Bahnson, HT

Griffith, B

Hardesty, R

Rabin, BS

Singh, G

Starzl, T

Thompson, ME

English

PubMed

D-Scholarship@Pitt

p 564 TRANSPLANTATION Vol. 33. No.5 TABLE 2. Viability of encapsulated LEW spleen cells incubated with ACI anti-LEW antiserum ';' viability" Time Encapsulated Free Ab + C- Ab NS' + C' NS Ab + C' Ab NS+ C' NS 0 85 85 92 92 16h 8 79 81 83 0 89 88 94 " ~ean of three determinations; O.I-ml microcapsules (4 x 10' cellsl ml) or 4 x 10" free LEW splenocytes were incubated in 0.9 ml of M199 or supplemented with Ab or NS. h NS. normal ACI serum: C'. rabbit complement. host sensitization. Even with intact microcapsular membrane the intra capsular splenocytes were still destroyed in vitro by their respective antiserum. thus indicating that the membrane was permeable to y-globulins. In vivo implantation of empty microcapsules i.p. also induced an inflammatory response in the host animal. Therefore. in order to realize the theoretical advantage of microencapsulation over that of the hollow fiber unit and micro pore chamber. improvements of the encapsulation process with biocompatible polymers of high tensile strength and durability and with a better defined molecular weight cut-off will have to be achieved. WAH JUN TZE .JOSEPH TAl G. F. Strong Laboratory for Medical Research Department ol Pediatrics and Pathology University of British Columbia Vancouver. British Columbia. Canada LITERATURE CITED 1. Barker CF. Naji A. Silvers WK. 1980 Diabetes 29(Supp\): 86. 2. Tze WJ. Wong FC. Chen LM. 1979 Diabetologia 16: 247. :3. Theodorou NA. Vrbova H. Tyhurst M. et al. 1980 Diabetologia 18: 313. 4. Lim F. Sun AM. 1980 Science 210: 908. 5. Amos DB. 1965. P 151 Histocompatibility testing. Munksg:l:ird, Copenhagen. Received 15 December 1981. Accepted 20.January 1982. POSITIVE. WARM T CELL CROSSMATCH IN CARDIAC TRANSPLANTATION: WITH TRANSIENT VASCC'LITIS AND WITHOUT HYPERACUTE REJECTION Testing for cytotoxic antibodies in recipient serum. against donor lymphocytes. is perhaps the most important test done prior to allotransplantation. With the possible exception of liver transplants. a positive T cell crossmatch at 37 C is generally considered to be an absolute contraindication for organ trans-plantation (I, 2). The status of other types of positive cross-matches remains to be clarified (3.4). Allotransplantation done in the presence of a positive, warm T cell crossmatch does not always result in immediate graft loss. although hyperacute rejection or accelerated rejection is frequent. Both hyperacute rejection and graft survival have been de-scribed in cardiac transplants done in the presence of a. positive crossmatch (5. 6). We observed an intennediate outcome with early graft injury without immediate loss of the organ in a patient who received a cardiac transplant from a crossmatch-positive donor. The relevant clinical data are presented below: MB. a 48-year-old white woman, presented with severe con-gestive heart failure requiring vasopressors immediately follow-ing cardiac catheterization. Coronary arteries were normal and a diagnosis of cardiomyopathy with biventricular dysfunction was made. Cardiac transplantation was done on the 5th hospital day (recipient HLA type A2,A24 (9),B7,B27,DR2; donor HLA type A3,A26(10),BI5,B22,DR2,DR7). The recipient suffered a cardiac arrest within 2 hr of trans-plantation, but she was successfully resuscitated. She was treated with cyclosporin A and prednisolone. Cyclosporin A was given orally at a dose of 17.5 mg/kg/day for 19 days when a rise in bilirubin was observed and the dose reduced to 6.5 mg/ kg/day. She received 1 g of methylprednisolone at surgery and 1 g of cortisol i.v., every 4 hr for 24 hr. On the 2nd postoperative day, she received 200 mg of prednisone orally. The prednisone dose was reduced by 20 mg/day until 30 mg/day was reached. The donor heart was obtained from a distant city and the crossmatch between donor lymph node cells and the recipient serum was completed after transplantation. The crossmatch was carried out by the two-color fluorescence method at 4 and :37 C (7; Rabin et al .• unpublished data). The crossmatch vas positive at both temperatures against both T and B lympho-cytes at a serum dilution of 1:4. Following a single platelet absorption of the patient's serum. the crossmatch was negative. The patient's pretransplant serum was tested for the presence oflymphocytotoxic antibodies using a 26-member random panel by the trypan blue dye exclusion method. The patient's serum was cytotoxic to T and B lymphocytes from 50% of the panel members. Three transvenous endomyocardial biopsies were done each month during the 2-month post-transplant period. The regults of biopsy findings are outlined in Figure 1. The biopsy taken 1 week after transplantation showed focal lymphocytic and neu-trophilic infIltration with edema. The changes were felt to be consistent with mild acute rejection. The biopsy taken 22 days after transplantation exhibited edema with mild neutrophilic infiltration and focal myocytolysis without any appreciable lymphocytic infiltration. The biopsy taken 53 days after trans-plantation showed moderate, acute cellular rejection with nod-ular aggregates of lymphocytes, focal myocytolysis. and endo-cardial fibrosis. Four of the biopsies were examined by direct immunofluo-rescence for IgG, IgM, IgA, C3, C4, Clq, properdin, and fibrin-ogen. Vascular deposition of IgG and C3 were present in the biopsy taken 1 week after transplantation (Fig. 2). IgG, IgM, C3, C4, and fibrinogen deposits were seen in the vessel walls in. the biopsy taken 22 days postoperatively. The succeeding biop-sies were negative by direct immunofluorescence (Fig. 1). Postoperative serum specimens were examined for the pres-ence of antibodies cytotoxic to donor cells by the same tech": nique used for the original crossmatch. The serum taken 9 days post-transplantation gave a positive, warm T cell crossmatch with a titer of 32, a specimen taken 24 days post-transplantation was positive only when tested undiluted. The serum specimens taken 27 and 53 days post-transplantation were negative for· donor lymphocyte-specific cytotoxic antibodies (Fig. 1). ., BRIEF COMMUNICATIONS 565 FIGURE 1. Biopsy findings: The type test done is indicated on the vertical . axis and the post-transplant days on which the tests were conducted are in-dicated on the horizontal axis. Necrosis myocardial fibers, edema, neutrophilic and lymphocytic infiltrate CELLULAR REACTIVITY (L1F 6 TRANSFORMATION) CROSS MATCH !TITER) DIRECT IF' 19 AND C' LYMPHOCYTIC INFILTRATE NECROSIS EDEMA NEUTROPHILIC INFILTRATE + + 141 (3Z) + + + + 7 BIOPSY FINDINGS + + -(II + + + + ++(NODULAR AGGREGATES) + + + + + + + + 21 35 49 63 78 DAYS POST - TRANSPLANT assessed from biopsy specimens processed for light microscopy. Direct immunofluorescence was done on sepa-rate specimens processed as frozen sec-. tions. Crossmatch titers indicate the se-rum dilution at which more than 80% of the donor lymphocytes were killed. Cel-lular reactivity and transformation per-tain to the in vitro tests with saline ex-tract of donor heart tissue. + • POSITIVE OR PRESENT - • NEGATIVE OR ABSENT The patient's lymphocytes were tested (62 days post-trans-plantation) for their reactivity to donor heart tissue. The stim-ulation index. on incubating the patient's lymphocytes with saline extract of donor heart tissue, was 5.7 whereas the index in response to third-party liver extract was 2.4. The patient's lymphoc~Dtes were also tested for their ability to produce leu-koC':,·te migration inhibition factor in an indirect assay using saline extract of donor heart and third-party liver extract. The migration inhibition index with the donor heart extract was 0.64. The patient's lymphocytes, on exposure to third-party liver extract. and lymphocytes from a normal donor on exposure to donor heart and third-party liver extract, failed to produce leukocyte migration inhibition factor. ---------- The patient's EKGs demonstrated reduction in voltage in the earlv post-transplant period. However, the output stabilized by the 30th post-transplant day. The patient was discharged in a stable, though not a satisfactory, condition 8 weeks post-trans-plantation. She expired 2 weeks later with a 7-cm mediastinal abscess and bilateral pneumonia. Histologically, the ventricular myocardium showed edema and mild focal individual cell ne-crosis. The vascular endothelial cells were prominent, but no evidence of accelerated atherosclerosis was present. The patient's course, especially the study of endomyocardial FIGURE 2. The endomyocardial biopsy was stained with fluorescein-conjugated antihuman C3 antibod~DK The deposition of C3 in small vessels is evident. IgG, Ig-M, C4, and fibrinogen deposition were seen in similar distribu tion. biopsies (Fig. 1), points to a transient vascular injury initiated by the deposition of alloantibodies in addition to acute cellular rejection episodes. The outcome, however, was not hyperacute rejection as is the usual case with preformed antibodies. Al-though the treatment may have altered the appearance of the lesions, the histological findings during the period when direct immunofluorescence was positive were not entirely consistent with cell-mediated acute rejection, especially around postoper-ative days 22 to 24. The early picture can thus be described as an alloantibody-mediated vasculitis with mild acute rejection, leading to transient graft injury with recovery. We have no explanation for the nonprogressive nature of the vasculitis or the disappearance of the preformed antidonor antibodies in the patient's serum. Hypogammaglobulinemia did not occur during the course of observation. Although we were unable to serially test the cellular response to donor tissue, the single test (mito-genic response and leukocyte migration inhibition factor pro-duction) was positive at a time when biopsy findings indicated acute cellular rejection. Despite general agreement that allotransplants should not be done when a warm T cell crossmatch is positive, there may not be enough time to wait for the results of the crossmatch because of limited tolerance of the organ to ischemia. The indication of --------~-~---566 TRANSPLANTATION the presence of antiallotypic antibodies in the patient's serum can be obtained by testing the serum against a panel of lym-phocytes. The use of a panel of lymphocytes frozen and stored in Terasaki tissue typing trays was found to expedite such screening (8). It is being suggested that quick screening should be carried out for all prospective recipients to estimate the probability of a positive crossmatch against a random donor. By using lymphocytes frozen in Terasaki trays, a quick screen-ing could be carried out for all patients within a matter of about 5 hr. A patient reacting with 50% of the panel members has roughly a 50% chance of having a positive crossmatch with a random donor. It may, at times, not be possible to wait for the crossmatch results before transplantation; under su'ch circum-stances, knowing the probability of a positive crossmatch against a random donor may facilitate the decision for under-taking or not undertaking a transplant. We are in agreement with the recommendation of Weil et al. (5) that cardiac trans-plantation should not be done in the presence of a positive, warm T cell crossmatch unless the patient is not likely to survive long enough to wait for another organ. In summary, we have described a patient who had cytotoxic antibodies to 50% of the members of a random panel and had a positive, warm T cell cross match with the donor lymphocytes. The cardiac transplant suffered transient vasculitis, but did not undergo hyperacute rejection. Evidence for humoral rejection. i.e., positive direct immunofluorescence and positive cross-matches. waned despite initial positivity. Humoral and cellular rejection were present simultaneously in the initial period, but were discordant after the initial 2 weeks. It is suggested that quick screening for lymphocytotoxic antibodies be carried out using lymphocytes frozen in Terasaki typing trays to determine the likelihood of a positive crossmatch. Whenever possible, transplantation in the face of a positive crossmatch should be avoided. Acknowledgments. We are grateful to Dr. N. R. Dunn and her staff for their kind cooperation. GURMUKH SINGH BRUCE S. RABIN MARK E. THOMPSON ROBERT HARDESTY BARTLEY GRIFFITH HENRY T. BAHNSON THOMAS STARZL Division of Clinical Immunopathology Department of Pathology Division of Cardiology Departments of Medicine and Surgery University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261 LITERATURE CITED 1. Iwatsuki S, Iwaki Y, Kano T, et al. Successful liver transplantation from crossmatch-positive donor. Transplant Proc 1981: 13: ~:CSK 2. Patel R, Terasaki PI. Significance of the positive crossmatcn in kidney transplantation. N Engl J Med 1969; 280: 735. 3. Guttmann RD. Renal transplantation. N Engl J Med 1979; 301: 975. 4 .. leannet M, Benzonana G, Ami I. Donor-specific B and T lympho-cyte antibodies and kidney graft survival. Transplantation 1981; 31: 160. 5. Wei! III R. Clarke DR, Iwaki y, et al. Hyperacute rejection of a transplanted human heart. Transplantation 1981; 32: 71. 6. Baumgartner WA, Reitz BA, Oyer PE. Stinson EB, Shumway NE. Cardiac homotransplantation. Curr Probl Surg 1979; 16: 1. 7. van Rood JJ, van Leeuwen A, Plovem JS. Simultaneous detection of two cell populations by two-colour fluorescence and applica-tion to the recognition of B-cell determinants. Nature 1976; 262: 795. 8. Goldstein AS, Hubbard MC, Barry JM. A simple method for freezing lymphocytes in microtest plates. Am J Clin Patho11981; 75: 221. Received 11 December 1981. Accepted 22 January 1982. VALUE OF A PHYSIOLOGICAL LIVER TRANSPLANT MODEL IN RATS INDUCTION OF SPECIFIC GRAFT TOLERANCE IN A FULLY ALLOGENEIC STRAIN COMBINATION' With the one known exception of spontaneous graft tolerance, DA -. PVG (1,2), fully allogeneic rat liver transplantation (RL T) still shows very poor results, whereas in more than one semiallogeneic combination specific transplantation tolerance has been achieved (3,4). RLT, whether performed.in an aux-iliary heterotopic model (4) or in variously modified orthotopic models (1,5-7), has, so far, not yet been immunologically analyzed after reanastomosis of the hepatic artery. Major com-plications after RLT, such as biliary peritonitis and bile duct necrosis, may be attributable to the lack of arterial blood supply. Improving the modified orthotopic technique described by Lee et ai. (8), by reanastomosing the hepatic artery, syngeneic LEW and fully allogeneic BN livers were grafted into LEW recipients. In comparison of the results of transplantations performed with reanastomosis of the hepatic artery (REART) to those performed under nonrearterialized (Non-REART) con-'This work was supported by the Deutsche Forschungsgemeinschaft in SFB 111, B 9. ditions, we will show: (1) dramatic increase of isograft survival using the optimized technique, (2) improvement of graft mor-phology, (3) elimination of unspecific cell-mediated in vitro reactivity, and (4) specific transplantation tolerance in the fully allogeneic BN -+ LEW combination, By using the inbred rat strains LEW (RT11) and BN (RT1 n), the following four combinations were studied: LEW -+ LEW Non-REART, n = 22, REART, n = 17 and BN -+ LEW Non-REART, n = 8, REART, n = 12. RL T (Non-REART) was perfonned with the modified orthotopic Lee technique (8) using a polyethylene cuff for bile duct anastomosis. In REART rats in addition to suprahepatic inferior vena cava anastomosis, portal vein anastomosis and anastomosis of the infrahepatic inferior vena cava, the aorta of the donor, bearing the celiac axis and the hepatic artery, was anastomosed in an end to side fashion to the recipient's infrarenal aorta. Mean operation time for Non-REART rats was 45 min, for REART rats 70 min. Survival rates were evaluated and morphological studies of the liver (open liver biopsies were taken at day 10 and then in 4-week intervals) were performed in every surviving animal. Cell-

Positive, warm T cell crossmatch in cardiac transplantation: With transient vasculitis and without hyperacute rejection

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Positive, warm T-cell crossmatch in cardiac transplantation: With transient vasculitis and without hyperacute rejection

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Positive, warm T cell crossmatch in cardiac transplantation: With transient vasculitis and without hyperacute rejection

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