A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

Mechanism of canine cyclic hematopoiesis: the role of prostaglandin E in feedback regulation

Prostaglandin E inhibits granulocyte-macrophage colony formation in vitro in man and mouse, suggesting that it plays a role in feedback regulation of granulocyte production in vivo. Therefore, we examined the role of PGE in normal canine hematopoiesis and its potential role in the pathogenesis of cyclic hematopoiesis in grey collie dogs. The prostaglandin synthesis inhibitors indomethacin and ibuprofen (10(-5) M) increased CFU-C growth to 194 and 160% of control, respectively, while PGE2 addition caused a dose-dependent inhibition of bone marrow CFU-C growth in both normal and grey collie dogs. These concentrations of indomethacin and ibuprofen decreased bone marrow cell elaboration of PGE measured by radioimmunoassay to less than 5% of control values. The levels of PGE in leukocyte conditioned medium prepared from grey collies correlated with the number of monocytes in the conditioning cell suspension (r = 0.78, n = 10, p less than 0.05) so that PGE production per monocyte was no different in normal and grey collie dogs. The effect of PGE2 upon CFU-C was to inhibit formation of macrophage, but not neutrophil colony subtypes. These findings make aberrant PGE-mediated inhibition of precursor cells an unlikely mechanism to cause cyclic hematopoiesis, and show that PGE produced by monocytes acts as a feedback inhibitor for precursor cells destined to produce monocytes but not for those destined to form neutrophils.     

Lithium augments GM-CSA generation in canine cyclic hematopoiesis

Cyclic hematopoiesis in gray collie dogs can be cured by lithium treatment. We examined the mechanism of lithium's effect by developing an assay for the canine equivalent of GM-CSF (called GM-CSA). Phytohemagglutinin (PHA)-stimulated canine blood mononuclear cells produce GM-CSA in a dose-dependent manner; this GM-CSA stimulates more neutrophil-containing colonies than does endotoxin-treated dog serum. Production of GM-CSA by PHA-stimulated normal dog cells was not altered by lithium. However, cells from gray collies during their neutrophilic period increased their GM-CSA when lithium (2 mEq/L) was added to low doses of PHA, whereas neutropenic gray collie cells did not. These data suggest that lithium could modulate cyclic hematopoiesis by increasing intramedullary GM-CSA at the time when marrow neutrophilic progenitor cells are at their nadir.     

Canine cyclic haematopoiesis: the effect of endotoxin on erythropoiesis

We have examined the effects of chronic endotoxin treatment on erythropoiesis in six grey collies with cyclic haematopoiesis. Blood reticulocytes, bone marrow erythroid colony (EC) forming cells, serum iron and erythropoietin (ESF) values showed regular periodic fluctuations in untreated grey collies. Daily endotoxin injections eliminated the cyclic fluctuations of reticulocytes and EC. The mean serum iron values were increased and recurrent hypoferraemia eliminated, while the mean serum ESF values were reduced. The cyclic fluctuations of serum ESF values were no longer apparent in the endotoxin treated grey collies. Tritiated thymidine suicide of the marrow EC forming cells failed to show cyclic changes either in untreated or endotoxin treated dogs. The ESF sensitivity of EC in the grey collie was unchanged during endotoxin treatment and was not different from normal dogs. Endotoxin appears to alter periodic erythropoiesis by stabilizing the flux of cells into the committed erythroid precursor cell pool from a more primitive stem cell compartment.     

A c-kit ligand, recombinant human stem cell factor, mediates reversible expansion of multiple CD34+ colony-forming cell types in blood and marrow of baboons.

The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte-monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.     

Effects of recombinant canine stem cell factor, a c-kit ligand, and recombinant granulocyte colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

The effects of recombinant canine stem cell factor (rcSCF) on hematopoiesis were studied in normal dogs and in dogs given otherwise lethal total body irradiation (TBI) without marrow transplant. Results were compared with previous and concurrent data with recombinant granulocyte colony-stimulating factor (rG-CSF). Four normal dogs received 200 micrograms rcSCF per kilogram body weight daily either by continuous intravenous infusion for 28 days (n = 2) or by subcutaneous (SC) injection in two divided doses for 20 days (n = 2). All dogs showed at least a twofold increase in peripheral blood neutrophil counts starting approximately 7 days after the initiation of treatment. Hematocrit level and monocyte, lymphocyte, eosinophil, reticulocyte, and platelet counts were not elevated. Marrow sections after rcSCF treatment showed panhyperplasia. The only toxicity was facial edema during the first few days of rcSCF administration, presumably caused by mast cell stimulation. Ten dogs were given 400 cGy TBI at 10 cGy/min from two opposing 60Co sources. They were given no marrow infusion and received 200 micrograms/kg/d rcSCF SC in two divided doses for 21 days starting within 2 hours of TBI. Five of the 10 dogs showed complete and sustained hematopoietic recovery and survived as compared with 1 of 28 control dogs not receiving growth factor (P < .005). RcSCF treatment allowed for hematopoietic recovery in two of seven dogs administered 500 cGy of TBI but in none of five dogs given 600 cGy of TBI. Results with rcSCF are similar to those obtained with rG-CSF. The rate of neutrophil recovery in rcSCF-treated dogs after 400 cGy TBI was not different from that of rG-CSF-treated dogs (P = .65), but the rate of platelet recovery was faster (P = .06) in the rcSCF-treated animals. Combined treatment with rcSCF and rcG-CSF after 500 cGy TBI did not result in strongly improved survival as compared with results obtained with either factor alone.     

DLA-identical bone marrow grafts after low-dose total body irradiation: the effect of canine recombinant hematopoietic growth factors

Previous studies found that bone marrow (BM) allografts from DLA- identical littermates resulted in survival of two thirds of recipient dogs after otherwise lethal doses of 450 to 600 cGy of total body irradiation (TBI) because of successful allografts or autologous recovery after rejection of the allografts. The current study asked whether survival could be further improved by treating allograft recipients with recombinant canine granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), or G-CSF/SCF. Of 21 dogs, 14 (67%) receiving allografts but no growth factors survived, 10 with successful allografts (including 5 mixed chimeras) and 4 with autologous recovery; whereas 7 animals died, 5 from infections during BM aplasia and 2 from acute graft-versus-host disease. By comparison, 30 of 34 dogs (88%) receiving hematopoietic growth factors in addition to the BM graft survived, 17 with successful allografts (including 10 mixed chimeras) and 13 with autologous recovery; whereas 4 died, all with infection related to BM aplasia after rejection of the allograft. Survival was similar for recipients of G-CSF, SCF, or the combination of G-CSF and SCF. Logistic regression analyses, which accounted for possible effects of TBI dose, showed a trend for improved survival in dogs receiving growth factors (P = .09), no change in allogeneic engraftment (P = .74), and a slight increase in autologous recovery (P = .22). In agreement with previous data, we found that grafts of BM from DLA-identical littermates improved survival of recipient dogs exposed to low but otherwise lethal doses of TBI. A further improvement in survival could be achieved by additional treatment with G-CSF, SCF, or G-CSF/SCF. Results suggest that treatment by hematopoietic growth factors along with BM grafts should be considered for victims of radiation accidents.     

Effects of granulocyte colony-stimulating factor and stem cell factor, alone and in combination, on the mobilization of peripheral blood cells that engraft lethally irradiated dogs

The effects of recombinant canine granulocyte colony-stimulating factor (rcG-CSF) and recombinant canine stem cell factor (rcSCF), a c-kit ligand, on the circulation of hematopoietic progenitor and stem cells were studied in a canine model. Administration of rcG-CSF (10 micrograms/kg) for 7 days led to a 5.4-fold increase in CFU-GM/mL of blood, while 7 days of rcSCF (200 micrograms/kg) led to an 8.2-fold increase. Although treatment with low-dose rcSCF (25 micrograms/kg) had no effect on the level of peripheral blood progenitors, 7-day exposure to a combination of G-CSF plus low dose SCF led to a 21.6-fold increase (P = .03). To assess the ability of these factors to increase the circulation of cells capable of rescuing animals after lethal total body irradiation (TBI), 1 x 10(8) peripheral blood mononuclear cells (PBMC)/kg were collected and cryopreserved from animals after 7 days of treatment with G-CSF, SCF or a combination of the two. One month later, animals were exposed to 9.2 Gy TBI and transplanted with the previously collected cells. Control animals transplanted with 1 x 10(8) PBMC/kg collected without pretreatment died with marrow aplasia 11 to 29 days after TBI as did animals treated with only low-dose SCF before cell collection. In contrast, all animals given PBMC collected after G-CSF, high-dose SCF, or a combination of G-CSF plus low-dose SCF recovered granulocyte function. Recovery to 500 granulocytes/microL after transplant took 17, 18.8, and 13.6 days, respectively, (P = .056 for the difference between the combination G-CSF-SCF group and the other two groups). In both the G-CSF and SCF groups, 4 of 5 animals completely recovered while 1 of 5 in each group died with prolonged thrombocytopenia. In the combination group, all 5 animals became long- term survivors. These studies demonstrate that both G-CSF and SCF dramatically increase the level of peripheral blood hematopoietic progenitor and stem cells and support the view that these factors can act synergistically.     

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol

The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.     

Defective polymorphonuclear leukocyte metabolism and function in canine cyclic neutropenia.

Humans and grey collie dogs with cyclic neutropenia are known to suffer from an increased rate of bacterial infection. Because of the previously described microanatomic abnormalities of lysosome formation found in the polymorphonuclear leukocytes (PMNs) of dogs with canine cyclic neutropenia, studies of these cells were undertaken. PMNs from grey collie dogs were found to have significant metabolic and functional abnormalities when compared with normal collie PMNs. These included abnormally increased postphagocytic C1-glucose oxidation, decreased iodination of trichloroacetic acid-precipitable protein in the resting and phagocytizing state, decreased levels of intracellular myeloperoxidase,and a bactericidal defect against a variety of bacteria. Phagocytosis was normal. These abnormalities appear to differ from those previously described in the PMNs of patients with chronic granulomatous disease of childhood and the Chediak-Higashi syndrome and more closely resemble those seen in hereditary myeloperoxidase deficiency. Thus, the studies reported here demonstrate defective PMN function in a disease state previously believed to be a model only of periodic hematopoiesis.     

Effect of recombinant canine granulocyte-macrophage colony-stimulating factor on hematopoietic recovery after otherwise lethal total body irradiation

Recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) was studied in normal dogs and in dogs receiving otherwise lethal total body irradiation (TBI) without marrow transplant. Five normal dogs receiving 25 micrograms/kg of rcGM-CSF by subcutaneous (SC) injection twice daily (BID) for 14 days showed increases in peripheral blood neutrophil counts of three to five times the baseline. Platelet counts decreased during administration of rcGM-CSF to a mean nadir of 52,800. Ten dogs received 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow graft. Within 2 hours of TBI, rcGM-CSF was begun at a dose of 50 micrograms/kg SC BID for 5 doses and then continued at 25 micrograms/kg SC BID for 21 days. Only 1 of the 10 dogs receiving rcGM-CSF survived with complete and sustained recovery of hematopoiesis. One of 13 historical control dogs survived after 400 cGy with no hematopoietic growth factor or marrow infusion. Results with rcGM-CSF were compared with previous and concurrent data with G-CSF studied in the same model. Of 10 dogs receiving G-CSF, 8 survived with complete and sustained hematopoietic recovery, a significantly better survival than that seen with rcGM-CSF (P = .006). Neutrophil counts were sustained at higher levels after TBI for the first 18 days in the G-CSF group (P < .016) and the neutrophil nadirs were higher. No differences in neutrophil nadirs were noted between the rcGM-CSF and control groups. Dogs treated with rcGM-CSF experienced a more rapid decline of platelet counts than G-CSF-treated or control dogs over the first 18 days (P < .001). The nadir of the platelet count was higher in the control group than in either the G-CSF or rcGM-CSF group and no significant difference was observed between the G-CSF and rcGM-CSF groups. After otherwise lethal TBI (400 cGy) in dogs, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.     

Effects of recombinant granulocyte colony-stimulating factor treatment on hematopoietic cycles and cellular defects associated with canine cyclic hematopoiesis.

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by 14-day cycles of neutropenia, monocytosis, thrombocytosis, and reticulocytosis. Platelets from CH dogs have decreased dense-granule serotonin pools and decreased aggregation responses to collagen, platelet-activating factor (PAF), and thrombin. Recombinant granulocyte colony-stimulating factor (rG-CSF) was administered (5 micrograms/kg, b.i.d.) to four CH and six normal dogs to determine if G-CSF therapy corrected qualitative platelet defects in CH dogs. Neutrophil counts increase to greater than 25,000 cells/microliters within 24 h after starting treatment in all dogs. Treatment with G-CSF blocked neutropenic episodes in the CH dogs. Platelet aggregation, and serotonin content and secretion were significantly (p less than 0.05) decreased in the CH dogs both before and during recombinant human (rh) G-CSF treatment compared to normal dogs. Neutrophil myeloperoxidase, a primary granule enzyme, was significantly (p less than 0.05) decreased in CH dogs and was not corrected by rhG-CSF treatment. Administration of rG-CSF to CH dogs eliminated cell cycles but apparently did not correct cellular defects in CH dogs. Identification of primary biochemical defects in cells from CH dogs may be crucial to investigating the biochemical basis for cyclic hematopoiesis.     

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.     

Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential

Splenectomized mice treated for 7 days with pegylated recombinant rat stem cell factor (rrSCF-PEG) showed a dose-dependent increase in peripheral blood progenitor cells (PBPC) that have enhanced in vivo repopulating potential. A dose of rrSCF-PEG at 25 micrograms/kg/d for 7 days produced no significant increase in PBPC. However, when this dose of rrSCF-PEG was combined with an optimal dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 200 micrograms/kg/d), a synergistic increase in PBPC was observed. Compared with treatment with rhG-CSF alone, the combination of rrSCF-PEG plus rhG-CSF resulted in a synergistic increase in peripheral white blood cells, in the incidence and absolute numbers of PBPC, and in the incidence and absolute numbers of circulating cells with in vivo repopulating potential. These data suggest that low doses of SCF, which would have minimal, if any, effects in vivo, can synergize with optimal doses of rhG-CSF to enhance the mobilization of PBPC stimulated by rhG-CSF alone.     

Correction of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by neutropenic episodes at 14- day intervals. The biochemical basis for CH is not known but may involve a regulatory defect of the response to or production of a hematopoietic growth factor. Administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to two CH and one normal dog caused a marked leukocytosis (greater than 50,000 WBCs) in all three dogs. The leukocytosis was due largely to a greater than tenfold increase in neutrophils. Less pronounced but significant elevations in monocytes occurred during G-CSF treatment. The elevated WBC count was maintained for more than 20 days in all three dogs, and two predicted neutropenic episodes were prevented in both CH dogs during rhG-CSF treatment. A decline in the WBC count occurred simultaneously in all three dogs during the last five treatment days and was presumably associated with the development of neutralizing antibodies to the heterologous rhG-CSF protein. Bone marrow evaluation indicated that the swings in the myeloid/erythroid progenitor cells that are characteristic of CH were eliminated by rhG-CSF treatment in both CH dogs. These results suggest that the regulatory defect in canine CH can be temporarily alleviated by treatment with rhG-CSF and point to the potential treatment of human cyclic neutropenia with this agent.     

Acceleration of recombinant tissue-type plasminogen activator-induced reperfusion and prevention of reocclusion by recombinant antistasin, a selective factor Xa inhibitor, in a canine model of femoral arterial thrombosis.

Antistasin is a 119-amino acid protein initially isolated from salivary glands of the Mexican leech, Haementeria officinalis, that exhibits potent anticoagulant properties resulting from selective inhibition of blood coagulation factor Xa. The comparative antithrombotic efficacies of recombinant antistasin (rATS), standard heparin (Hep), and aspirin (ASA) administered adjunctly with recombinant tissue-type plasminogen activator (tPA) on thrombolytic reperfusion and reocclusion were determined in a canine model of femoral arterial thrombosis. An occlusive thrombus was formed by insertion of a thrombogenic copper coil into the femoral artery, and blood flow velocity was monitored directly and continuously by Doppler flowmetry. Sixty minutes after occlusion, dogs received an intravenous infusion of either saline (vehicle) or rATS (0.31, 1.25, or 2.5 micrograms/kg/min), intravenous boluses of Hep (100 units/kg + 50 units/kg/hr or 200 units/kg + 150 units/kg/hr), or a single intravenous bolus of ASA (2.0 mg/kg), followed 45 minutes later by tPA (0.8 mg/kg i.v. over 90 minutes). The saline and rATS infusions were discontinued 60 minutes after termination of tPA, and the last Hep boluses were given 105 minutes after termination of tPA. All dogs achieved reperfusion. The time to reperfusion in the ASA group was similar to that in the vehicle group (50 +/- 9 versus 50 +/- 6 minutes, respectively). Reperfusion times were slightly decreased by the low and high doses of Hep (34 +/- 6 and 31 +/- 4 minutes, respectively) and the rATS doses of 0.31 and 1.25 micrograms/kg/min (37 +/- 4 and 36 +/- 5 minutes, respectively). However, the time to reperfusion was dramatically reduced with the 2.5 micrograms/kg/min rATS dose (15 +/- 3 minutes, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Allogenic transplant of canine peripheral blood stem cells mobilized by recombinant canine hematopoietic growth factors

We have studied graft-versus-host disease (GVHD) after transplantation of allogeneic peripheral blood stem cells (PBSC) mobilized by either recombinant canine granulocyte colony-stimulating factor (rcG-CSF) alone or combined with stem cell factor (rcSCF). These studies were prompted by the observation of extremely rapid and sustained engraftment of growth factor-mobilized PBSC in the autologous setting using genetically marked cells and changes in function of T lymphocytes from donors that had undergone mobilization. Specifically, lymphocytes from growth factor-treated donors were hyporesponsive in mixed leukocyte culture and in response to Con A, raising hopes that GVHD in dogs given growth factor mobilized allogenic PBSC might be altered in a beneficial way. Eighteen dogs were given a median of 17.1 x 10(8) PBSC/kg from littermate donors after 920 cGy of total body irradiation without postgrafting immunosuppression. Donors were either genotypically DLA-identical (n = 9) or DLA-haploidentical (n = 9). The median number of colony-forming unit-granulocyte macrophage (CFU-GM) infused was 27 x 10(4)/kg, and the number of CD34+ cells in the transplant was on the order of 4.6 x 10(6)/kg. The dogs received a median of 52.8 x 10(7) CD4 cells/kg and 13.7 X 10(7) CD8 cells/kg. All 18 dogs had prompt hematopoietic engraftment of donor cells as assessed by chimerism studies using variable number tandem repeat, as well as cytogenetic markers. Three of the nine dogs given grafts from DLA- identical littermates had fatal GVHD, five had transient GVHD, and one had no GVHD. All nine DLA-haploidentical recipients of PBSC developed fatal hyperacute GVHD. In conclusion, the expectation about rapid engraftment was fulfilled. However, incidence and severity of acute GVHD after transplantation of mobilized PBSC were not different than previously reported for nonmobilized PBSC or marrow. This model will allow for further studies, including T-cell depletion to minimize GVHD without increasing graft rejection.     

Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis of normal dogs and on hematopoietic recovery after otherwise lethal total body irradiation

This study was designed to test whether recombinant human G-CSF (rh G-CSF) affects hematopoiesis in normal dogs and, if so, to test the effects of G-CSF in dogs given otherwise lethal total body irradiation (TBI). Rh G-CSF given subcutaneously at 10 or 100 micrograms/kg/d for 14 days to two normal dogs increased peripheral blood neutrophils eight to tenfold and monocytes four to sixfold above controls. Lymphocyte counts remained unchanged at the lower dose and increased threefold at the higher dose of rh G-CSF. No significant changes were observed in eosinophil, platelet, reticulocyte, or hematocrit levels. After 2 weeks of treatment with rh G-CSF, bone marrow displayed myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rh G-CSF, peripheral leukocyte counts returned to control levels within three days. Five dogs administered 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow infusion or growth factor, all developed profound pancytopenia and died between 17 and 23 days after TBI with infections secondary to marrow aplasia. Four of five dogs treated within two hours after 400 cGy TBI with 100 micrograms rh G-CSF/kg/d subcutaneously twice a day for 21 days showed complete and sustained endogenous hematopoietic recovery. In contrast, five dogs irradiated with 400 cGy TBI and treated with 100 micrograms rh G-CSF/kg/d starting on day 7 after TBI, all died between days 17 and 20 after TBI with infections secondary to marrow aplasia. Rh G-CSF, if administered shortly after irradiation, can reverse the otherwise lethal myelosuppressive effect of radiation exposure.     

Bromocriptine treatment of digitalis-induced ventricular tachyarrhythmias: studies in a canine model

Ventricular tachyarrhythmias associated with digitalis toxicity are believed to be due, in part, to cardiac glycoside-mediated increased central sympathetic neural activity. Because dopaminergic receptor agonists reduce sympathetic outflow, this study assessed effectiveness of the available dopaminergic agonist, bromocriptine, in slowing or terminating ouabain-induced ventricular tachycardia in anesthetized dogs. In all experiments, ouabain was administered intravenously (20 micrograms/kg body weight bolus injection, followed by 2.5 micrograms/kg per min infusion) until the onset of stable ventricular tachycardia. Of seven untreated dogs (Group 1), ouabain-induced ventricular tachyarrhythmias resulted in ventricular fibrillation in three, while in four dogs tachycardia persisted without significant change in rate until the study was terminated. Fourteen dogs (Group 2) received bromocriptine, either 30 micrograms/kg (Group 2A) or 50 micrograms/kg (Group 2B), after the onset of ventricular tachycardia. Tachycardia slowed in all 14 dogs and terminated with resumption of sinus rhythm in 8 of the 14. In all six dogs pretreated with the peripheral dopaminergic antagonist domperidone (Group 3), bromocriptine, 50 micrograms/kg, slowed ventricular tachycardia and in three of the six, tachycardia terminated. In contrast, of five dogs pretreated with haloperidol, a central and peripheral dopaminergic receptor antagonist (Group 4), bromocriptine, 50 micrograms/kg, failed to slow ventricular tachycardia in three, and two of the three developed ventricular fibrillation. In summary, the dopaminergic receptor agonist, bromocriptine, presumably acting at central dopaminergic receptor sites, consistently slowed and in most cases reversed ouabain-induced ventricular tachycardia in a canine model.     

The effects of SCF/G-CSF prestimulation on radiation sensitivity and engraftment in nonmyeloablated murine hosts

OBJECTIVE: Previous studies have shown improved engraftment in a murine model when granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) were administered for 5 days prior to irradiation, with significant levels of engraftment in the growth factor-preconditioned group even at very low radiation doses. We sought to explore the mechanisms behind this effect. METHODS: The radiation sensitivity of mice with or without 5 days of prestimulation with G-CSF (200 microg/kg/d) and SCF (50 microg/kg/d) was compared. To further evaluate whether growth factor prestimulation enhances engraftment by mobilization of hematopoietic progenitors into peripheral blood, thus creating less endogenous competition within the marrow compartment, female mice were pretreated with 5 days of G-CSF/SCF or control diluent. Engraftment of 40 x 10(6) peripheral blood stem cells (PBSCs) harvested from G-CSF/SCF-mobilized male mice was compared in the two recipient groups. RESULTS: There was no difference in survival between the pretreated and control mice at the radiation doses tested. Additionally, there was no significant difference in the recovery of blood counts, bone marrow cellularity, colony-forming unit (CFU) content, or stem cell numbers assessed 4 months later in a competitive repopulation model. Engraftment levels of male cells did not differ between G-CSF/SCF-pretreated and control recipients, and could be detected in 30% of recipients at 20-24 weeks (4/12 in each group) at overall levels of 0.1-1%. CONCLUSIONS: The enhanced engraftment in cytokine pretreated recipients is unlikely to be due to increased endogenous stem-cell killing or to the creation of endogenous marrow "space" by egress of endogenous stem cells after cytokine prestimulation.