Prolonged lung allograft survival with a short course of FK 506

Volume 105,

Number 1

January 1993

THORACIC AND CARDIOVASCULAR SURGERY The Journal of

Cardiac and Pulmonary Transplantation

Prolonged lung allograft survival with a short course of FK 506 We examined the hypothesis that FK 506 would induce graft acceptance after lung transplantation. Left lung allotransplantation was performed in size-matched mongrel dogs allocated to control (no immunosuppression, n = 3) and FK 506 (n = 5) groups. FK 506 (1.2 mgjkg intramuscularly every day) was given on posttransplantation days 0, 1, and 2. No other immunosuppressive agents were administered to either group. Chest x-ray and transplant lung physiologic assessments were performed on the fifth day and weekly thereafter. On day 29 an open lung biopsy and a third-party skin graft were performed. Lymphocytes were harvested and frozen from the recipient peripheral blood before transplantation and on days 8 and 29 afterwards for assessment in mixed lymphocyte reaction. Dogs were killed when their chest x-ray films showed allograft opacification or when the skin graft was rejected. Control lungs were all rejected after a median of 5 days. In the FK 506 group, one of five dogs aspirated during the fifteenth-day assessment and was killed, on the twenty-ninth day, because of severe rejection. At day 29, in the other four dogs, the transplanted lung yielded an arterial oxygen tension of 613 ± 25 mm Hg (mean ± standard deviation) and lung biopsy specimens showed no abnormalities histologically. These four dogs rejected third-party skin grafts after a median of 10 days. In two FK 506 dogs, mixed lymphocyte reaction at day 8 showed suppression of proliferation responses against donor and third-party lymphocytes. By day 29 responses against third-party lymphocytes had returned to almost preoperative levels, whereas antidonor responses were still suppressed. After skin graft rejection and killing, one of four dogs showed no sign of rejection, and the other three showed minimal to mild lung rejection at the time they were killed. We conclude that a 3-day course of 1.2 mgjkg of FK 506 induced prolonged graft acceptance after lung transplantation in dogs. (J THORAe CARDIOVASC SURG 1993;105:1-8)

Takashi Hirai, MD,* Thomas K. Waddell, MD, John D. Puskas, MD, Hiromi Wada, MD,* Shigeki Hitomi, MD, * Reginald M. Gorczynski, MD, Arthur S. Slutsky, MD, and G. Alexander Patterson, MD, FRCS(C),** Toronto, Ontario, Canada

From the Division of Thoracic Surgery, Department of Surgery, University of Toronto, Toronto General Hospital, Toronto, Ontario, Canada.

Address for reprints: G. A. Patterson, MD, FRCSe C), Division of Cardiothoracic Surgery, Suite 3108-Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110-1013.

Supported by the J. P. Bickell Foundation Grant 8237.

*Present address: The Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, 53 Kawahara-Cho Shogoin Sakyo-Ku, Kyoto, 606 Japan.

Received for publication Jan. 2, 1992. Accepted for publication May 25, 1992.

12/1/41625 0022-5223/93/$1.00/ + 0.10

**Present address: The Division of Cardiothoracic Surgery, Washington University School of Medicine, St. Louis, Mo.

2 Hirai et al.

Although the first clinical attempt at lung transplantation was in 1963, pulmonary transplantation has only recently become a widely applied and successful therapeutic modality for patients with end-stage lung disease. 1-3 A number of technical advances have contributed to achieving this recent success. The most important advance, however, has been the availability of the potent immunosuppressive agent cyclosporine.l-' Despite universal use ofcyclosporine, rejection remains a major cause of early and late morbidity and mortality after clinical lung transplantation." In addition, cyclosporine is associated with a significant prevalence of side effects such as nephrotoxicity and hypertension. 5 FK 506 is a powerful immunosuppressive agent isolated in 1984 from Streptomyces tsukubaensis strain No. 9993. It is an antibiotic of the macrolide family, has a molecular weight of822, and is structurally different from cyclosporine.s-? The mechanisms of immunosuppression mediated by FK 506 and cyclosporine, however, appear to be remarkably similar.f In vitro experiments have demonstrated that FK 506 suppresses interleukin-2 and interferon-v (lFN-/,,) secretion as well as interleukin-2 receptor expression by T lymphocytes. It inhibits the expression of interleukin-2 receptor, mixed lymphocyte culture responses, and cytotoxic T cell generation at about 100 times lower concentration than cyclosporine.P- 7 It has been shown to have potent immunosuppressive activity in vivo and to prolong graft survival in skin, heart, kidney, or liver allografts in rodents, dogs, primates, and human beings. 9-13 We previously examined the immunosuppressive effect of FK 506 in a canine single lung transplantation model. Histologic assessment of the lung and the bronchial anastomosis revealed that FK 506 was as effective as

cyclosporine.!" In addition to its efficacy in long-term use, a short

course of high-dose FK 506 has been shown to induce graft acceptance after liver, heart, and multivisceral transplantations in rats. 15-18 A number of investigators have reported similar drug-induced long-term graft survival with cyclosporine in the rat. 19, 20Induction of tolerance with cyclosporine, however, appears to be much more difficult in dogs.I': 22Yet Ueda and colleagues have reported some degree of canine renal and hepatic graft acceptance with use of a short course of FK 506. 23 The present study was undertaken in a canine left lung allotransplantation model to study the effect on the subsequent survival and function of lung allografts, of a short course of high-dose FK 506 treatment.

Methods Lung transplantation. Donor and recipient procedures were performed simultaneously in adult mongrel dogs. All dogs

The Journal of Thoracic and Cardiovascular Surgery

received standard intravenous anesthetic sequence of meperidine (50 mg) and acepromazine maleate (I mg), followed by atropine (0.5 mg), cefazolin (I gm), and Pentothal (thiopental sodium) (10 rug/kg). The dogs were then intubated (8.5F) and their lungs ventilated with tidal volumes of 25 ml/kg at a rate of 12 breaths per minute, with 5 em H 20 positive end-expiratory pressure. Halothane (0.5% to 1.5%) was used to maintain anesthesia. Donor lung harvest and left lung allotransplantation were performed between size-matched mongrel dogs as described previously-" except that pulmonary vascular flush was not performed. An inflatable silicone rubber cuff was placed around the recipient proximal right pulmonary artery (PA), allowing independent assessment of transplanted lung function. 24.25 Drug administration and experimental design. Control animals were given no immunosuppressive therapy (n = 3). FK 506 was provided by Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan, through Professor S. Hitomi, Chest Disease Research Institute, Kyoto University. It was dissolved in saline and injected in the thigh at a dosage of 1.2 mg/kg intramuscularly once a day on postoperative days 0 through 2 (n = 5). To prevent vomiting caused by FK 506, 0.5 mg of atropine sulfate was given once a day for 3 days intramuscularly. Evaluation of graft function. Lung allograft function was evaluated on the fifth day and weekly thereafter by plain chest roentgenograms and functional analysis of the left lung with use of the inflatable PA cuff. The dog was anesthetized and lungs were ventilated in the supine position, with 5 cm H20 PEEP and forced inspiratory oxygen of 1.0; arterial blood gases and systemic and pulmonary hemodynamics were recorded. Similar determinations were repeated after a 1O-minuteocclusion of the right PA. Lung biopsy and skin transplantation. If the lung grafts survived for 29 days, recipient animals were anesthetized and biopsy specimens were taken from both the transplanted and the native lung via left thoracotomy. After closing the chest, full-thickness skin grafts (3 X 3 em) from an unrelated dog (third party) were performed. These skin grafts were harvested the day before skin transplantation. The graft was placed on the right lateral chest wall and was held in place with interrupted sutures of 2-0 silk. Rejection of the skin grafts was assessed by changes in color and texture, the end point being necrosis of the graft epithelium. At the time of killing, rejection was confirmed histologically in all cases. Mixed lymphocyte reaction (MLR) and concanavalin A responses. Stimulator cells were obtained from the donor spleen and an unrelated (third party) dog spleen with the use of standard Ficoll-Hypaque (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) gradient techniques-" Responder cells were collected from 60 ml of peripheral blood of the recipient before transplantation, on day 8, and immediately before skin transplantation on the twenty-ninth day. Both spleen and peripheral blood monocytes were isolated and frozen in RPMI 1640 (Gibco Laboratories, New York, N.Y.), supplemented with 10% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) and 30%fetal bovine serum (Flow Laboratories Inc., McLean, Va.), at -70°. All cultures were set up in RPMI 1640 medium supplemented with 2% pooled, heat-inactivated dog serum, penicillin (100 IU /ml), streptomycin (100 /oLg/ml), HEPES buffer (25 mmol/ L), L-glutamine (2 mmol/L), and 2-mercaptoethanol (5 X 10- 5 mmol/L) (Schwarz/Mann Biotech, Cleveland, Ohio) (RPMI complete) at 37° C in 5% carbon dioxide. MLR was performed

Volume 105 Number 1 January 1993

in quadruplicate in 96-well U-bottom microtiter plates (Linbro, Flow). Recipient lymphocytes were incubated with irradiated (2000 rad) stimulator cells (cryopreserved donor or cryopreserved third-party cells). While the third-party cells were different for individuals, multiple tests performed on any individual dog used the same third-party source. Responder cells (1 X 105) were cultured with stimulator cells (1 X 105) in each well. Cryopreserved recipient cells, isolated preoperatively, on day 8 or on day 29, were plated on the same plate. The plate was pulsedwith 3H-thymidine (I ~Ci per well) on day 6. Wells were harvested 18 hours later onto glass fiber filter papers (Cambridge Technology, Inc., Watertown, Mass.) and were counted in a well-type scintillation counter (Rackbeta, LKB-Wallac, Stockholm, Sweden). For concanavalin A stimulation assays, 1 X 105 responder cells per well were cultured under the same conditions as those described for the MLR except that concanavalin A was added at a concentration of 5 ~g/ml. Cultures were pulsed with 3H-thymidine (I ~Ci per well) at day 3. Wells were harvested in the same way as for MLR. Assessment of side effects of FK 506. Weekly blood samples for complete blood count, glucose, amylase, creatinine, urea, protein, total bilirubin, aspartate, aminotransferase, alanine aminotransferase, and alkaline phosphatase were taken to monitor the side effects of FK 506. Dogs were weighed and checked for signs of illness daily. Killing and histologic assessment. When the skin graft from a third party was rejected or complete opacification of the transplanted lung occurred, the dogs were killed and all the anastomoses were carefully examined. The bronchial anastomotic site, the upper and lower lobes of the transplanted and native lungs, as well as heart, liver, pancreas, and kidney, were preserved in 10% formalin, sectioned, and stained with hematoxylin and eosin. The histologic appearance of the transplanted lung was classifiedaccording to the grading of acute rejection by the International Society for Heart Transplantation as follows-": grade 0, no significant abnormality; grade 1, minimal abnormality; grade 2, mild abnormality; grade 3, moderate abnormality; grade 4, severe rejection. The donor bronchus was examinedin the same fashion as described before.14 Findings in the bronchial epithelium were classified according to the following criteria: 0, no pathologic change; 1, few or no cylindric cells, many cuboidal cells; 2, no cylindric cells and few cuboidalcells;3, no epithelial cells.The submucosal of the donor bronchi were examined and divided into fivegroups: group 0, no lymphocyticinfiltration; group 1, mild lymphocytic infiltration; group 2, moderate lymphocytic infiltration; group 3, severe lymphocytic infiltration; group 4, necrosis. All dogs receivedhumane care in compliance with the "Principlesof Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Statistics. Graft survivalbetweengroups was compared with the use of an accelerated failure time model with a Weibull distribution (LIFEREG procedure, SAS Institutes, Cary, N.C.). All data are presented as mean ± standard deviation. Results Twelve dogs underwent left single lung transplantation and all survived the operation. Preliminary studies were

Prolonged lung allograft survival with FK 506 3

Lung Biopsy Skin Graft

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15 22 29 Days Post Transplant

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Fig. 1. Transplanted lung function (Pao-). In control animals (dotted lines), the Pao- before harvest and immediately after lung transplantation was normal. Within 8 days however, the Pa02 of the transplanted lung declined to less than 50 mm Hg. For four of fiveFK 506-treated dogs (solid lines) the mean Pao, was 613 ± 25 mm Hg on the twenty-ninth day. The Pao2 of one FK 506-treated dog who aspirated on the fifteenth day was 48 mm Hg on the twenty-ninth day. At the time of killing the Pao2 was variable (76.7, 365.8, 569.7, and 558.9 mm Hg). performed in two dogs to select the FK 506 dose and timing of administration. In the first dog, FK 506 was given on days 0, I, and 2 at 1.5 mg/kg intramuscularly; allograft function was excellent, but toxicity due to FK 506 was severe (jaundice, long-lasting anorexia, vomiting, and emaciation). Because of these adverse effects, we reduced the FK 506 dose from 1.5 to 1.2 rug/kg subsequently for all dogs. An other dog given FK 506 on days 4, 5, and 6 at 1.2 mg/kg intramuscularly, showed severe lung rejection at the time of killing on the fifth day. We concluded that treatment started on day 4 was too late. We therefore chose to study FK 506 (1.2 rug/kg intramuscularly given on days 0, I, and 2. Two additional dogs were excluded from analysis because of technical problems with the inflatable cuff. All subsequent data (reported below) were obtained from measurements made with the cuff inflated, and hence only the transplanted lung was perfused and ventilated with aid of a forced inspiratory oxygen of 1.0. Control animals (n = 3). Lungs were rejected after a median of 5 days in untreated control dogs (Table I). Chest roentgenograms showed complete opacification in all three animals within 8 days (8, 5, and 5 days postoperatively, respectively). The mean arterial oxygen tensions (Pao-) before harvest, immediately after transplantation, and at the time of killing were 630 ± 14.2, 577 ± 25.2, and 47 ± 1.6 mm Hg, respectively. (Fig. 1 summarizes the functional performance in all animals.) Histologically, severe pulmonary rejection was observed in two animals and moderate rejection in one. At the

4

The Journal of Thoracic and Cardiovascular Surgery

Hirai et al.

Fig. 2. Histology of the transplanted lung at the twenty-ninth postoperative day (A)and at the time of killing (fortieth postoperative day) (B)of an FK 506-treated dog (F-4). No sign of rejection is seen on the twenty-ninth day. At the time of killing a few perivascular cuffs are visible (minimal rejection). (Hematoxylin and eosin stain; Xl 00.)

Table I. Histologic findings of transplanted lung and bronchial anastomotic site Graft survival (days) Group

Dog No.

Control

C-1 C-2 C-3

8

F-I F-2 F-3 F-4 F-5

>41 43 29 >40 >40

FK 506

Lung

Skin

Transplanted lung* (grade) Biopsy (29th POD)

5 5 12 9

ND II II

0 0 4 0 0

Bronchial anastomosis (grade)

Killed

Epithelium'[

Submucosal

4 4 3

3 3 3

4 4 4

I 2 4 I 0

0 3 0 0 0

3 3 2 2 2

POD, Postoperative day; N D, not done.

*Grade 0, no significant abnormality; grade I, minimal rejection; grade 2, mild rejection; grade 3, moderate rejection; grade 4, severe rejection. pathologic change; I, few or no cylindric cells, many cuboidal cells; 2, no cylindric cells, few cuboidal cells; 3, no epithelial cells. :1:0, No lymphocytic infiltration; 1, mild lymphocytic infiltration; 2, moderate lymphocytic infiltration; 3, severe lymphocytic infiltration; 4, necrosis.

to, No

bronchial anastomotic site, desquamation of epithelial cells and submucosal necrosis were observed in all three dogs (see Table I). FK 506 animals (n = 5). Median survival of lung grafts in the FK 506 group was more than 40 days (see Table I), significantly longer than in control dogs

(p = 0.0001). In four of five dogs, on day 29 chest roentgenograms were clear and the lung biopsy specimens showed no pathologic change (Fig. 2, A), mean Pao- was 613 ± 25 mm Hg, and mean PA pressure was 18 ± 4 mm Hg (see Fig. 1). One other dog aspirated on the fifteenth day, and progressiveleukocytosisdeveloped. In this

Volume 105 Number 1 January 1993

Prolonged lung allograft survival with FK 506

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Fig. 3. MLR in two FK 506-treated dogs (F-4 and F-5). Recipient lymphocytes wereincubated incomplete RPMI 1640 with irradiated splenocytes fromdonoror third-party animals. Wells were pulsed for 18hours with 1 ~Ci per well of 3H-thymidine at 6 days. Lymphocytes fromthe recipient responded to both irradiated donor andthird-party splenocytes preoperatively. Antidonor andanti-third-party responses are bothdecreased onday8. Byday29anti-third-partyresponses havereturned to almost the preoperative level, whereas the antidonor responses are still substantially suppressed. animal at 29 days, complete opacification of the transplanted lung, associated with significant impairment of gas exchange (Pao- 48 mm Hg), was noted on chest x-ray examination. The animal was killed at that time. Histologically, severe rejection was noted in the transplanted lung, but bronchial epithelial cellswere wellpreservedand moderate submucosal mononuclear cell infiltration was observed.Thus only one of fivedogs in the FK 506 group had any sign of rejection on the twenty-ninth day. Third-party skin grafts placed on the four surviving dogswere all rejected after a median of 10 days. The dogs were killed (41, 43, 40, and 40 days after lung transplantation) when the skin graft was rejected. During this interval (29 days to the time of killing), pulmonary rejection appeared to develop in three of the four dogs. At the time of killing the Pao- was variable (76.7,365.8,569.7, 558.9 mm Hg, respectively), and mean PA pressure was 35 ± 10.9 mm Hg. Histologically, one of four dogs showed no sign of rejection, but the other three showed minimal to mild rejection at the time of killing (Fig. 2, B). Bronchial epithelial cells were well preserved in three dogs, but desquamation of epithelial cells was seen in the dog with the very low Pao- (76.7 mm Hg).

F-2 F-3 Dog Number Preop ~ 8 POD

0

29 POD

Fig. 4. Concanavalin A assayfor FK 506-treateddogs. Concanavalin A responses were evaluated at 3 days of culture. Responses weresuppressed on the eighthday but recovered by thetwenty-ninth day.Unstimulated (background) proliferation wasless than 2000cpm.

MLR and concanavalin A responses. There were significant technical difficultiesin obtaining canine MLR data. We titrated the concentration of dog serum and used 2% of pooled heat-inactivated dog serum. Sensitivity to the serum was variable between dogs, however, and we found a high and variable background level of proliferation of unstimulated cells in some dogs and poor response to stimulation in other dogs. In two animals in the FK 506 group, lymphocytes obtained preoperatively from the recipients responded strongly to both irradiated donor and third-party splenocytes. Antidonor and antithird-party responseswere decreased on day 8. By day 29 responses to third-party cells had returned to the preoperative level, whereas the antidonor response was still markedly suppressed (Fig. 3). This suggests the in vitro unresponsiveness induced by a 3-day course of FK 506 lasted at least 1 month and was specifictoward the donor. Concanavalin A responses (a nonspecificindex of lymphocyte function) were markedly suppressed on the eighth day but had recovered by the twenty-ninth day in all dogs receiving FK 506 (Fig. 4). Therefore a generalized immunosuppressive effect of FK 506 may have been present on the eighth postoperative day but had reversed by the twenty-ninth day. Side effects of FK 506. One dog, given FK 506 on days 0, 1, and 2 at 1.5 mg /kg intramuscularly, exhibitedjaundice, severeanorexia, and vomiting for more than 2 weeks. At 1.2 rug/kg intramuscularly, the dogs given FK 506 appeared to be healthy and had normal hepatic and renal function (Table 11).Histologically no pathologic changes of the heart, liver, kidney, or pancreas were seen. Loss of appetite and vomiting were common in the first week after

6

The Journal of Thoracic and Cardiovascular Surgery

Hirai et al.

Table II. Preoperative and postoperative leukocyte, creatinine, and bilirubin measurements Dog No. WBC

(XI09/L)

F-I F-2 F-3 F-4 F-5

Creatinine (umol/L)

F-I F-2 F-3 F-4 F-5

Total bilirubin (umol/L)

F-I F-2 F-3 F-4 F-5 P_I*

Preop 9.1 5.0 9.9 6.0 6.6

8th POD 4.3 6.2 9.8 7.3 6.1

15th POD 10.8 5.6 9.0 5.7 7.6

22nd POD 7.7 9.0 13.7 6.1 11.5

29th POD 5.6 6.7 18.6 4.8 12.7

III 107 94 III 106

91 94 84 82 105

114 109 71 93 III

105 104 75 98 98

108 99 77 93 98

5 3 4 7 5 3

4 4 4 7 5 3

4 4 7 8 5 43

3 3 3 6 II 46

I 6 7 6 3 18

POD. Postoperative day; WBC, white blood count.

'Preliminary FK 506-treated dog (FK 506, 1.5 rug/kg intramuscularly days 0, 1, and 2).

administration of FK 506. Mild weight losswas observed in all animals given FK 506, but by the twenty-ninth day most lost weight had been regained. Discussion Progress in lung transplantation has been difficult in comparison with other vital organs because of specific problems with preservation, bronchial healing, infection, and rejection.' Rejection remains a major cause of mortality despite the use of cyclosporine." Cyclosporineis also associated with a number of side effects, including nephrotoxicity and hypertension. In addition, increased levels of immunosuppression employed to reduce rejection are associated with infectious complications.P Donor-specific tolerance or graft acceptance without continuous immunosuppression would solvethe problems of chronic rejection and decrease the risk of infection and side effects related to treatment. Recently a short course of high-dose FK 506 has been used to induce cardiac and liver graft acceptance in rats l 5- 18 and after canine renal or hepatic transplantation.23 There have been no previous reports, however, concerning induction of tolerance or graft acceptance with a short course of FK 506 in lung transplantation. In the present study four of fivedogs given FK 506 (1.2 rug/kg) on days 0, 1, and 2 showedno signsof pulmonary rejection on the twenty-ninth day, yet these animals were all healthy and rejected third-party skin grafts a median of 10 days after implantation. Concanavalin A responses of the recipient cells were markedly suppressed on the eighth day but recovered to the preoperative value on the

twenty-ninth day. Thus it appears that FK 506 induced a generalized immunosuppressive effect, which resolved by the twenty-ninth day. Pharmacokinetic studies of FK 506 in dogs have shown that FK 506 is eliminated very rapidly, with a mean half-life around 10 hours.28 Based on the rejections of skin grafts, concanavalin A assays, and known pharmacokinetics, it seems unlikely that a generalized immunosuppressiveeffect due to FK 506 still existed on the twenty-ninth day. MLR responsesagainst donor splenocytesremained subnormal through the twenty-ninth postoperative day, while responsesagainst thirdparty cells had recovered by this time. The MLR data suggest some donor-specificimmunomodulation. One of the four dogs showed no sign of rejection at the time of killing (40 postoperative days), but three of the four long-term survivors showed minimal to mild rejection at time of killing.Therefore the state of graft acceptance was variable and probably not permanent. It is interesting to speculate on the possiblemechanism of "early rejection" in the dog that aspirated on day 15. This infectious episode may have stimulated the dog's immune system. There is clinical evidence from a small retrospective series that the prevalence of chronic rejection ishigher in patients with posttransplantation cytomegalovirus illness.i"The precise mechanism is unclear, but infectious stimuli are associated with the systemic release of cytokines, notably IFN-'Y. 3o INF-'Y has effects on the immune systemof the recipient and, more importantly, on the parenchymal cellsof the grafted tissue.'! It can induce the expression of both class I and class II major histocompatibility complex antigens on a wide variety of cells

Volume 105 Number 1 January 1993

that can stimulate both helper and killer T cell responses, accelerating the rejection process. There are a number of possible immunologic mechanisms that have been proposed to explain prolonged graft acceptance, including modulation of graft antigen expression, clonal deletion of active cells after immunosuppression and exposure to the graft, induction of anergy, antigen/antibody blockade ofeffector cells, and the activation of suppressor cells. 32-34 The mechanism behind graft prolongation in the model described herein, however, remains to be elucidated. FK 506-related toxicity appears to be more prominent in dogs compared with other species.P A dose of 1.2 rug/kg is 8 to 12 times the ordinary daily dose (0.1 to 0.15 mg/kg per day) used in our previous study.!" Veda and colleagues-' reported appreciable mortality resulting from FK 506, when given at a dose of greater than 2.0 mg/kg. Our findings are similar. In the one dog in which FK 506 was given on days 0, 1, and 2 at 1.5 rug/kg intramuscularly, jaundice, severe emaciation, long-lasting anorexia, and vomiting developed. Anorexia and vomiting in the first week were the only side effects after administration of FK 506 at 1.2 rug/kg. No liver toxicity, nephrotoxicity, or histologic abnormality of the heart or pancreas was observed in these dogs. The optimum dosage to maximize graft acceptance and minimize side effects has not been determined. In summary, we have shown that FK 506, given at a dosage of 1.2 rug/kg for 3 days immediately postoperatively, was successful in producing a state of graft acceptance in 4 of 5 dogs for at least up to 4 weeks with evidence for immunomodulation of responses directed to graft antigens. The precise mechanism remains unknown, but these data suggest that it may be possible to induce longterm specific tolerance with appropriate agents when we more fully understand the fundamental immunologic nature of rejection and tolerance. We would like to acknowledge the valuable assistance of Y. Matsuzaki, MD, and N. Christie, MD, and the superb technical assistance of J. Mates, S. Diamant, and Y. Wang. We also acknowledge S. Hashimoto, PhD, A. de Hoyos, MD, and J. Nelsonfor their instructionand suggestionsin MLR techniques. REFERENCES I. Cooper JD, Pearson FG, Patterson GA, et al. Technique of successful lung transplantation in humans. J THORAC CARDIOVASC SURG 1987;93:173-81. 2. The Toronto Lung Transplantation Group. Experience with single-lung transplantation for pulmonary fibrosis. JAMA 1988;259:2258-62. 3. Cooper JD. Current status of lung transplantation. Transplant Proc 1991;23:2107-14. 4. Kriett JM, Kaye MP. The registry of the International

Prolonged lung allograft survival with FK 506 7

Society for Heart and Lung Transplantation: eighth official report-1991. J Heart Transplant 1991;10:491-8. 5. Maurer JR. Therapeutic challenges following lung transplantation. In: Grossman RF, Maurer JR, ed. Clinics in chest medicine. Philadelphia: WB Saunders, 1990;11:27989. 6. Kino T, Hatanaka H, Hashimoto M, et al. FK506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot (Tokyo) 1987;40:1249-55. 7. Kino T, Hatanaka H, Miyata S, et al. FK506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK506 in vitro. J Antibiot (Tokyo) 1987;40:1256-65. 8. DeFranco AL. Immunosuppressants at work. Nature 1991;352:754-5. 9. Inamura N, Nakamura K, Kino T, et al. Prolongation of skin allograft survivalin rats by a novelimmunosuppressive agent, FK506. Transplantation 1988;45:206-9. 10. Todo S, Veda Y, Demetris JA, et al. Immunosuppression of canine, monkey, and baboon allografts by FK506: with special reference to synergism with other drugs and to tolerance induction. Surgery 1988;104:239-49. 11. Thomson AW. FK506-How much potential? Immunol Today 1989;10:6-9. 12. Starz1 TE, Todo S, Fung JJ, Demetris A, Venkataramanan R, Jain A. FK506 for liver, kidney, and pancreas transplantation. Lancet 1989;2: 1000-4. 13. Todo S, Fung JJ, Starzl TE, et al. Liver, kidney, and thoracic organ transplantation under FK506. Ann Surg 1990;212:295-307. 14. Hirai T, Wada H, Hasegawa S, et al. Immunosuppressive effect of FK 506 in canine lung transplantation. J THORAC CARDIOVASC SURG 1992;103:1127-35. 15. Murase N, Todo S, Lee PH, et al. Heterotopic heart transplantation in the rat receiving FK-506 alone or with cyclosporine. Transplant Proc 1987;19(suppI6):71-5. 16. Ochiai T, Nakajima K, Nagata M, Hori S, Asano T, Isono K. Studies of the induction and maintenance of long term graft acceptance by treatment with FK506 in heterotopic cardiac allotransplantation in rats. Transplantation 1987;44:734-8. 17. Murase N, Kim DG, Todo S, Cramer DV, Fung JJ, Starzl TE. Suppression of allograft rejection with FK506. I. Prolonged cardiac and liver survival in rats following shortcourse therapy. Transplantation 1990;50:186-9. 18. MuraseN, Kim DG, TodoS, Cramer DV, FungJJ, Starzl TE. FK506 suppression of heart and liver allograft rejection. II. The induction of graft acceptance in rats. Transplantation 1990;50:739-44. 19. Nagao T, White DJG, Caine RY. Kinetics of unresponsiveness induced by a short course of cyclosporin A. Transplantation 1982;33:31-5. 20. Kasahara K, White DJG, Caine R Y. Antigen dependence of cyclosporinA-induced allograft acceptance. Transplantation 1982;34:216-8. 21. Deeg HJ, Storb R, Miller LG, Shulman HM, Weiden PL,

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The Journalof Thoracic and Cardiovascular Surgery

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