Thrombocytopenia in dogs induced by granulocyte-macrophage colony- stimulating factor: increased destruction of circulating platelets

Administration of recombinant canine granulocyte-macrophage colony- stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose- dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In- labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM- CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of fractionated to single-dose total body irradiation in conditioning canine littermates for DLA-identical marrow grafts

We explored the ability of fractionated total body irradiation (TBI) given at a rate of 7 cGy/min from opposing dual 60Co sources at otherwise lethal doses of 450, 600, 700, 800, and 920 cGy to condition dogs for marrow grafts from DLA-identical littermates. Results were compared with those of a previously reported study using single-dose TBI administered under otherwise identical conditions. Fractionated TBI was less immunosuppressive than single-dose TBI, as evidenced by a significantly higher rate of graft rejection (P = .001). Specifically, sustained allogeneic engraftment was observed in only two of 18 (11%) dogs that received 600 to 800 cGy fractionated TBI as compared with 11 of 17 (65%) dogs that received comparable doses of single-dose TBI. Only at 450 cGy (none of the ten dogs studied had sustained engraftment) and at 920 cGy (four of five dogs that received fractionated and 20 of 21 dogs that received single-dose TBI engrafted) were we unable to find differences between the two modes of radiation. Most dogs that rejected their graft survived with autologous recovery (13 of 22 that received fractionated and eight of 12 that received single-dose TBI; P = .49), presumably the result of extended support provided by the transient allogeneic grafts. We conclude that at equivalent doses fractionated TBI is significantly less effective than single-dose TBI to condition DLA-identical littermate dogs for marrow transplantation. These findings have implications for the design of conditioning programs in clinical transplantation, especially when T- cell-depleted marrow grafts are used.     

Transfusion of autologous cytotoxic cells leads to failure of unrelated, DLA-nonidentical marrow grafts

Dogs conditioned with 9.2 Gy total body irradiation (TBI) and given histoincompatible marrow transplants have a high rate of graft failure. Engraftment can be achieved by using 18 Gy fractionated TBI as preparative regimen. In this study, we tested the effects of infusing, at the time of histoincompatible marrow transplantation, autologous cells that had been stored before beginning high-dose (18 Gy) TBI. Our aim was to identify the peripheral blood mononuclear cells (PBMC) that contribute to failure of marrow grafts. Marrow graft failure was observed in three of three dogs receiving a mean of 2.1 x 10(8) unfractionated autologous PBMC/kg body weight as well as in two of two dogs receiving a mean dose of 0.075 x 10(8) PBMC/kg. When the dose of PBMC was decreased to 0.01 x 10(8)/kg, engraftment was seen in two of two dogs. These experiments thus established a cell dose response for causing marrow graft failure; further studies evaluated which subset of cells mediated this effect. Infusion of 0.09 x 10(8) nylon wool-nonadherent, plastic-nonadherent PBMC/kg was effective in causing marrow graft failure in three of three dogs. In contrast, infusion of 0.03 x 10(8) autologous monocytes/kg, enriched threefold above the number contained in the lower dose of PBMC causing graft failure, was associated with engraftment in four of six dogs. Infusion of 0.13 x 10(8) PBMC/kg treated with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), a drug that depletes canine cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, and monocytes, permitted engraftment in three of four dogs. These data suggest that cytotoxic lymphocytes mediate failure of histoincompatible marrow grafts.     

Facilitation of engraftment of DLA-nonidentical marrow by treatment of recipients with monoclonal antibody directed against marrow cells surviving radiation

Past studies in dogs have suggested that marrow graft rejection was mediated by major histocompatibility complex (MHC) class II antigen-positive non-T cells that survived standard doses of total-body irradiation (TBI). We have now raised 4 monoclonal antibodies (mAbs) against marrow cells harvested 6 days after TBI. The mAbs are highly reactive (greater than 70%) with marrow cells surviving radiation and also bind strongly (greater than 50%) to normal marrow cells, lymphocytes, monocytes, and granulocytes. One of the mAbs (34-S3) reacted strongly with NK-like cells. In vitro treatment of marrow with mAb and rabbit complement (C') did not affect erythroid colony-forming unit (CFU-E) growth, whereas 2 of the 4 mAbs inhibited granulocyte-macrophage colony-forming unit (CFU-GM) growth, and all 3 mAbs tested suppressed autologous marrow engraftment. One of the mAbs, 69-S5 (IgG1), bound to a 95,000 dalton antigen. It crossreacted with human cells, but not with cells from Rhesus monkeys, baboons, and cats. We administered this mAb intravenously at 0.2 mg/kg/day on days -5 to 0 to dogs given 9.2 Gy TBI on day 0 followed by marrow grafts (less than or equal to 4 x 10(8) cells/kg) from DLA-nonidentical unrelated donors. Three of five dogs had sustained grafts. Increasing the dose of mAb ten-fold (2 mg/kg/day) resulted in graft failure (2 of 2 dogs). Treatment with a dose of 0.2 mg/kg/day from day -7 to -2 showed sustained engraftment in 7 of 10 dogs. This result is in contrast to sustained grafts in 3 of 36 dogs not given mAb, and in 1 of 7 dogs treated with an irrelevant mAb (P = 0.0002 and 0.04, respectively). We conclude that treatment of recipients with a mAb raised against marrow cells surviving radiation and not directed at major histocompatibility complex (MHC) class II antigens and NK-like cells can also facilitate engraftment of DLA-nonidentical canine marrow. These results may be relevant for the transplantation of HLA-incompatible marrow in man, particularly after in vivo T cell depletion, where graft failure is frequent.     

In vivo radioprotective effect of AcSDKP on canine myelopoiesis

 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. Received: 9 October 1996 / Accepted: 18 December 1996     

Gene therapy with drug resistance genes

A major side effect of cancer chemotherapy is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner.     

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.