (tacrolimus and steroids), long-termimmunosuppression and different induction therapies, depending on the era of transplantation

Patient Tacrolimus Steroids ATG/ TGB Daclizumab Alemtuzumab Sirolimus MMF

1 X X X X X2 X X X X X X3 X X X4 X X X X5 X X X X6 X X X X X7 X X X X X8 X X X X9 X X X X

ATG, antihuman T-lymphocyte immunglobulin; TGB, antithymocyte globulin.

all patients with late-onset allograft alterations underwent this algorithm,their unresponsiveness to conventional therapy served as internal control.Hence, all patients who did not respond to the approach of escalation ofimmunosuppression were subject to infliximab treatment. Details of inflix-imab administration are listed in Table 4.

Exclusion criteria: The following studies were undertaken to exclude con-traindications for infliximab therapy, such as active systemic or focal infec-tions, and chronic rejection.

Former or active tuberculosis was excluded by chest x-ray, Tine-test, andy-interferon-ELISPOT-assay. To determine reversible alterations of the allo-graft from chronic rejection, all patients underwent graft biopsy, contrast-enhanced magnetic resonance imaging and ultrasonography to excludethickening of the mesentery and the intestinal wall, and rarefication ofmesenteric vasculature. Transmural biopsies were not performed becausenone of the patients showed ample signs of chronic rejection.

Sera were screened for Epstein–Barr virus (EBV), cytomegalovirus (CMV),herpes simplex virus (HSV), varicella zoster virus (VZV), human herpesvirus 6, 7 (HHV 6, 7), adenovirus, parvovirus B19. Enterovirus, adenovirus,

norovirus, rotavirus and bacterial infections (Clostridium difficile, Salmonellaspecies, Campylobacter species, Vibrio cholerae, Yersinia enterocolitica)were ruled out by stoolsample analysis. CMV, EBV and adenovirus infec-tions were further excluded by molecular biological analysis of graft biop-sies. Samples derived from stool, peripheral blood, urine and throat werescreened for adenovirus infection (DNA, Antigen).

Mycotic infections (Aspergillus, Candida) were excluded by sera-screeningand galactomannan analysis.

Endpoint of therapy: The endpoint of infliximab therapy was denotedas stable complete remission. Remission was defined as complete disap-pearance of clinical symptoms, decline in infection parameters, recoveryof citrulline levels, absence of histological and inflammatory alterations onendoscopy or intestinal power Doppler sonography over three months.

Immunoassays

TNFa and LBP serum levels were determined using an automatic randomaccess immunoassay analyzer according to the manufacturer’s instruc-tions (Immulite, DPC Biermann, Germany). Infliximab levels and humanantichimeric antibodies (HACA), e.g. antibodies to infliximab (ATI), were

Table 3: Characteristics of anti-TNFa therapy in each patient with relation to the initial indication for anti-TNFa therapy, the date oftreatment initiation after Tx, the number of infliximab applications and the outcome; in addition, documented adverse events are listed

Indication for Months InfliximabPatient anti-TNFa therapy post-TX applications Outcome Adverse events

1 Late-onsetOKT 3 resistant ACR

27 20 Repeated transientremission,ongoing treatment

EBV-infection

2 Late-onsetOKT 3 resistant ACR

10 17 Sustained remission Pneumonia EBV-infection

3 Early-onset OKT 3 resistantACR and humoral rejection

0, 5 7 Graft loss,death of recipient inMOF

None

4 Steroid-resistant ACR 8 14 Sustained remission Local cutaneousmycosis5 Chronic ulcerations of distal

Tx-ileum and anastomoticregion

17 4 Repeated transientremission,ongoing treatment

None

6 Steroid-resistant ACR 40 5 Sustained remission None7 Steroid-resistant ACR 39 4 Sustained remission EBV-infection8 Chronic ulcerations of distal

Tx-ileum and anastomoticregion

24 2 Sustained remission None

9 Early-onset OKT3-resistantACR

1 3 Sustained remission None

x ACR, acute cellular rejection; TX, transplantation; MOF, multiorgan failure; EBV, Epstein–Barr Virus.

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Table 4: Characteristics of infliximab application with time oftreatment initiation and overall number of infliximab applications.Patients underwent certain treatment periods, in which they re-ceived approximately two applications of infliximab. Number oftreatment periods and time between treatment periods are listedas well as the time between infliximab applications within onetreatment period (mean ± SEM)

Infliximab dose 5 mg/kg BW

Start of infliximab therapy aftertransplantation

18.2 ± 14.1 months

Number of applications per patient 8.4 ± 6.7Number of treatment periods per patient 4.8 ± 5.1Number of applications per treatment

period1.7 ± 1.2

Time between applications within onetreatment period

7.6 ± 6.8 days

Time between treatment periods 5.8 ± 8.3 months

determined every 4 weeks using Sanquin Diagnostic Services R©, Nether-lands to guide the frequency of infliximab administrations and to monitor apotential loss of efficacy. Infliximab cut-off levels were 10 lg/mL becauseTNFa-inhibition with infliximab was achieved at concentrations of 0.2–10lg/mL in vitro (19). The detection limit of ITA was 12 AU/mL.

Data analysis

Data were collected prospectively and analyzed descriptively because oflimited patient numbers. Results are provided as mean ± SEM.

Results

Indications for infliximab treatment

Five patients (nos. 1, 2, 4, 6, 7) received infliximab forlate-onset OKT3- or steroid-resistant rejection and chroniculcerative graft alterations at 24.8 ± 15.3 months after ITX.Patients 5 and 8 received infiximab for late-onset inflam-matory graft alterations without histological rejection signs(ileitis/anastomositis) at 20.5 ± 4.9 months after transplan-tation; patients 3 and 9 were treated for early-onset OKT3-resistant ACR 15 and 26 days respectively after ITX.

Table 5: General outcome after infliximab application. Outcomeis differentiated into repeated transient remission with ongoingtreatment, (applies for two patients, who are on a regular treat-ment schedule) and sustained remission without any further anti-TNFa therapy (applies for six patients)

Number of patients with completeremission n = 6

Total treatment duration until completeremission

471.5 ± 634.4 days

Number of applications until completeremission

7.5 ± 6.3

Follow up after onset of completeremission

746.8 ± 570.3 days

Number of patients still on infliximab n = 2Treatment duration in patients still on

infliximab1548 ± 1624.2 days

Number of applications in patients stillon infliximab

12 ± 11.3

Figure 1: Treatment circles of infliximab. Dotted lines repre-

sent the clinical course after transplantation in years. Filled

circles show treatment periods. Green circles illustrate patientswith sustained remission after a transient infliximab therapy. Redcircles represent patients with repeated transient remission, whoare still on infliximab therapy due to recurrent allograft alterations.

Treatment characteristics

Each patient underwent treatment cycles in which they re-ceived repeated infliximab administrations. The treatmentcharacteristics (average time of treatment initiation, num-ber of administrations, treatment cycle, administrationsper treatment cycle, administration intervals within andbetween treatment cycles) are shown in Table 4. Patient3 died despite infliximab treatment (Table 3, Figure 1), pa-tients 1 and 5 were still undergoing regular infliximab treat-ment when we submitted this study and patients 2, 4, 6,7, 8, 9 had overcome the episodes of rejection and graftinflammation, showed complete remission for more than3 months and achieved the defined endpoint. There was aduration of 471.5 ± 634.4 days of treatment until completeremission, and the mean number of infliximab administra-tions was 7.5 ± 6.3. The average duration of complete re-mission until the end of this study was 746.8 ± 570.3 days(Table 5). In two patients, who were still undergoing in-fliximab therapy when this study was written, the mediantreatment duration was 1548.5 ± 1624.2 days and theaverage number of infliximab administrations was 12 ±11.3. Of the latter, patient 1 had reached completeremission after 1 year of treatment, but relapsed threeyears later. None of the patients developed chronicrejection.

Figure 3 shows the administration algorithm of patient2. Infliximab was administered based on biopsy-provenallograft alterations and rapidly and significantly increasingTNFa and LBP levels (POD 306–325). Infliximab administra-tion was occasionally based on certain response patterns



Figure 2: Small bowel power doppler ultrasonography (a+b) and colour duplex sonography (c+d) in patient 1, showing stratified

thickening and intense hyperemia of the submucosa, indicating inflammatory alterations of the distal graft. 1: muscularis;2: submucosa; 3: luminal reflex; 4: mucosa (thickened); 5: mesenterial lymphnodes; 6: thickened gutwall; 7: intestinal loop with disturbedmotility.

of TNFa and LBP changes alone (POD 390–392), when clin-ical symptoms and endoscopical findings appeared typicalfor allograft alterations. Because TNFa and LBP elevationsare not specific to intestinal allograft alterations, infectionsthat cause these elevations had to be excluded (POD 340—community-aquired pneumonia).

Side effects of infliximab treatment

Three patients, who were EBV carriers before transplan-tation, showed EBV infections after infliximab treatment.They were successfully treated through a reduction in themaintenance immunosuppression. One patient developeda leftsided prepatellar cutaneous mycosis with the sootymold Alternaria alternata, which was treated with local ex-cision and subsequent oral itraconazole for 4 weeks. An-other patient presented with two episodes of community-acquired pneumonia, and was treated by calculated antibi-otic therapy (oral levofloxacine) for 7 days.

There were no cases of post-transplant lymphoproliferativedisease (PTLD) or other malgnancies.

Infliximab serum levels and antibodies to infliximab

Infliximab serum levels and ATI were determined every 4weeks during treatment periods to identifiy a reduction inthe efficacy of TNFa inhibition and to potentially readminis-ter the drug. Infliximab serum levels were confirmed to beabove 20 lg/mL in all patients at all times during treatmentcycles, the average serum level was 40.55 ± 19.03 lg/mL.Infliximab antibody levels were below the limit of detection(<12 AU/mL) in all patients.

Clinical case descriptions

I. Infliximab for late-onset OKT3-resistant rejection: In-fliximab was first initiated as an individually tailored rescuetherapy for patients 1 and 2, who presented with a steroid-and OKT3-resistant rejection 27 and 10 months after ITX,


Gerlach et al.

Figure 3: Course of TNF alpha

and LBP in patient no. 2 during the

first treatment period with inflix-

imab (six applications in 3 months;

10 months after ITX). The applica-tion algorithm is shown exemplarily.Infliximab was given on account ofbiopsy-proven allograft alterations andrapidly and distinctively rising serum-TNF alpha levels as well as increasedLBP-levels (post-transplant days 306–325). In the case of specific responsepatterns of clinical signs/symptomsas well as TNF-alpha, LBP and CRPwith allograft alterations in individualpatients, infliximab treatment wasoccasionally only based on typicalTNF-alpha and LBP changes (e.g. POD390–392). Since both TNF-alpha andLBP are not specific for intestinalallograft alterations, infections causingtheir elevation had to be excluded(e.g. POD 340—community-aquiredpneumonia). Dotted horizontal linesdemonstrate the reference levels ofTNFa (8.1 pg/mL) and LBP (15 lg/mL).Dotted vertical lines demonstrate eachapplication of infliximab (5 mg/kg BW),followed by a decrease of TNF alphaand LBP.

respectively (21) Both patients developed graft changes in-dicative of indeterminate for rejection, which progressedto severe exfoliative rejection within days despite steroidtherapy. OKT3 was administered on 10 and 5 consecutive

Figure 4: Recurrent inflammatory alterations of the distal ileal

graft in patient no. 2 on endoscopy.

days. There was a resolution of graft alterations in the prox-imal intestine, but not in the distal portion. Graft biopsiesobtained from this portion were still indicative of persis-tent ACR. LBP, TNFa and CRP levels were continuouslyelevated (15–25 lg/mL, 60–170 pg/mL, 11–18 mg/dL), cit-rulline decreased (<8 lmol/L). To prevent graft loss, bothpatients received infliximab repeatedly as a rescue ther-apy, which led to a complete resolution of graft ulcerationsand recovery of graft function. There was a decline of CRP,LBP and TNFa below cut-off and citrulline recovered to>30 lmol/L.

After this initial period, patient 1 developed recurrentepisodes of ACR (four within 3 months), which was fol-lowed by persistent inflammatory alterations in the distalgraft. All episodes were successfully treated with inflix-imab. A complete remission was achieved 10 months af-ter the first infliximab administration. Three years later, thepatient presented with diarrhea and weight loss due to vi-ral enteritis, which led to acute renal failure. Graft biopsieswere obtained and yielded evidence of ACR. Treatmentwith steroids and thymoglobuline did not achieve sustainedsuccess, and infliximab was applied twice, which led to asuccessful restoration of intestinal graft function. Further-more, infliximab administration allowed for a reduction intacrolimus trough levels to 5 ng/L, which avoided nephro-toxicity and haemodialysis. Due to a malrotation of therecipient colon, endoscopies were no longer performedroutinely instead contrast-enhanced power Doppler



sonographies were performed, to quantify inflammatoryhyperperfusion of the gut wall and the mucosa (Fig-ure 2). Hereafter, infliximab has been regularly adminis-tered, based on clinical or sonographic evidence of inflam-matory graft alterations (Table 3 and Figure 1). Tacrolimusand sirolimus trough levels have been adjusted between3–4 ng/L and 2–3 ng/L, respectively, to preserve kidneyfunction.

Patient 2 also continued to show recurrent episodes ofACR and inflammatory alterations of the distal graft. In theinitial period the patient received six infliximab adminis-trations within 3 months (Figure 3). The following endo-scopies showed no further histological evidence of ACR.However, inflammatory alterations of the distal ileal graftrecurred (Figure 4) and the patient repeatedly receivedinfliximab until complete remission (Figure 1). Graft func-tion was entirely restored with low tacrolimus levels (3–4ng/L) and no further evidence of rejection or inflammationwas observed.

II. Infliximab for steroid-resistant rejection avoiding

OKT3: Due to the successful treatment of OKT3-resistantrejection, the indications for infliximab were expanded toinclude patients, who presented with steroid-resistant re-jection and similar inflammatory alterations of the distalgraft. This reduced the need for OKT3.

Three male patients (nos. 4, 6, 7) developed steroid-resistant rejections with recurrent graft alterations in thedistal 20–40 cm of the graft. They showed relapsing eleva-tions of CRP (8–15 mg/dL), LBP (16–22 lg/mL) and TNFa(80–140 pg/mL), which indicated mucosal damage and bac-terial translocation; citrulline decreased to <7 lmol/L. Graftalterations showed signs of ongoing rejection and chronicinflammation in the form of a mononuclear dominatedinflammatory infiltrate with circumscript erosions, cryptdropout and multiple apoptotic bodies (Figure 5). Based onthe assumption that the inflammatory manifestations weresimilar to those of the two previously described patients,and to reduce OKT3 requirements, all patients received in-fliximab repeatedly. The initiation of infliximab allowed forthe maintenance of low tacrolimus levels (5 ng/L) despiteACR. All patients recovered with sustained remission of re-jection and inflammation episodes and resumption of graftfunction (Figure 1). CRP, LBP, TNFa and citrulline recoveredto reference levels.

III. Infliximab for patients with ileitis/anastomositis

without ACR: Two patients with ulcerative distal graft in-flammation that resembled terminal ileitis and inflamma-tion of the ileocolic anastomosis were also treated with in-fliximab. Unlike the other cases, histological signs of ACRwere not detected. Both patients (nos. 5 and 8) presentedwith permanent clinical signs and lab parameters of in-flammation (CRP:3–9 mg/dL, LBP:18–25 lg/mL, TNFa:60–114 pg/mL, subfebrile temperatures, weight loss), which

suggested chronic inflammation and bacterial transloca-tion. Diagnostic imaging and clinical investigations couldnot determine the location of inflammatory sites or malig-nancies. Endoscopic and histological examination showedan ulcerative inflammatory process of the distal ileal graft,which affected graft portions adjacent to the ileocolic anas-tomosis and was referred to as an anastomositis. Histo-logical examination showed an ulcer with granulation tis-sue and fibrinoid necrosis on the surface. There was noincrease in apoptosis and no infiltration of lymphocytes,which would have accounted for ACR (Figure 6).

There was a rapid recovery of clinical signs and lab markersafter the first infliximab administration; however, the ther-apy is still ongoing for patient 5 due to recurring episodes ofananastomositis (Figure 1). Therefore, only transient com-plete remissions were achieved, and patient 5 was placedon a regular treatment schedule with infliximab infusionsevery 6 months. Patient 8 achieved sustained completeremission (Tables 3 and 4).

IV. Infliximab in early-onset OKT3-resistant rejection:

Patients 3 and 9 received infliximab for OKT3-resistantearly-onset rejections 15 and 26 days after transplanta-tion, respectively. Patient 3 developed severe exfoliativeOKT3-resistant ACR 15 days after transplantation that wasaccompanied by a humoral rejection. Infliximab was ad-ministered as rescue therapy (seven applications within11 days), because neither thymoglobuline and OKT3 norplasmapheresis and subsequent intravenous immunoglob-uline were successful. Mucosal exfoliation was inevitableand graft removal was performed 4 weeks after trans-plantation. The patient subsequently died of multiorganfailure.

Patient 9 presented with graft alterations indicative ofindeterminate for rejection the day after transplantation(Table 2). Despite repeated administration of steroids andOKT3, ACR proceeded to grade I and mycophenolatemofetil (MMF) commenced on POD 18. CRP, LBP andTNFa were increased (9–17 mg/dL, 18–27 lg/mL, 100–167 pg/mL), and citrulline levels were <8 lmol/L. Despitethe onset of sirolimus on POD 24, a grade I rejection wasstill detectable, and infliximab was administered 5 dayslater.

Histological signs resolved after the first infliximab infu-sion. After three administrations the patient was symp-tomfree and achieved sustained complete remission (CRP<0.5 mg/dL, TNFa <10 pg/mL, LBP <12 lg/mL, citrulline>30 lmol/L).

Discussion

In 2003 we reported on two patients with late-onsetOKT3-resistant rejection, who presented with ulcerative


Gerlach et al.

Figure 5: Steroid-resistent rejection in patient no. 6. A: acute intestinal rejection. A flat mucosa with atrophy of the villi, focalerosions and severe crypt injury. A mononuclear dominated infiltrate of inflammatory cells is widly scattered within lamina propria. B:(high power field of left side): one crypt with confluent apoptotic bodies, loss of goblet cells and basophilic cytoplasm. H&E 100x(a), H&E400x(b).

alterations of the graft and benefitted from infliximab. Theconcept of treating patients with infliximab was based ontwo assumptions. First, because TNFa serum levels wereelevated during rejection episodes, TNFa was hypothe-sized to be involved in the process of steroid- or OKT3-refractory rejection after ITX. This appeared to be similarto the inflammatory process and immunological mecha-nisms of IBD, where infliximab is used as an effective op-tion for inadequate response to steroid therapy. Second,the Crohn’s-like cobblestone pattern that was detected inthe distal portion of the graft in the two initial patientsresembled the process of persistent inflammation in IBD(21). A few weeks after the initial infliximab administration

in the two patients, which resulted in prompt improve-ment in clinical symptoms and graft histology, both pa-tients experienced a relapse of less severe symptoms andreceived further infusions. This was also consistent withthe experience of IBD patients, who tend to relapse if notretreated. The optimized long-term treatment in IBD pa-tients is regular retreatment every 8 weeks. Episodic ther-apy upon relapse was shown to be less effective and oftenassociated with the formation of ATI unless maintenanceimmunosuppression was concomitantly applied (25).Recurring episodes after the first infusion of inflx-imab were also observed in steroid-refractory GvHD,in which the drug was successfully administered on a

Figure 6: Inflammatory alterations of the distal graft including the anastomotic region, without histological signs of rejection in

patient no. 5. A: acute ulcer in an Anastomositis. Granulation tissue with fibrinoid necrosis on the surface. B: normal intestinal mucosaapart from the anastomosis without evidence of rejection. H&E 100x (both).



once-weekly-basis until clinical improvement (26). Becausesingle infusions almost certainly require additional sup-plementation with elevated maintenance immunosuppres-sion and/or steroids we adapted this treatment policy. Todate, six of the nine patients achieved a sustained com-plete remission, whereas two were on a regular treatmentschedule. The latter two patients received infliximab in-fusions every 6 months, which was in accordance withthe average interval of treatment periods in our study(Table 4). Following our successful experience with inflix-imab for late-onset OKT3-resistant rejection, we expandedthat approach to patients with late-onset steroid-refractoryrejection. First, because they showed similar signs ofchronic inflammation and second, to reduce depleting an-tibodies. Because TNFa inhibition was also successful inlong-term surveillance, we eventually administered inflix-imab to patients, who showed inflammatory alterationsof the ileocolic anastomosis referred to as anastomositis.These changes were not accompanied by histological re-jection signs and responded well to repeated infliximabadministrations, leading to complete remission in one ofthe two patients.

Data on infliximab administration in early-onset rejectionshave been limited and reported failure (15,27) which wassimilar to our experience with patient 3. However, thisepisode was a severe form of early-onset rejection unre-sponsive to antirejection regimen and also a combinationof humoral and cellular rejection, which made it difficultto determine the reason for treatment failure. One mayspeculate about the different dynamics of early and lateoccuring rejections and different immunological processesengaged in different phases of rejection after ITX. Interest-ingly, we recently used inflixmab to successfully treat onepatient for steroid-, thymoglobuline- and OKT3-resistantearly-onset rejection. This indicates that TNFa-dependentmechanisms play an important role in early-onset rejectionand may be influenced by early intervention with TNFa in-hibitors. Thus, successful TNFa inhibition as antirejectiontreatment may also depend on early intervention beforerejection has proceeded to a severe form.

Following our initial report (21), administration of TNFa in-ibitors in late-onset rejections after ITX, was also describedby other authors (15,24). They used infliximab in patientsfor late ileal graft ulcerations nonresponsive to steroidsand augmented immunosuppression. Two characteristicsof ITX recipients, who were successfully treated with in-fliximab were late-onset rejection, which was refractoryto standard antirejection regimen and the appearance ofmucosal ulcerations that were similar to the inflammatorymanifestations of IBD. These characteristics were rarelyconsistent with the standard criteria for moderate to se-vere intestinal graft rejection and they still persisted in theabsence of ACR.

This indicates that late organ impairment with ileal ulcer-ation after ITX may present some form of inflammatory

graft enteropathy related to IBD, which might be moreappropriate for long-term TNFa inhibition than the stan-dard antirejection regimen. Because further enhancementof immunosuppression was not effective in these cases,the infliximab administration may avoid the use of deplet-ing antibodies and reduce side effects of conventional im-munosuppressives like calcineurin inhibitors. The role ofTNFa inhibitors in early-onset rejection, however, requiresfurther investigation.

In a subset of patients TNFa-inhibitors may be ineffective,which could be due to an initial lack of response or loss ofan initial response over time (29), particularly when treatedin an episodic manner. Two relevant clinical questions are,whether switching to a different TNFa-antagonist wouldbe effective if the first treatment failed (28) and whether astrict treatment schedule is more purposeful for patientswho are on long-term treatment (29). The formation of an-tibodies against infliximab has been found to reduce itseffectiveness (30), with the interval between infusions in-fluencing the development of HACA (25). In the presentstudy, none of the patients developed HACA, and they allhad sufficient infliximab serum levels upon testing. For pa-tients on a regular treatment-schedule, clinicians should beaware of the potential development of a lack of responseto infliximab over time.

Adverse events upon TNFa inhibition, particularly infec-tious complications and immunogenicity should be con-sidered. Although the new generation monoclonals, likeinfliximab, have a longer half-life, reduced immunogenic-ity and improved specificity, all monoclonal antibodies areimmunogenic to some extent. The predominant adverseevents upon infliximab that appeared in the present studywere mainly EBV-infections, which indicates that exposureto infliximab together with the previous extensive immuno-suppression may represent a risk for the development oflymphoma. In long-term surveillance, however, there wasno development of PTLD and no CMV-infections were re-ported upon infliximab, which suggest that infliximab is asave therapeutic option for treatment-refractory late-onsetrejections and inflammatory graft alterations that manifestas graft enteropathy.

Conclusion

Although the present study was limited to a small num-ber of patients, we can conclude, that TNFa inhibitorscan expand the therapeutic options for late-onset OKT3- orsteroid-refractory rejection in ITX recipients. Infliximab maybe especially relevant for patients with additional persistentsubacute inflammatory alterations of the distal ileal graft.Furthermore, the introduction of infliximab in the treatmentof ACR may allow a reduction in the total CNI-burden andmay avoid the use of depleting antibodies, which wouldhelp to minimize toxicity. In addition, TNFa inhibition playsan important role in early-onset rejections. Indeed, TNFa


Gerlach et al.

inhibition may serve as a new approach to expand ther-apeutic options for early-onset treatment-refractory rejec-tion after ITX when applied in a timely fashion.

Disclosure

The authors of this manuscript have no conflicts of inter-est to disclose as described by the American Journal ofTransplantation.

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Tumor Necrosis Factor Alpha Inhibitors as Immunomodulatory Antirejection Agents after Intestinal Transplantation