Fg Schuening

FK-506 and methotrexate prevent graft-versus-host disease in dogs given 9.2 Gy total body irradiation and marrow grafts from unrelated dog leukocyte antigen-nonidentical donors.

FK-506 was evaluated either alone or combined with methotrexate (MTX) for prevention of graft-versus-host disease (GVHD) in dogs given 9.2 Gy total body irradiation and dog leukocyte antigen-nonidentical unrelated marrow grafts. Studies with marrow autografts showed gut toxicity and weight loss to be major side effects of FK-506. There was no hematopoietic toxicity with FK-506. In an initial allograft study, 5 dogs were given FK-506 intramuscularly at 0.3 mg/kg/day from days 0 to 8 and then orally at 0.5 mg/kg/day. All 5 died, 3 with intussusception most likely due to FK-506 toxicity, 1 with graft failure, and 1 with GVHD. Subsequently, the FK-506 dose was reduced and these drug schedules were used: FK-506 days 0–8 at 0.15 mg/kg/day i.m. and then orally at 0.5 mg/kg/day until day 90, with or without MTX intravenously at 0.4 mg/kg days 1, 3, 6, and 11. Twenty allografts were done, 10 with FK-506 alone, and 10 with MTX/FK-506. Results were compared with those in concurrent and historical controls given either no immunosuppression (n=64), MTX (n=114), CsA (n=15), or MTX/CsA (n=17). Five of 20 current dogs died with intussusception, too early to be evaluated for GVHD. The 10 dogs given FK-506 alone survived significantly better than those not given immunosuppression but not differently from those given short-term MTX or CsA alone. Three died from toxicity, 2 with graft failure, and 4 with GVHD. Only 1 dog became a long-term survivor, and this dog inadvertently received a single dose of MTX on day 7. Two of 10 dogs given MTX/FK-506 died from toxicity, 1 died with graft failure, 2 died with GVHD, and 5 became long-term survivors, a result that is significantly better than seen with either drug alone and similar to that seen with MTX/CsA. Four of the 5 survivors had no clinical GVHD. FK-506 blood levels were 15–35 ng/ml between days 8 and 15, when gut toxicity was most severe. Thereafter, levels were approximately 5 ng/ml. In conclusion, FK-506 prolonged survival of recipients of dog leukocyte antigen-nonidentical unrelated marrow grafts. When FK-506 was combined with MTX, graft-host tolerance was induced in 50% of dogs, even though FK-506 was stopped on day 90.

γ-irradiation of pretransplant blood transfusions from unrelated donors prevents sensitization to minor histocompatibility antigens on dog leukocyte antigen-identical canine marrow grafts

Pretransplant blood transfusions from a dog leukocyte antigen (DLA)-identical canine littermate marrow donor will sensitize the recipient to non-DLA-linked polymorphic minor histocompatibility antigens, which uniformly results in graft rejection. We observed previously that 2000 cGy gamma-irradiation of marrow donor blood transfusions prevented this sensitization and subsequent marrow graft rejection. The purpose of the present study was to determine whether treatment of unrelated blood transfusions with gamma-irradiation would also prevent sensitization. Conceivably, sensitization to minor histocompatibility antigens might be more efficient or potent and thus more difficult to prevent when those antigens are seen in the context of disparity for DLA antigens. Furthermore, this model, in which sensitization to DLA-identical littermate marrow is caused by unrelated blood transfusions, is directly relevant to the clinical circumstances of human marrow transplantation. We assessed sensitization caused by unrelated blood transfusions by monitoring graft outcome in recipients transplanted with DLA-identical littermate marrow after conditioning with 920 cGy total body irradiation. Two thousand cGy gamma-irradiation of unrelated blood transfusions significantly reduced the incidence of transfusion-induced sensitization of recipients. There was successful marrow engraftment in 15 of 16 (94%, P < 0.003) of these animals in contrast to the previous study in which only 7 of 16 (44%) animals engrafted after they were transfused with unmodified blood on the same schedule. These results suggest that blood transfusions for use in humans, especially for patients with aplastic anemia, should be gamma-irradiated in order to reduce the incidence of marrow graft rejection caused by sensitization to minor histocompatibility antigens.

In vivo radioprotective effect of AcSDKP on canine myelopoiesis

Abstract The tetrapeptide acetyl-N–Ser-Asp-Lys-Pro (AcSDKP) interferes with G1/S-phase progression, and the resulting cell cycle arrest is thought to protect hematopoietic stem cells against injury by cycle-active cytotoxic agents. We investigated the radioprotective effect of AcSDKP in a canine radiation model. Dogs were given total-body irradiation (TBI) at an exposure rate of 10 cGy/min, either without further therapy (control) or with administration of AcSDKP at 0.05–500 μg/kg/24 h beginning before and continuing until after completion of TBI. At 400 cGy of TBI, one of 28 control dogs and one of eight AcSDKP-treated dogs recovered hematopoiesis (p=0.40). At 300 cGy, seven of 21 control dogs recovered hematopoiesis compared with five of five AcSDKP-treated dogs (p=0.01). In dogs given 300 cGy and AcSDKP, the granulocyte nadirs were less profound (p=0.04) and occurred later (p=0.04) than among controls; platelet kinetics did not differ. These data suggest, therefore, that AcSDKP provides a radioprotective effect in dogs exposed to 300 cGy TBI. Such an effect might be beneficial in recipients of intensive radiation therapy. Conceivably, the effect on hematopoietic recovery could be amplified by growth factor administration after irradiation.

L-leucyl-l-leucine methyl ester treatment of canine marrow and peripheral blood cells: Inhibition of proliferative responses with maintenance of the capacity for autologous marrow engraftment

Incubation of canine marrow and peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester resulted in the inhibition of mitogen- and alloantigen-induced blastogenesis, the elimination of allosensitized CTL and NK activity, and prevented the development of CTL from pCTL. The effects of these incubations were similar to those described in mice and humans. Additionally, in vitro CFU-GM growth from treated canine marrow was reduced, but could be regained when the Leu-Leu-OMe-treated marrow was cocultured with either untreated autologous peripheral blood mononuclear cells or monocyte-enriched PBMC but not with untreated monocyte-depleted PBMC. Six of seven dogs conditioned with 920 cGy total-body irradiation engrafted successfully after receiving autologous marrow that was incubated with Leu-Leu-OMe prior to infusion. These cumulative results indicate that incubation with Leu-Leu-OMe is a feasible method to deplete canine marrows of alloreactive and cytotoxic T cells prior to transplantation.

Pharmacologic, toxicologic, and marrow transplantation studies in dogs given succinyl acetone

A novel immunosuppressant, succinyl acetone (4,6-dioxoheptanoic acid), was studied in dogs. Results with bolus intravenous injections at doses ranging from 50 to 1600 mg/kg showed dose-dependent α and β half-lives, ranging from 30 to 80 min and 7 to 27 hr, respectively. Results suggested that continuous i.v. infusion was necessary to maintain constant plasma levels. Four dogs were given 9.2 Gy total-body irradiation and autologous marrow transplants along with continuous i.v. infusion of succinyl acetone at 50, 100, 200, or 400 mg/kg/day for 21 days, and all four had rapid, sustained hematopoietic engraftment. However, two of the four dogs receiving 200 and 400 mg succinyl acetone/kg/day, respectively, developed bilateral hind-limb ataxia, with histologically confirmed cerebellar lesions in the dog given the higher dose, thus establishing a potential doselimiting neurotoxicity. Prevention of graft-versus-host disease was studied in recipients of allogeneic marrow. Dogs were given 9.2 Gy TBI, followed by hematopoietic grafts from unrelated DLA-nonidentical or DLA-haploidentical littermate dogs. Succinyl acetone was given as continuous infusion for 21 days after transplant at doses of 100–300 mg/kg/day. Starting succinyl acetone on the day of marrow infusion in four dogs failed to prevent rapid onset of acute GVHD, and dogs survived no longer than controls. Starting succinyl acetone 3 days before transplant delayed the onset of acute GVHD and prolonged survival significantly compared with that of dogs not given postgrafting immunosuppression (P=0.008); survival was comparable to that in previously reported dogs given either methotrexate or cyclosporine as postgrafting immunosuppression (P=0.88 and 0.99, respectively). Seven of the sixteen allogeneic recipients developed evidence of neurotoxicity during succinyl-acetone infusion. Neurological dysfunctions were manifested by hind-limb ataxia and posterior paresis. In conclusion, succinyl acetone significantly delayed the onset of GVHD and prolonged survival of DLA-nonidentical marrow graft recipients but did not induce graft-host tolerance and was associated with dose-limiting neurotoxicity.

Toxicity trial of prophylactic 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine (ganciclovir) after marrow transplantation in dogs.

The effects of 9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine, or ganciclovir, administered during the immediate posttransplant period to dogs given total-body irradiation and autologous marrow transplants were studied. Doses of ganciclovir of 3.0 mg/kg (body weight) per day were well tolerated without detectable delay in hematopoietic recovery, whereas doses of 5.0 mg/kg per day were associated with delayed platelet recovery.

Implanted Right Atrial Catheters for Continuous Infusion of Solutions into Dogs

Silastic catheters were fabricated and aseptically implanted through the skin into the jugular vein of 64 dogs with the intravascular tip located in the right atrium. Solutions were infused through the catheter at 2 to 2.5 mL/h by a portable pump worn by the dog. Following 9.2 Gy total body irradiation (TBI) and allogeneic bone marrow transplantation (BMT), succinyl acetone, an experimental chemotherapeutic agent, was infused into 34 dogs. Hematopoietic growth factors were infused into an additional 30 dogs, two of which had 9.2 Gy TBI and an autologous BMT, and four of which had 4.0 Gy TBI and no BMT. All dogs received continuous oral and parenteral antibiotics while the catheters were in place. All catheters functioned well until electively removed (n = 28) or until the dogs died or were euthanized (n = 36) at 12 to 68 days after implantation. Mean length of catheter function was 30.3 +/- 1.5 (SEM) days. No catheters were dislodged and there was no evidence of catheter-related blood loss or sepsis. Semiquantitative cultures of 5 catheters were negative, but Staphylococcus epidermidis was isolated from 3 of 7 catheters cultured in broth. Six dogs had thrombosis adjacent to the intravascular catheter tip. The catheters were well tolerated and facilitated successful long-term infusion of solutions into dogs.