Graham Tc

Hemopoietic grafts between DLA-identical canine littermates following dimethyl myleran. Evidence for resistance to grafts not associated with DLA and abrogated by antithymocyte serum.

Dogs were given a single (supralethal) dose of 10 mg of dimethyl myleran (DMM) per kg i.v. followed by infusions of marrow, 0.4 ± 0.2 × 109 (SD) cells/kg, and blood leukocytes, 1.4 ± 0.5 × 109 (SD)/kg, from DLA-identical littermates of opposite sex. DLA identity was determined by serological histocompability typing and mutual nonreactivity in mixed leukocyte culture. Sixteen dogs were given only DMM before the hemopoietic graft: two never showed evidence of marrow engraftment, six had initial engraftment but subsequently rejected the graft, and eight had sustained engraftment. Four of the eight were alive between days 557 and 739. Ninety-seven, 70, and 65%, respectively, of metaphases analyzed in their marrows, peripheral blood, and lymph nodes showed the donor karyotype. Ten dogs were given antithymocyte serum (ATS) for 6 days before DMM and the hemopoietic graft: one had initial engraftment but subsequently rejected the graft and nine had sustained engraftment. Eight of the nine were alive between 174 and 382 days. Between 82 and 97% of their hemopoietic cells showed the donor karyotype. Three dogs were given a combination of procarbazine and ATS for 6 days before DMM and the hemopoietic graft: all three had sustained engraftment, and 99 to 100% of their hemopoietic cells showed the donor karyotype. In conclusion, a dose of 10 mg of DMM per kg was sufficiently immunosuppressive to permit sustained engraftment of DLA-identical marrow in 50% of the dogs studied. Fifty percent of the dogs rejected the graft, presumably as a result of resistance to “minor” histocompability systems not associated with DLA. The addition of ATS or procarbazine and ATS to DMM successfully abrogated resistance. Its profound antitumor activity with marrow toxicity reversible by marrow transplantation combined with its moderate immunosuppressive properties suggest that DMM is useful in combination with other agents to condition human patients with hematologic malignancy for marrow transplantation.

TREATMENT OF CANINE GRAFT-VERSUS-HOST DISEASE WITH METHOTREXATE AND CYCLOPHOSPHAMIDE FOLLOWING BONE MARROW TRANSPLANTATION FROM HISTOINCOMPATIBLE DONORS

SUMMARY The effectiveness of immunosuppressive therapy with methotrexate was compared to that with cyclophosphamide in preventing or delaying the lethal graft-versus-host disease in dogs following bone marrow transplantation from unrelated donors that were mismatched with their recipients by canine histocompatibility testing. Recipients were prepared for transplantation by 1200 E. of total body irradiation followed by combined infusions of donor marrow and peripheral blood leukocytes. Immunosuppressive drugs were administered on days 1, 3, 6, and 11 posttransplantation and once weekly thereafter until day 102. Four of 6 recipients in group 1 given 0.5 mg methotrexateAg died between days 20 and 33 with acute graft-versus-host disease. One died on day 76 with pneumonia, and one is alive more than 124 days posttransplantation. All 8 recipients in group 2 given 5 mg cyclophosphamide/kg died between 11 and 20 days with graft-versus-host disease. Four of 6 dogs in group 3 treated with 7.5 mg cyclophosphamide/kg died with graft-versus-host disease between days 11 and 13. Two of the. 6 failed to show evidence of marrow repopulation and died on day 13 with pneumonia. Three of 4 dogs in group 4 given 10 mg cyclophosphamide/kg died with marrow aplasia between days 8 and 10. One of the 4 died on day 20 with graft-versus-host disease. Statistical analysis of the survival data demonstrated the superiority of the methotrexate regimen (P<0.01)

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.

Batten's disease : failure of allogeneic bone marrow transplantation to arrest disease progression in a canine model

We investigated whether the course of canine ceroid lipofuscinosis (CCL), a model of Battens disease in man, was affected by allogeneic bone marrow transplantation. Four English setters with CCL, 4 months of age, were given 9.2 Gy of total body irradiation, followed by the infusion of bone marrow cells from healthy DLA identical sibling donors. All transplanted dogs had complete hematologic reconstitution. However, at 12–13 months posttransplant, all dogs developed characteristic and progressive signs of CCL. Autopsies revealed cerebral atrophy and findings of ceroid storage not different from those in non‐transplanted controls. These findings suggest that bone marrow cells do not contain or release the gene product(s) necessary to correct the disease. It appears unlikely that with our current knowledge, allogeneic marrow transplantation would be beneficial in the treatment of Battens disease.

Marrow grafts between canine litter-mates homozygous or heterozygous for lymphocyte-defined histocompatibility antigens.

Hemopoietic grafts following 1,200 R of total body irradiation were carried out between canine littermates homozygous or heterozygous for lymphocyte defined (LD) antigens of the major histocompatibility complex (MHC). In all but five of the 25 pairs studied, LD homozygous dogs were also homozygous for serologically defined (SD) antigens of the MHC. Results of transplants were compared with previous results obtained in littermate pairs matched or mismatched for the MHC. Two groups of recipients were studied. Of 14 LD homozygous recipients in group 1 given grafts from LD heterozygous donors, 12 died between 8 and 193 days and two survived more than 238 and 627 days. The most frequent cause of death was graft-versus-host disease (GVHD). Survival was significantly shorter (P <0.005) than that of dogs given grafts from matched littermates and not different (P < 0.5) from that of dogs given grafts from mismatched littermates. Survival of 11 LD heterozygous recipients in group 2 given grafts from LD homozygous donors was not different from that of dogs in group 1 (P < 0.5). The results indicate that the LD loci detected by mixed leukocyte culture are not the principal determinants within the MHC that are responsible for GVHD.

Genetic variation of red blood cell enzymes in the dog. Use of soluble glutamic oxaloacetic transaminase as proof of chimerism.

One hundred ten dogs, of whom at least 63 were unrelated, were examined for electrophoretically distinguishable, genetic variants of 22 different red blood cell enzymes and of hemoglobin. A variant of soluble glutamic oxalaocetic transaminase (S-GOT) was found to occur with high frequency in dogs of the Basenji breed, and infrequent variants of indophenol oxidase and acid phosphatase were found in other breeds. The S-GOT variant was utilized to establish the presence of donor- and the absence of recipient-type cells following marrow transplantation.

The effect of prior blood transfusions on hemopoietic grafts from histoincompatible canine littermates.

SUMMARY The present study investigated the influence of preceding blood transfusions from randomly selected unrelated dogs on the outcome of marrow grafts between histoincompatible littermates. Two groups of recipients were conditioned for marrow grafting by 1,200 R of whole body irradiation followed within 4 hr by infusion of hemopoietic cells from the incompatible littermate. Group 1 consisted of 12 dogs not given a preceding blood transfusion. Group 2 was made up of 9 dogs given 3 transfusions on each of 3 occasions: 24, 17, and 10 days before grafting. All 12 dogs in group 1 showed prompt and sustained marrow engraftment. Six of nine dogs in group 2 showed acute marrow graft rejection and died with marrow hypoplasia. Three showed sustained hemopoietic engraftment. It was concluded that multiple prior transfusions from unrelated dogs in most instances immunized a recipient and prevented subsequent marrow engraftment from a histoincompatible littermate

THE EFFECT OF BUFFY COAT-POOR BLOOD TRANSFUSION ON SUBSEQUENT HEMOPOIETIC GRAFTS

SUMMARY Previous studies in dogs have shown that a single preceding transfusion with whole blood from the intended marrow donor can immunize a recipient and lead to rejection of a subsequent marrow graft even when donor and recipient are littermates compatible for the major dog leukocyte antigen (DL-A) locus. The present study investigated two approaches to diminish the chance of marrow graft rejection in transfused canine recipients: (1) the use of the immunosuppressive drug methotrexate (MTX) after grafting, and (2) the use of buffy coat-poor blood instead of whole blood for the preceding transfusion. Recipients were conditioned for marrow grafting by 1,200 R of total body irradiation followed within 4 hr by infusion of hemopoietic cells from DL-A-incompatible unrelated donors. MTX, 0.4–0.5 mg/kg, was administered i.v. on days 1, 3, 6, and 11 after irradiation and grafting and then once weekly until day 102. A single preceding transfusion of whole blood from the intended DL-A-incompatible marrow donor in most instances led to rejection of the marrow graft. Treatment with MTX did not diminish the incidence of graft rejection. Substituting buffy coat-poor blood (reduction in white blood cells: 99.8 ± 0.2% (SD) and platelets: 99.1 ± 0.7% (SD)) for whole blood did not significantly change the incidence of immunization of recipients.

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.