Multivisceral transplantation (MVT) is the concurrent transplantation of the small bowel and liver with one or more of the following organs: stomach, pancreaticoduodenal complex, jejunum, ileum, and/or colon. Rejection after small bowel transplantation (SBT) and MVT is a serious and common complication that affects both patient and graft survival. The early posttransplant phase can be particularly vulnerable and close surveillance is necessary during this period so that potentially disastrous complications can be promptly diagnosed and treated. After discharge, intestinal transplant patients require more readmission than other solid organ transplant recipients because of the high incidence of acute rejection (AR), the difficulty in accurately diagnosing AR in its earliest stage, and the life-threatening consequences associated with unrecognized rejection. Because there are no reliable serum markers for AR in small intestine allografts, frequent endoscopic biopsies are necessary to identify it early, before it becomes severe and irreversible (1).
AR in the intestinal allograft seems to originate in similar fashion to that in other solid organ allografts, with primary involvement of the cell-mediated and humoral-based arms of the immune response. However, there is little known regarding humoral immunity and SBT. When recognized in the early stages of involvement in the SBT, AR can often be successfully managed through an appropriate adjustment of immunosuppression. Occasionally, AR proves to be refractory and requires more aggressive interventions (2). This is likely due to the fact that current anti-rejection therapies are focused primarily on the cellular mechanisms rather than on the humoral component. In many intestinal transplant cases, the recipients become sensitized and form alloantibodies that probably activate the classical pathway of the complement cascade resulting in graft injury (3, 4). In kidney transplantation, the presence of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) at the time of AR diagnosis is a poor prognostic factor for allograft survival (5–8). Similarly, in our previous studies, we found that the frequency and severity of mucosal vascular injury in small intestine allograft is associated with humoral presensitization of the recipient and may be a form of AR (3, 4). High panel reactive antibody (PRA) levels are found in 18% to 30% of patients in the waiting list for intestinal transplantation (9–11), but few studies in the past have addressed the incidence and possible role of DSA in intestinal transplantation. Here, we have hypothesized that the frequency and severity of AR in small intestine allograft may result, at least in part, from the presence of DSA. We designed a prospective study in SBT and MVT recipients, to seek and establish the significance and potential of DSA to serve as an effector mechanism of AR.
Thirteen patients with 15 grafts (two retransplants) were available for analysis. The median age of pediatric patients was 11.5 months (4 months to 15 years), with a median of 43.8 years (19–57 years) for adults. The clinical characteristics of patients are shown in Table 1. There were five adult recipients and eight pediatric recipients. Five allografts, three SBT and two MVT, in the adult group were primary transplants. Ten allografts, six primary transplants (1 SBT and 5 MVT) and two retransplants (both MVT followed by SBT) were in the pediatric group. Two of the 15 grafts were performed with a positive cytotoxic crossmatch. Two of the six SBT grafts (33.3%) and seven of nine MVT grafts (77.8%) experienced clinical rejection episode. The clinical rejection episodes were significantly associated with the presence of DSA (P=0.041) (Table 2). At a mean follow-up of 7.97±9.37 months, there were three mortalities in this study. All of the mortalities were in adult patients; the causes of death were sepsis (n=1) and aortic pseudoaneurysm rupture (n=2).
In adult recipients, three had preformed DSA. In pediatric recipients, two grafts had preformed DSA and four grafts developed de novo DSA. Two adult grafts (1 SBT and 1 MVT) and four pediatric grafts (1 SBT and 3 MVT) never developed DSA. The preformed DSA were against class II (n=3) and both class I and class II (n=2). The de novo DSA were against class I (n=1) and both class I and class II (n=4). The first detectable de novo DSA was 15.25±4.72 days after transplantation.
We obtained 291 biopsy samples from graft ileum and date-matched DSA assay pairs for protocol (n=191, and DSA incidence=6.28%) and for clinical indication (n=100, and DSA incidence=59%). The mean number of biopsies per patient was 19.4 ± 11.89 (range, 5–37). Sixty-three (21.65%) of the biopsies was diagnosed with AR in mild grade or above. The distribution of pathology diagnosis was shown in Figure 1(A). The mean time to first AR biopsy was 23.27±32.22 days after transplantation. Among the 63 AR proved biopsy specimens, 30 (46.7%) had positive DSA. The ratio of severe AR was higher in patients with coincident positive DSA (43.33% vs. 12.12%) compared with patients without demonstrable DSA. Indeed, the presence of DSA is significantly related to severe AR grading (P=0.009) (Fig. 1B).
The correlations between the AR status of the biopsy and the presence of DSA are shown in Table 2. The sensitivity and specificity of presence of DSA for detecting the presence of AR were 47.62% and 82.90%, respectively. The positive predictive value (PPV) and negative predictive value were 43.48% and 85.14%, respectively. Positive DSA showed an association with AR biopsy (kappa=0.30, P<0.001). In the pediatric group (Table 3), positive DSA were more likely to coincide with a rejection positive biopsy (kappa=0.34, P<0.001) and had a higher PPV (47.92%).
The DSA fluorescence intensity (FI) showed correlation to treatment response in six patients (Table 4). The DSA vanished after successful treatment in four recipients. One of these four was successfully rescued by novel agent bortezomib. The remaining two grafts were lost due to refractory rejection (n=1) and rejection during concurrent disseminated adenovirus invasion (n=1) (Fig. 2).
AR is an adaptive immune response mediated through both T-cell and antibody immune mechanisms. Different immune mechanisms tend to act against different types of grafts. Among the unique characteristics of intestine are an abundant amount of lymphoid tissue in the mesenteric lymph nodes, Peyer's patches, and lamina propria and resident bacterial flora, which make SBT particularly prone to AR and infection. Data from the 2003 report of the Intestine Transplant Registry, the rejection rates were 57% for SBT, 39% for combined intestine-liver grafts, and 48% for MVT in the 989 registered grafts by six international participants (12).
The treatment of allograft rejection has historically focused on T-cell mediated processes. For that reason, antibody-mediated rejection (AMR) is typically unresponsive to conventional anti-rejection therapy. Hyperacute rejection is rare but steroid-resistant AR definitely contributes to high graft loss in SBT (13, 14). Therefore, there is considerable interest in the role of AMR in intestinal allograft AR. We have previously shown an association of PRA and AR in SBT recipients (3). Furthermore, Gondolesi et al. (15) reported an intravenous immunoglobulin (IVIG)-based pretransplant desensitization protocol significantly improved the PRA levels and obtained better outcomes in sensitized SBT candidates although Serre et al. (16) found no evidence of correlation of C4d deposition to outcome in SBT which has been well documented in renal transplantation. Current knowledge shows that DSA are specific antibodies against class I and class II HLA. In renal transplantation, the presence of DSA is significantly associated with AMR. Several studies also clearly delineate the role of AMR and DSA in heart and lung transplantation (17, 18). In SBT and MVT, only a limited amount of literature exists to clarify the relationship between DSA and AMR. Serre et al. (16) screened de novo DSA by enzyme-linked immunoabsorbent assay kit but none of the tested serum samples was positive for DSA, which is contrary to our results acquired by Luminex. In our study, preformed and de novo DSA were detected in 5 (33.33%) and 4 (26.67%) of the 15 grafts, respectively. Lethal hyperacute rejection has been reported in presensitized recipients in rat SBT model (13). In addition, we recently reported a successful therapy of hyperacute AMR in a SBT allograft recipient with high titers of preformed DSA with intense immunosuppression and plasmapheresis (4). Of the five grafts in our study that were presensitized, four of them lost their grafts [refractory rejection (n=2), sepsis (n=1), and aortic pseudoaneurysm rupture (n=1)] and one has no clinical rejection episode. Our data indicate that patients with pre-formed antibody could potentially receive SBT or MVT. However, a positive preformed DSA was associated with more frequent and severe AR episodes, graft loss, and mortality.
Early diagnosis of AR of the intestine allograft is essential to control the rejection process. Monitoring d-xylose (19), citrulline (20), and immune parameters such as lipopolysaccharide-binding protein (21) and calprotectin (22) are used as a potential marker for intestine allograft rejection. For example, serum citrulline level declines with intestinal allograft dysfunction; however, it is still not able to differentiate immune or nonimmune causes. To date, despite considerable effort to find one, no minimally invasive marker reliably predicts AR and our study either. Endoscopic surveillance biopsy remains the standard for diagnosing AR (1). Our results show concordance of DSA with biopsy proven AR in which the kappa score was 0.30, furthermore the kappa score increased to 0.34 in pediatric subgroup. This study compares, for the first time, the DSA with synchronized biopsies. We found an agreement between DSA and biopsy in AR detection. The presence of DSA should alert the clinical team to a higher potential for risk of rejection and higher rejection grade. Serial DSA monitoring in the early posttransplantation period offer a safe and reliable method for assisting in diagnosing AR. Endoscopy guided biopsies combined with simultaneous DSA measurement in posttransplantation follow-up are useful to assess the risk of AR and discriminate the possible mechanism.
In this study, DSA incidence was much higher in clinical rejection episode (i.e., indication biopsy) than in regular follow-up circumstance (i.e., protocol biopsy). Our results show DSA is probably a component of AR in the posttransplant period and may work synergistically with cell-mediated immunity against bowel grafts. The sample analysis herein is made to clarify the relevance between DSA and AR in each survey. Some biopsies were pathologically diagnosed AR, but not compatible with clinical condition and vice versa. In this situation, DSA is a useful referee to tell and monitor. That is to say that the clinical team can be more confident to judge the possible mechanism resulting to AR and make decision in treatment.
We selected cut-off values 3000 FI for positivity, because it has been reported that when a FI of more than 1000 was used as the cutoff and anti-HLA antibody were detected in 63% of non-immunized healthy male blood donors (23). The value of FI in 3000 may be not the optimal cutoff values for AR, but it does make the clinician aware in earlier timing. Further study is warranted how to differentiate these natural antibodies from alloantibodies developed pretransplantation or posttransplantation and establish appropriate cutoff value for determining early AR.
In the case of AMR, the therapeutic strategies are based on elimination of circulating antibodies by plasmapheresis, inhibition of residual antibody by IVIG and depletion of B cells by rituximab (24). These therapies do not target the major antibody-producing mature plasma cell. In the setting of refractory renal transplant rejection, treatment with 26S proteasome inhibitor bortezomib depletes plasma cells and provides a potential means for rapid DSA elimination (25). One of our pediatric MVT recipients in this study was first reported treated successfully with bortezomib for his intractable mixed cellular and humoral rejection (26). Close surveillance of humoral immunity is mandatory for precise adjustment of anti-humoral therapy and optimal therapeutic outcomes (27). Recent experience has described that DSA should be considered a potentially new end point for rejection therapy (28).
The limitation of this study was the small size. Further larger multicenter prospective studies to investigate the role of DSA and therapeutic effect of desensitization protocols are necessary to establish the proper role of humoral alloreactivity in SBT. This is the first report of the use of DSA as a marker for the monitoring of intestinal allograft rejection. The measurement of DSA, preformed or de novo, may emerge as a diagnostic tool to monitor and to treat AR in intestinal allografts.
MATERIALS AND METHODS
All patients who underwent SBT or MVT at our center from June 2009 to April 2010 were prospectively evaluated. Patients who died immediately (n=2) from postoperative surgical complications were excluded. The data of the three patients with four grafts who participated in pilot study done in 2008 were included in this serial analysis. Donors and recipients were HLA typed for class I and class II loci by serological and molecular methods. Pretransplant crossmatch tests by cytotoxicity and DSA for HLA class I and II were performed in all patients. DSA were studied by Luminex Flow Beads (LABScreen products; One Lambda, Canoga Park, CA) that uses a panel of fluorescent beads coated with single HLA antigens on a Luminex platform. The results were interpreted as FI. FI less than 3000 was considered negative, and FI more than or equal to 3000 considered positive. ABO blood group identical grafts are used regardless of the results of crossmatch and titer of DSA. The pretransplant induction immunosuppressants were alemtuzumab (Campath-1H), anti-CD20 monoclonal antibody (rituximab), and steroid in adult recipients, whereas thymoglobulin and steroids are used in pediatric recipients. The postoperative immunosuppressants consisted of a therapy with tacrolimus and steroids. Surveillance endoscopies were performed twice a week for the first 30 days after transplantation, followed by once weekly for the following 2 months, then monthly. Additional endoscopies (and biopsies) were performed if the clinical suspicion of AR included fever, abdominal pain, abdominal distention, increased or decreased or bloody output and changes in the stomal appearance. AR were classified and scored according to the 2003 grading criteria established at the VIII International Small Bowel Transplant Symposium and a semi-quantitative scoring of vascular changes. The grading system was no evidence of rejection, indeterminate, mild, moderate, and severe. If a biopsy did show rejection, treatment was started with steroid boluses and adjustments of tacrolimus dose and supplemental prednisone. Steroid-resistant rejection was defined as no response to pulsed steroids. Patients with steroid resistant rejection were further aimed at depleting DSA with rituximab, plasmapheresis, or IVIG and muromonab-CD3 (ORTHOCLONE OKT3), for depressed cell-mediated immunity. 26S proteasome inhibitor, bortezomib (Velcade), was used as a rescue therapy for refractory rejection. Matched serum samples for DSA were collected serially at the time of protocol endoscopic biopsies. Their clinical characteristics, time of presence of DSA, association between matched biopsy proven rejection episodes and DSA sample pairs, and graft status during the following posttransplant period were analyzed.
All continuous data were expressed as mean ± SD except age. Chi-square was used to investigate any differences in severity of rejection between the DSA positive and negative. The correlation between biopsy showing AR and presence of DSA was assessed by Kappa statistical analysis. The sensitivity, specificity, negative predictive value, PPV of DSA for detecting the presence of AR was calculated. Significance was indicated when P was less than 0.05.
1. Fishbein TM. Intestinal transplantation. N Engl J Med
2009; 361: 998.
2. Ruiz P, Garcia M, Pappas P, et al. Mucosal vascular alterations in isolated small-bowel allografts: Relationship to humoral sensitization. Am J Transplant
2003; 3: 43.
3. Kato T, Mizutani K, Terasaki P, et al. Association of emergence of HLA antibody and acute rejection in intestinal transplant recipients: A possible evidence of acute humoral sensitization. Transplant Proc
2006; 38: 1735.
4. Ruiz P, Carreno M, Weppler D, et al. Immediate antibody-mediated (hyperacute) rejection in small-bowel transplantation and relationship to cross-match status and donor-specific C4d-binding antibodies: Case report. Transplant Proc
2010; 42: 95.
5. Gloor JM, Winters JL, Cornell LD, et al. Baseline donor-specific antibody levels and outcomes in positive crossmatch kidney transplantation. Am J Transplant
2010; 10: 582.
6. Lachmann N, Terasaki PI, Budde K, et al. Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts. Transplantation
2009; 87: 1505.
7. Gibney EM, Cagle LR, Freed B, et al. Detection of donor-specific antibodies using HLA-coated microspheres: Another tool for kidney transplant risk stratification. Nephrol Dial Transplant
2006; 21: 2625.
8. Hidalgo LG, Campbell PM, Sis B, et al. De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure. Am J Transplant
2009; 9: 2532.
9. Wu T, Abu-Elmagd K, Bond G, et al. A clinicopathologic study of isolated intestinal allografts with preformed IgG lymphocytotoxic antibodies. Transplant Proc
2002; 34: 878.
10. Wu T, Abu-Elmagd K, Bond G, et al. A clinicopathologic study of isolated intestinal allografts with preformed IgG lymphocytotoxic antibodies. Hum Pathol
2004; 35: 1332.
11. Bond G, Reyes J, Mazariegos G, et al. The impact of positive T-cell lymphocytotoxic crossmatch on intestinal allograft rejection and survival. Transplant Proc
2000; 32: 1197.
12. Grant D, Abu-Elmagd K, Reyes J, et al. 2003 Report of the intestine transplant registry a new era has dawned. Ann Surg
2005; 241: 607.
13. Toyama N, Kobayashi E, Yamada S, et al. Fulminant second-set allograft rejection and endoscopic findings following small bowel transplantation in the rat. J Gastroenterol
1995; 30: 465.
14. Abu-Elmagd K, Reyes J, Todo S, et al. Clinical intestinal transplantation: New perspectives and immunologic considerations. J Am Coll Surg
1998; 186: 512.
15. Gondolesi G, Blondeau B, Maurette R, et al. Pretransplant immunomodulation of highly sensitized small bowel transplant candidates with intravenous immune globulin. Transplantation
2006; 81: 1743.
16. de Serre NP, Canioni D, Lacaille F, et al. Evaluation of C4d deposition and circulating antibody in small bowel transplantation. Am J Transplant
2008; 8: 1290.
17. Rodriguez ER, Skojec DV, Tan CD, et al. Antibody-mediated rejection in human cardiac allografts: Evaluation of immunoglobulins and complement activation products C4d and C3d as markers. Am J Transplant
2005; 5: 2778.
18. Martinu T, Chen DF, Palmer SM. Acute rejection and humoral sensitization in lung transplant recipients. Proc Am Thorac Soc
2009; 6: 54.
19. Ruiz JO, Uchida H, Schultz LS, et al. Problems in absorption and immunosuppression after entire intestinal allotransplantation. Am J Surg
1972; 123: 297.
20. Pappas PA, Saudubary JM, Tzakis AG, et al. Serum citrulline and rejection in small bowel transplantation: A preliminary report. Transplantation
2001; 15: 1212.
21. Cicalese L, Freeswick PD, Watkins SC, et al. Use of CD14 and lipopolysaccharide binding protein mRNA expression as markers for acute rejection in rat small bowel transplantation. Transplant Proc
1996; 28: 2470.
22. Sudan D, Vargas L, Sun Y, et al. Calprotectin a novel noninvasive marker for intestinal allograft monitoring. Ann Surg
2007; 246: 311.
23. Morales-Buenrostro LE, Terasaki PI, Marino-Vázquez LA, et al. “Natural” human leukocyte antigen antibodies found in nonalloimmunized healthy males. Transplantation
2008; 86: 1111.
24. Singh N, Pirsch J, Samaniego M. Antibody-mediated rejection: Treatment alternatives and outcomes. Transplant Rev
2009; 23: 34.
25. Walsh RC, Everly JJ, Brailey P, et al. Proteasome inhibitor-based primary therapy for antibody-mediated renal allograft rejection. Transplantation
2010; 89: 277.
26. Island ER, Gonzalez-Pinto IM, Tsai HL, et al. Successful treatment with bortezomib of a refractory humoral rejection of the intestine after multivisceral transplantation
. Clin Transpl.
27. Zeevi A, Lunz JG III, Shapiro R, et al. Emerging role of donor-specific anti-human leukocyte antigen antibody determination for clinical management after solid organ transplantation. Hum Immunol
2009; 70: 645.
28. Everly MJ, Everly JJ, Arend LJ, et al. Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss. Am J Transplant
2009; 9: 1063.