Epstein Rb

Methotrexate regimens for control of graft-versus-host disease in dogs with allogeneic marrow grafts

SUMMARY Immunosuppressive therapy with high doses of methotrexate beginning 1 day after allogeneic bone marrow transplantation was evaluated for its effectiveness in preventing or delaying the lethal graftversus-host disease in dogs. A short-term regimen of methotrexate for 6 days after transplantation was compared with a long-term regimen during 102 days postgrafting. Unrelated donor-recipient pairs mismatched by canine histocompatibility testing were used. The recipients were given combined infusions of donor marrow and leukocytes to provide a test system in which the graft-versus-host syndrome is acute and rapidly fatal. Recipients were prepared for transplantation by 1200 R of total body irradiation. Five control dogs, not receiving immunosuppressive therapy, died between 9 and 14 days after transplantation with graft-versus-host disease. Eight of the 9 dogs receiving short-term methotrexate treatment died between 14 and 21 days with graft-versus-host disease, and 1 dog lived 136 days. Seven of the 10 dogs given long-term methotrexate treatment died between 18 and 68 days, and 3 dogs are alive more than 180 days after transplantation. Statistical analysis of the survival data demonstrated the superiority of the long-term methotrexate treatment. The results show that stable long-term chimerism can be achieved in mismatched recipient dogs when intensive methotrexate is begun immediately after marrow transplantation and continued for a prolonged period of time.

High‐dose cyclophosphamide therapy for malignant disease. Toxicity, tumor response, and the effects of stored autologous marrow

Twenty‐five patients with disseminated malignant disease received single or multiple courses of cyclophosphamide (CY) 60 to 120 mg/kg. Stored autologous marrow was infused following 120 mg/kg of CY in 6 instances. One patient received 240 mg/kg of CY over a 4‐day period followed by autologous marrow infusion. Patients receiving 60 mg/kg showed a moderate leukopenia (mean nadir of 500/mm3), anemia (mean hematocrit decline of 8.2 vol%) and variable thrombocytopenia that ranged from no change to 34,000/mm3 with a mean nadir of 127,000/mm3. Increasing the dose to 120 mg/kg produced leukopenia to below 500 cells/mm3 with a mean nadir of 120 cells/mm3. Thrombocytopenia was severe with a mean nadir of 37,000/mm3. Hematocrit values in this group fell by 15.2 vol%. Infectious complications occurred following 1 of 10 courses at 60 mg/kg and 18 of 35 courses at 120 mg/kg. In the latter group, there were 7 episodes of septicemia including one death from pseudomonas septicemia. A second patient, with cerebral metastases, died of a CNS hemorrhage. The patient receiving 240 mg/kg died as a consequence of myocardial necrosis. The infusion of autologous marrow had no apparent effect on hemopoietic recovery or infectious complications. Of the responding patients, 3 had ovarian carcinoma, 3 had testicular tumors, 1 had adenocarcinoma of the bowel, and 1 had an undifferentiated malignancy.

CYTOTOXIC TYPING ANTISERA FOR MARROW GRAFTING IN LITTERMATE DOGS.

SUMMARY Immunization of dogs with buffy coat cells from unrelated dogs consistently produced cytotoxic antisera which gave strong reactions but were too complex for suitable analysis. Cross immunization of littermate pairs produced cytotoxic antisera only infrequently, and these antisera gave weaker reactions. Four antisera were obtained by sibling immunization. They showed limited specificity as determined by cross absorption studies and typing of canine families. Genetic studies indicated that these antisera recognized independent antigens of dominant inheritance. Testing of lymphocytes from 62 unrelated dogs with these four antisera gave positive reactions with frequencies of 0.55, 0.53, 0.31, and 0.26. In order to evaluate the usefulness of these sera for histocompatibility typing, six litters were typed with the four antisera. A donor was selected from each litter, and an aliquot of marrow was infused into a matched and into a mismatched sibling following 1500r of whole-body irradiation. None of the mismatched recipients lived longer than 14 days. All of the matched recipients lived beyond 40 days, and 4 of the 6 lived more than 100 days. It was concluded that cross immunization of litter mates produced reasonably specific typing sera. These sera were of value in selecting donor-recipient pairs of canine siblings for marrow grafting.

Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing.

Consistent and rapid bone marrow grafts were produced in lethally irradiated dogs by infusions of marrow cells and peripheral blood leukocytes obtained from single living donors. Canine histocompatibility typing by lymphocyte cytotoxic antisera was evaluated in selecting donor-recipient pairs of unrelated dogs for marrow grafting. Significantly longer survival was observed in recipients that received marrow grafts from matched donors (P > 0.01 < 0.02).

Allogeneic canine bone marrow transplantation following cyclophosphamide

Allogeneic bone marrow transplantation following a lethal dose of cyclophosphamide was studied in 20 donor-recipient pairs of dogs. Eighteen of these were littermate pairs matched with four canine histocompatibility typing sera, and two were pairs of unrelated mismatched dogs. Ten dogs died in the 1st week from infection, bleeding, and nonhemopoietic drug toxicity. Their survival was too short to evaluate the success or failure of the marrow graft. Evidence for allogeneic bone marrow engraftment was demonstrated in 8 of the 10 recipients that lived beyond 7 days. Five of the dogs with marrow engraftment died within 36 days, 1 with evidence of graft-versus-host disease, 1 with pneumonia, and 3 after rejection of the marrow graft. Three of the 8 dogs with marrow grafts became long-term chimeras. Two are alive 350 and 450 days after transplantation. Peripheral blood, bone marrow, and lymph nodes in these 2 dogs showed a mixed population of host and donor cells. One additional dog survived over 8 months with evidence of a persistent change to donor red cell type and with prolonged survival of a skin graft from the marrow donor. In addition, this dog developed an isoantibody against the former host red cell type. It was concluded that persistent hemopoietic chimerism can be obtained in outbred animals following a lethal dose of cyclophosphamide.

Hemophilia: Role of Organ Homografts

The concentration of factor VIII and partial thromboplastin times became normal and have remained normal for 140 days after orthotopic tranplantation of a normal liver to a hemophilic dog. Transplantation of a normal spleen into a hemophilic recipient did not result in a significant increase in factor VIII although the splenic graft was viable for at least 47 days. Transplantation of normal marrow to a lethally irradiated hemophilic dog did not result in an increase in factor VIII during 34 days of observation.

Transplantation of stored allogeneic bone marrow in dogs selected by histocompatibility typing

SUMMARY Allogeneic canine marrow preserved at —80 C in 10% dimethyl sulfoxide was used for transplantation in unrelated dogs following 1200 R whole body irradiation. Lymphocytes of donors and recipients were typed with four canine cytotoxic isoantisera. The results in 10 recipients that were matched with their donors were compared to the results in 10 mismatched recipients. A successful marrow graft was observed in all 10 matched recipients and in 9 of 10 mismatched recipients. Graft rejection occurred in 2 of 10 matched and 7 of 10 mismatched recipients. The matched recipients survived significantly longer than did the mismatched ones (P < 0.005). Two of the matched dogs are alive more than 135 and 160 days after transplantation. The cryopreservation of the marrow did not appear to lessen its ability to produce lethal graft-versus-host disease. The results d emonstrate the importance of histocompatibility matching in successful grafting with cryopreserved marrow

CANINE MIXED LEUKOCYTE REACTIVITY AND TRANSPLANTATION ANTIGENS

SUMMARY A mixed leukocyte culture method was adapted for study of canine cells. The uptake of tritiated thymidine by reacting cells was measured following stimulation by cells inactivated by radiation. The results of histoeompatibility typing with this method bore a significant correlation (P < 0.01) to results obtained from typing with four cytotoxic antisera known to identify important transplantation antigens in this species.

Rescue from canine graft-versus-host reaction by autologous or DL-A-compatible marrow.

The present study is concerned with the termination of chimerism in canine marrow recipients threatened by lethal graft-versus-host (GVH) disease with use of an infusion of stored autologous marrow cells or cells from serotypically histocompatible littermates. All recipients were given 1,200 R of total body irradiation followed by a hemopoietic graft from a histoincompatible unrelated donor. Methotrexate was given on days 1, 3, 6, and 9 to delay the onset of GVH disease. Five dogs given no additional therapy died between 16 and 39 days with GVH disease. An attempt at rescue was made in 10 dogs by giving an infusion of stored autologous marrow on day 14 either with or without exposure of the recipient to 400 R. Rescue failed, and dogs died between 18 and 37 days either with GVH disease or marrow aplasia. Rescue was successful in three of seven dogs given 600 R on day 14 followed by infusion of marrow from a compatible littermate. The remaining four dogs either rejected the littermate marrow or died of infection. In conclusion, attempts at rescue from canine GVH disease are exceedingly difficult, and only possible in the present study after renewed exposure of the recipient to a high dose of total body irradiation followed by large numbers of hemopoietic cells from a compatible littermate.

Clinical and Hematologic Effects of Cross Circulation in Baboons

Potential clinical and hematologic complications were studied in 18 baboons subjected to cross circulation for periods of five to 31 days. Cross circulation was a safe procedure during the first four days. After that time, anaphylactoid reactions potentially fatal for one or both partners, thrombocytopenia, leukopenia, hemolytic anemia, and in one case, marrow depression were seen. These reactions were ameliorated but not entirely controlled by administration of methotrexate. When cross circulation was discontinued after five to 31 days, eight of nine baboons showed prompt clinical and hematologic recovery without late sequelae.