Allogeneic fetal intestinal transplantation with FK506 immunosuppression

Allogeneic

Fetal Intestinal Transplantation Immunosuppression

By Whitney

J. McBride,

Kennith

Burlington,

H. Sartorelli,

and

With

Dennis

FK506

W. Vane

Vermont

Purpose: Small intestinal transplantation remains a significant clinical problem. Allogeneic fetal intestinal (AFI) transplantation shows promise, particularly regarding procurement; however, no studies to date have evaluated the potential success of true allogeneic loci implantation. The authors hypothesized that isolated segments of AFI could be heterotopically transplanted but would require immunosuppression to survive.

with 10 days of FK506; and the entire 5-week maturation day 35.

Methods: Donor tissue was obtained from late-gestation Brown Norway rat fetuses with a histo-locus RTN and Fischer fetuses with a histo-locus RTIL. The recipients were adolescent male Fischer rats with a histo-locus RTIL. A 1.2-cm segment of fetal small bowel was implanted in the omentum of the recipient rat and allowed to mature for 5 weeks. Animals were then separated into five groups. Group A served as controls with syngeneic fetal intestinal (SFI) transplant. Group B received AFI with no immunosuppression; group C, AFI transplant with five days of FK506; group D, AFI

Conclusions: FK506 allows for normal intestinal development for use in allogeneic fetal bowel transplantation. With this observation, the use of fetal intestine transplanted into the portal circulation emerges as a potentially viable alternative to present intestinal transplant models. J Pediatr Surg 33:932-934. Copyright o 1998 by WB. Saunders Company.

S

ing the histocompatability advantages of fetal tissue in intestinal transplantation have not been answered yet. This report attempts to evaluate the issues regarding histocompatability in this model and describe potential treatment options using allogeneic fetal transplantation. We suspected that an allogeneic transplant model would still require immunosuppression to prevent rejection. But the question remained, would immunosuppression compromise the ability of the fetal tissue to grow and develop into viable intestinal segments? We hypothesized that heterotopically transplanted fetal bowel segments would mature in the presence of FK.506 immunosuppression and that rejection would occur in the absence on immunosuppression.

HORT GUT SYNDROME continues to be a challenging management problem. The limited availability of organs as well as the complications associated with small bowel transplantation have precluded the widespread use of small bowel transplantation as a treatment option. The use of fetal small bowel has been demonstrated to be effective donor material for functional small bowel transp1ants.l Previous work has demonstrated that implantation of short segments of fetal intestine allow nutritional improvement in animals with short gut syndrome.2 The theoretical advantage of multiple recipients from a single donor and additional donor pool of fetuses may allow improved use of intestinal transplantation in short gut syndrome. Additionally, the lack of lumenal bacterial colonization in fetal tissue could potentially assist in reducing septic complications currently associated with small bowel transplantation. Questions regard-

From the Division of Pediatric Surgery, Universi~ of Vermont College of Medicine, Burlington. VT Presented at the 24th Annual Meeting oj the Canadian Assoczation of Paediatric Surgeons, BamjJ Alberta, Canada, October 3-6, 1997. Address reprint requests to Dennis W Vane, MD, Professor and I/ice Chairman, Department of Surgery, Chau-man, Division of Pediatric Surgery, Unwersity of Vermont College of Medicine, E-309 Given Budding, Burlington. V7’05405. Copyright 0 I998 by WB. Saunders Company 0022.3468/98/3306-0030$03.00/O

932

Group E, AFI with period. Animals

daily FK506 were killed

for on

Results: All animals gained weight over the maturation period. Groups B, C, and D had no viable transplant segments at day 35. Groups A and E both had well-developed viable segments confirmed by gross and histological evaluation.

INDEX

WORDS:

Fetal

intestinal

MATERIALS

transplantation.

AND

METHODS

Fetal intestinal ttssue was obtained from late-gestation BrownNorway (histo-locus RTN) rat fetuses at 21 days gestation. The fetuses underwent laparotomy and enterectomy; the small bowel was debrided of any residual mesentery and &vided into 1.25~cm lengths with surgical clips at each end. The recipients were adolescent male Fischer (histo-locus RTlL) rats weighing 75 to 100 g. This model would allow for an allogeneic transplant because histolocus RTN to RTlL ts a histoincompatable match. The recipients were anesthetized with an intraperitoneal injection of sodium pentobarbital and underwent a midline laparotomy. The greater omentum was identified, an isolated fetal small bowel segment was placed in an omental sling, and the abdomen was closed. Immediately after surgery, the recipient rats were started on an immunosuppression regimen of daily FK506 injections, JournalofPediatric

Surgery,Vol33,

No 6 (June),

1998: pp 932-934

FETAL

INTESTINAL

933

TRANSPLANTATION

0.5 mg/kg of body weight. This dosage was based on dosages used in previous work with FK506 in bowel transplantatron m the rat model. Currently, in human transplantation. mrmunosuppression FK506 is administered at a dose of 0.1 mglkg. FK506 levels were not assessed during this protocol because of limited availability of FK506 assays at our facility at the time of this study. The FK506 was dissolved in a sterile olive oil vehicle and subcutaneous injections were administered daily. After a 5- to 6-week interval. a second laparotomy was performed, and the graft site was identified. There were five subgroups tested. Group A was a syngeneic fetal intestinal (SFI) transplant without FK506 immunosuppression that served as a control. Group B was an allogeneic fetal intestinal (AFI) transplant without FK506 tmmunosuppression. Group C was an AFI with an mittal 5-day course of FK506 immunosuppressron. Group D was an AFI with an initial lo-day course of FK506 immunosuppression. Group E was an AFI with daily FK506 immunosuppression for the enttre 5- to 6-week interval. Histological studies of surviving segments were performed usmg H & E stains. Statistical analysts was performed using a Fischer’s Exact test to determine significance (P < .05).

RESULTS

Fetal tissue transplantation was successfully performed in 42 adolescent male rats. In 32 of these animals, an allogeneic transplantation was performed using the Brown-Norway fetal tissue and a Fischer recipient. The remaining 10 Fischer recipients each received a syngeneic Fischer segment. Group A (n = 10) all survived the 5-week interval and had viable graft at the second laparotomy. Histological study results demonstrated normal small bowel morphology. Group B (n = 16) all survived to the 5-week interval but had no identifiable graft segment noted in the omental sling. In several animals the surgical clips were easily identified in the omental sling. Group C (n = 3), received a 5-day course of immunosuppression, all survived the 5-week interval but had no identifiable graft segments. Group D (n = 3), received a lo-day course of immunosuppression, all survived the interval period but also had no identifiable graft segment. Group E (n = lo), received daily immunosuppression, all survived the 5-week interval, and all had an identifiable graft segment. Normal small bowel morphology was verified by histological studies. Nine of 10 (90%) allogeneic bowel transplants demonstrated a tubular structure with a patent lumen and would be acceptable for an interposition type transplant to native bowel. One of 10 (10%) allogeneic bowel transplants showed a noncontiguous lumen and therefore would be unacceptable for an interposition grafting. In comparison, 8 of 10 (80%) syngeneic bowel transplants demonstrated a widely patent lumen, and 2 of 10 (20%) syngeneic bowel transplants had noncontiguous lumens. All animals demonstrated growth and weight gain during the 5-week interval with a slightly more rapid increase in weight noted in the nonimmunosuppressed group. The immunosuppression with FK506 was well tolerated in the test groups. The only apparent side effect was some intermittent diarrhea in three animals, and this resolved without

any sequelae. The fetal intestinal segment also showed growth; all initial graft lengths were 1.25 cm with a diameter of 0.15 cm. Group A had an average final length of 2.0 cm and a diameter of 0.8 cm. Group E had an average final length of 2.3 cm and a diameter of 1.0 cm (Table 1). The comparison of long-term immunosuppressed AFI to nonimmunosuppressed AFI or short-term immunosuppressed show a significant graft survival with the use of FK506 P value of <.OOl. The comparison of graft growth and patency between the SF1 and the immunsuppressed AFI showed no significant difference. DISCUSSION

The use of organ transplantation to treat a variety of solid organ failure syndromes is well established. In contrast, intestinal transplantation continues to be problematic because of the difficulties of procurement, preservation, infection, and rejection.3 The large quantity of gut-associated lymphoid tissue precipitates graft-versushost disease (GVHD) and rejection in the sensitized individual. Despite these daunting problems, a small bowel transplant may offer the only hope of long-term survival in patients with short gut syndrome. Small bowel transplantation in the rat model has been well established using adult rats with orthotopic placement of the graft.4.5 After this transplant, functional ability was demonstrated by survival and weight gain in rats with a lethal gut resection before transplant. These transplants were initially performed in a syngeneic model, and posttransplant immunosuppression was not required. When an allogeneic transplant was performed, rejection was noted in the orthotopic adult small bowel transplant if immunosuppression was not used. A slight improvement in graft survival with a nonimmunosuppressed model was noted if a portal rather than caval anastomosis was performed with the allogeneic transplant.6 The use of cyclosporine for immunosuppression demonstrated a significant improvement in rejection-free small bowel graft survival with long-term treatment.7 Despite the success with orthotopic transplant and immunosuppression, the procedure remains technically difficult. and the risk of infectious and neoplastic complications persist. The use of small bowel transplant was extended to fetal tissue after the success of heterotopic transplant segments. Fetal small bowel was implanted into the greater Table 1. Clinical Group

n

Graft Survwal

A

10

10

B C 0

16 3 3

0 0 0

E

10

10

Data FK lmmunosuppression

None None 5-day course IO-day course Daily

934

MCBRIDE,

omentum of syngeneic adolescent rats and developed a vascular supply (with portal venous drainage), enzymatic function, and growth of the fetal g~afts.1~8~9 This demonstrated the ability of a viable fetal bowel transplant model. Syngeneic heterotopic fetal grafts were then transplanted into adolescent rats, and after a 5-week maturation interval, the recipient’s native bowel was resected, and the graft was placed in continuity with the native bowel. This allowed for survival and weight gain in the recipients. 1,2~10 In addition, fetal graft lengths were found to be a determinate factor because myoelectric functionality and innervation was found to be delayed in the graft segments. ls2 Despite the use of fetal tissue, allogeneic transplants still required the use of immunosuppression to promote graft survival. The use of cyclosporine for posttransplant AFI has demonstrated a survival benefit.l’ Although the use of FK506 in orthotopic small bowel transplants in the adult rat model has been established, the use of FK506 for AFI transplant immunosuppression has not been reported previously.L2,13 We investigated the use of FK506 on the growth of AFI using a heterotopic model. This model permits the use of fetal tissue that would preclude vascular anastomosis because of the small size and facilitates a simple transplant technique. The vascular supply develops from the greater omentum to permit bowel growth with portal venous

SARTORELLI,

AND

VANE

drainage. The use of FK506 allowed for prevention of rejection and GVHD, all animals gained weight and were healthy at the end of the 5-week interval. The only adverse effects noted were intermittent diarrhea in three of the animals. At the end of the 5-week maturation period, all 10 immunosuppressed animals had viable graft segments. In the animals not receiving FK506, complete graft rejection and resorption occurred. This shows a significant survival benefit of FK506 for AFI grafts with a P value of less than .OOl. The animals receiving a brief course of immunosuppression demonstrated rejection. This suggests that immune tolerance cannot be induced by a short course of immunosuppression and that long-term immunosuppression is required to maintain graft viability. FK506 promotes significant fetal graft survival while allowing for growth with normal bowel morphology. The immunosuppression is required on a long-term basis for graft survival. This model demonstrates a possible treatment option for short gut syndrome. This would provide an increased availability of bowel because of the acquisition of multiple grafts from one donor and also facilitates a technically feasible procedure using fetal tissue. ACKNOWLEDGMENT FK506

was provided

by the Fujisawa

Corporanon.

REFERENCES 1. Seekri I, Rescorla F, Katz S, et al: Growth, myoelectiic function and enzymatic activity of fetal intestinal transplants placed in continuity with normal bowel. Surg Forum XLII: 577-579,199l 2. Keller MS, Vane DW: Graft length impacts outcome in fetal small bowel transplants. J Invest Surg (in press) 3. Todo S, Tzakis AG, Abu-Elmagd, et al: Intestinal transplantation in composite visceral grafts and alone. Ann Surg 216:223-234, 1992 4. Harmel RP: A simplified technique of small intestinal transplantation in the rat. J Pediatr Surg 19:400-403, 1984 5. Sonino RE. Teitelbaum DH, Dunway DJ, et al: Small bowel transplantation permits survival in rats with lethal short gut syndrome. J Pediatr Surg 24:959-962. 1989 6. Schraut WH, Rosemurgy AS, Riddell RM: Prolongation of intestinal allograft survival without immunosuppressive drug therapy. J Surg Res 34~597-607, 1983 7. Harmel RP, Stanley M: Improved survival after allogeneic small intestinal transplantation in the rat using cyclosporin immunosuppression. J Pediatr Surg 21:2114-217, 1986

8. Harmel RP, Tutschka PJ. Sonino RE, et al: Immune tolerance of intestinal allografts in the rat. Transpl Proc 21:2883-2884, 1989 9. Tisinai K, Shedd F, Harris R, et al: Comparison of growth, neovascularization and enzymatic function of fetal intestinal graft in the omentum and renal capsule. J Pediatr Surg 25:914-916, 1990 10. Kellnar S, Scbriber C, Rattansouwan T. et al: Fetal intestinal transplantation: A new therapeutic approach in short bowel syndrome. J Pediatr Surg 27:799-801, 1992 11. Kellnar S, Herkomer C, Bae S, et al: Allogeneic transplantation of fetal rat intestine: Anastomosis to normal bowel of the host. J Pediatr Surg 25:415-417, 1990 12. Yang R, Liu 0, Rescorla FJ, et al: Lack of GVHD after fetal intestinal transplantation. J Pediatr Surg 29:1157-1161, 1994 13. Utsunomiya H, Tanaka K, Uemoto S, et al: Effect of FK506 on orthotopic small bowel transplantation in rats. Trans Proc 24: 1191, 1992 14. Hatazawa C, Yamaguchi M, Kato T, et al: Effect of FK506 on bowel transplantation in rats. Trans Proc 24:1177. 1992