Noboru Kashiwagi

Clinical Reactions and Serologic Changes After the Administration of Heterologous Antilymphocyte Globulin to Human Recipients of Renal Homografts

Clinical reactions and serologic changes after intramuscular administration of horse anti-human lymphocyte globulin (ALG) were studied in 53 human recipients of renal homografts. The ALG was used as an adjuvant immunosuppressive drug. In the usual case 47 injections were given over a 4-month period. All patients had pain, tenderness, erythema, and swelling at the injection sites. Benign systemic side effects included fever in all cases, hives in eight cases, rash in five, pruritus in five, arthralgia in three, and periorbital edema in one. Anaphylactic reactions occurred in 11 cases. These were easily treated, and there was complete recovery in every instance within 90 min. In eight of these cases the ALG administration was discontinued. Subsequent injections were given in the other three. Four of 11 patients tested had positive skin tests to ALG before therapy. Antibodies against sheep red blood cells developed during therapy in 39 of 40 patients; 10 reached titers as high as 1:128 to 1:512. Precipitin antibodies as measured with an electroimmunodiffusion technique developed in 36 of 40 patients. All three immunologic tests were of value in predicting the probability of an anaphylactic reaction, but the discrimination was imperfect Immunoelectrophoretic studies of sera from 13 patients showed antibodies to horse beta globulins in all cases, to alpha globulins in 9 cases, and to gamma globulins in only 1. This finding indicates that a safer ALG could be made by removing the trace quantities of alpha and beta globulins from the immunologically more active gamma globulins.

Immunosuppressive effect of bredinin on cell-mediated and humoral immune reactions in experimental animals.

Bredinin (BR), an imidazole nucleoside isolated from Eupenicillium brefeldianum was previously reported to prolong kidney allograft survival in dogs. The immunosuppressive effect of BR was studied in experimental animals. In beagles, in vitro responses of lymphocytes stimulated by mitogens or allogeneic cells were suppressed by in vitro BR treatment. BR, given in vivo, also showed an inhibitory action against development of delayed hypersensitivity reaction to tubercle bacilli in guinea pigs or against hemagglutinin production following booster SRBC injection in rabbits. Of note may be the fact that BR was found to have an immunosuppressive potency comparable to that of azathioprine and, in addition, to show a decreased hepatotoxicity compared with the latter.

Changes in Serum Haptoglobin and Group Specific Component after Orthotopic Liver Homotransplantation in Humans

Summary In human recipients of orthotopic liver homografts with different haptoglobin (Hp) and group specific component (Gc) types than their respective donors, the donor phenotype permanently replaced that previously present. The findings prove that the liver is the sole source of Hp and Gc, and that it retains its metabolic specificity after transplantation to a new host.

Unsuccessful attempts to control hyperacute rejection of human renal homografts with F(ab') 2 and citrate organ pretreatment.

The presence of preformed cytotoxic antidonor antibodies in the serum of potential allograft recipients leads to the rapid destruction of the graft by the now well known events of hyperacute rejection (10, 21, 24-27). Experimental work in the past several years at our center and in other laboratories has been oriented to the solution of this difficult immunological problem, not only in the presensitized homotransplantation model but also in strongly incompatible xenograft combinations. Antibody and complement depletion (3-5, 7, 8, 13, 16, 19, 21, 23), or treatment by the chelating agents, sodium citrate (12, 14) and ethylenediaminetetraacetate (EDTA) (1), has been shown to delay hyperacute kidney rejection in both experimental models, whereas anticoagulation with heparin (15) or cobra venom (6) has yielded equivocal results. Even the most effective of these therapeutic procedures only delayed the destruction of the graft. More recently, encouraging results were obtained by several workers (11, 20, 22) with pretreatment of the organ with antidonor IgG fragments (F(ab′)2). It was suggested that F(ab′)2 fragments were protective by occupying the donor antigen receptor sites. Unsuccessful attempts to control hyperacute rejection in one of our patients who had preformed circulating cytotoxic antibodies are reported here, using homografts pretreated with sodium citrate or digested IgG.

Urine plasmin-like substances as an index of kidney allograft rejections.

Using solid state radioimmunoassays developed by the first author, changes in the urine level of plasmin-like substances (PLS) and fibrin degradation products (FDP) before and after human kidney transplantation were determined in 49 transplant patients. Averages of urine PLS and FDP in a normal population of 51 persons were 0.13+/-0.10 (SD) and 0.14+/-0.07 microng/ml, respectively. In all transplant patients there was an initial rise of both PLS and FDP in urine immediately after transplantation. This elevation peaked on days 4 and 5 and the PLS and FDP levels returned to normal range within 2 weeks in patients without evidence of rejeciton. A secondary rise of urine PLS was detected before or with a rise in serum creatinine in all of the patients experiencing rejections. Of 11 patients who showed a rejection episode within 2 weeks of transplantation, the secondary rise of urine PLS was detectable in 55% of the patients slightly before the serum creatinine level changes; of 6 patients with a rejection episode more than 2 weeks after transplantation, 100% showed a secondary PLS rise 6.7+/-2.3 (SE) days before the serum creatinine increased. The appearance of the secondary rise of urine FDP in the rejecting recipients was slightly later than the rise of PLS. Serial determination of urine PLS levels following human kidney transplantation appears to be an early index of rejections which occurs more than 2 weeks after transplantation, although the clinical usefulness of this measurement is probably limited.

Effects of phytohemagglutinin on humoral antibody response, delayed hyper sensitivity, and homograft rejection

Phytohemagglutinin (PHA), a mucoprotein derived from the red kidney bean, Phaseolus vulgaris, has erythroagglutinating and leukoagglutinating properties and stimulates the small lymphocytes of the peripheral blood to transform into large blast cells.4, 5, 16, 18 When administered parenterally, it can suppress the immune response in animals, if treatment is started several days before the antigenic stimulation.7, 8, 10, 12, 15, 18, 19 To our knowledge 6 studies have been reported concerning the effect of PHA on the survival of skin or renal homografts, with conflicting results.2, 3, 6, 13, 14, 17 Calne and associates2 and Casciani and Cortesini3 treated canine recipients of renal allografts solely with PHA and found a slight immunosuppressive activity; the effect was additive to that of azathioprine if the 2 agents were given together. Two of 4 publications concerned with skin grafts in rodents and rabbits reported no detectable effect of PHA,6,13 but the other 2 claimed slight prolongation of skin viability.15,17 In our laboratory the immunosuppressive qualities of a particular batch of PHA were evaluated by the ability of this agent to blunt humoral antibody formation in rabbits, to suppress the development of delayed hypersensitivity reactions in guinea pigs, and to mitigate the rejection of renal homografts in dogs. In addition the effect of red cell absorption on the biologic activity of PHA was assessed.

Interspecies reactivity and intraspecies specificity of antilymphoid globulin

It has been said that heterologous antilymphocyte serum is highly specific in that (a) it lyses or agglutinates equally well the white cells from all members of the species which provided the immunizing lymphoid tissue3 and (b) that it does not react with the white cells from other species.1, 3, 4, 6 The fore-going concepts were based upon studies employing anterisera of relatively low titer. These questions have been re-examined with antidog- and antihuman-lymphoid globulin of high titer raised in horses with repeated subcutaneous immunization with lymph node, thymus, and spleen tissue.5 It has been found that the white cells of individuals within the species population, against which the anti-bodies were directed, were variably affected by the immune globulin. Furthermore, it has been shown that there is not an absolute species specificity inasmuch as some reactivity against the white cells of at least seven widely divergent species is present in either antidog- or antihuman-lymphoid globulin.

The use of combined preservation techniques for extended storage of orthotopic liver homografts

During the 12 years since homotransplantation of the whole canine liver was first described by Welch, there have been reports of several techniques of hepatic homograft preservation in dogs. Goodrich and his colleagues showed that livers removed at normal temperatures became unsuitable for transplantation within 20 or 30 minutes. The first protective device used in our laboratories (15) and by Moore and his associates was hypothermia, induced first by whole body cooling of the donor to 30 degrees C. and then perfusing the excised liver with a chilled electrolyte solution. With a fall in the hepatic core temperature to approximately 15 degrees C., these organs could support the life of recipient dogs if revascularized as orthotopic homografts within 2 hours. After longer times, there was a high rate of acute failure due to outflow block of the transplants, a hemorrhagic diathesis, and acute liver failure. Almost identical conclusions about the efficacy of this simple approach were reached by Ono and his co-workers in experiments which did not involve homotransplantation. Subsequent efforts to extend the acceptable storage time have been disappointing. In dogs, Marchioro and his associates reported a method of hypothermic cadaveric perfusion with the use of an extracorporeal heart-lung apparatus into which a heat exchanger was incorporated. Either the entire corpse or the lower half of the dog was perfused. Orthotopic homotransplantation was performed with livers removed from 2 to 8 hours after the sacrifice of the donors. Eight of the 10 recipients which were treated with azathioprine survived operation, but all died within 1 to 5 days thereafter. Mikaeloff and Kestens and their associates used a similar principle in which hypothermic perfusion in situ was limited to the liver. They were able to obtain long term recipient survival after homotransplantation of canine livers removed as long as 6 hours after death. Their technique was an application of a method described several years earlier by Kestens and McDermott. More complex methods have been tried. Brown and his colleagues have evaluated a combination of freezing to − 6 degrees C., immersion into a dimethylsulfoxide or glycerol bath, and dehydration. After 1 to 5 days, the organs were viable but severely damaged and apparently incapable of supporting life as orthotopic homografts. When Moss and his co-workers cooled livers to −20 to −60 degrees C. for 1 to 14 days without dehydration, there was almost no function after the homografts were transplanted to the pelvis. Furthermore, all of the recipients died in 6 hours or less. Recently, there have been 2 reports of conservation of hepatic homografts for 8 to 24 hours, a combination of perfusion, hypothermia, and hyperbaric oxygenation being used. Slapak and his associates placed puppy livers preserved in this way in the neck of adult recipients and found that bile was produced in 8 of 19 experiments. Their perfusing fluid did not contain red blood cells. In a preliminary report from our laboratory (3), chronic survival was described of adult canine recipients that received orthotopic hepatic homografts which had been preserved for almost a day after sacrifice of the donor. In these experiments, the perfusate contained diluted blood. As will be documented, further experience has shown that a significant rate of survival can be attained with such conserved organs if attention is paid to several important details.