Effects of in vivo neuraminidase on the regulation of erythropoiesis. II: Modulation of erythroid colony formation by thymic regulatory cells

Effects of the enzyme vibrio-cholerae neuraminidase (VCN) on the marrow-derived erythropoietic progenitor CFU-E and thymic regulatory cells were examined in vitro 1 and 24 h after i.v. injection of the enzyme. An in vivo enzymatic modification of bone marrow and thymic helper regulatory cell function occurs within 1 h after i.v. injection of VCN and results in suppression of both CFU-E colony formation and thymic helper cell function. These inhibitory effects of neuraminidase, however, are no longer detectable by 24 h after injection. More importantly, these inhibitory effects can be reversed by adding thymocytes from control animals to cocultures of enzymatically modified marrow or thymic regulatory cells. These findings: 1) suggest that regulatory cells from the bone marrow and thymus may be enzymatically modified in vivo in a reversible manner, suggesting a noncytotoxic effect of the enzyme on accessory cells, and 2) confirm the importance of sialic acid for the helper function but not for the suppressor function of thymocytes and CFU-E colony formation in vitro.     

Engraftment of bone marrow transplants in W anemic mice measured by electronic determination of the red blood cell size profile.

Defective stem cells of WBB6F1-W/Wv mice produce macrocytic red blood cells (RBCs); stem cells of WBB6F1-+/+ mice produce normocytic RBCs. Utilization of the Coulter counter channelyzer permitted good dissociation between the size distribution of populations of +/+ and W/Wv RBCs. Peaks (mean cell volumes) for +/+ and W/Wv RBCs have been determined to be between the 30th and 40th channel and 50th and 60th channel, respectively. Variability of profiles for individual mice of both genotypes did not exceed the variability of separate determinations of the same cell suspension from a single mouse. Admixture (approximately 15%) of either type of erythrocytes could be quantitatively detected by this method. One week after transplant of 10(7) +/+ marrow cells into W/Wv recipients, 25% of donor type erythrocytes were detected. Eighteen days post-graft, concentration of +/- normocytes exceeded the concentration of macrocytes in the W/Wv recipients' circulation. Approximately 45 days post-transplant, the proportion of macrocytes decreased below the 10% detectable level. Calculation of the daily RBC production rate during repopulation and estimation of the number of RBCs produced by a single hematopoietic colony were determined. The RBC size profile was found to be a convenient method for studying the effect of implantation of W/Wv marrow into lethally irradiated +/+ mice. This method proved suitable for repetitive determination of the size population in individual transplanted mice.     

Polyamine biosynthesis is required for the maintenance of peripheral blood cell elements in the rat.

The specific ornithine decarboxylase inhibitor alpha-difluoromethylornithine, when given to adult rats in vivo for 5 wk, resulted in a decrease in peripheral blood cell elements in normal rats and a marked suppression of marrow recovery in rats with chemotherapy-induced marrow hypoplasia. In normal rats, alpha-difluoromethylornithine resulted in a reduction of the leukocyte count to 73% of control, erythrocyte count to 61% of control, and platelet count to 24% of control. The bleeding time was increased to twice normal and 67% of the animals had epistaxis and 42% had melena. In rats treated with the S phase-specific chemotherapeutic agent 1-beta-D-arabinofuranosylcytosine, the simultaneous administration of alpha-difluoromethylornithine prevented the recovery of the bone marrow. The peripheral blood cell counts remained low--leukocyte count was 10% of control, and erythrocyte and platelet counts were 6% of control. All the animals developed epistaxis and melena and there was a 72% mortality. The administration of putrescine (4 mmol/kg, intraperitoneally, daily), the specific polyamine product of ornithine decarboxylase, reversed these hematologic effects in both normal and recovering marrow and resulted in rapid clinical improvement. Thus, the maintenance of normal, adult rat hematologic parameters, as with the proliferation of neoplastic and transformed cells in culture, is critically dependent on continued polyamine biosynthesis.     

Inactivation of Friend erythroleukemia virus and Friend virus- transformed cells by merocyanine 540-mediated photosensitization

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus- transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.     

Elimination of acute myelogenous leukemic cells from marrow and tumor suspensions in the rat with 4-hydroperoxycyclophosphamide

Cell suspensions of normal rat marrow mixed with rat acute myelogenous leukemic cells were prepared and incubated in vitro with graded doses of 4-hydroperoxycyclophosphamide (4HC). The cell suspensions were injected into rats prepared with a lethal dose of total body irradiation. Animals injected with these cells survived fatal irradiation induced aplasia. In a dose related manner 4HC was able to "purge" tumor cells from the cell mixtures. Thus, animals given cell suspensions incubated with the lower doses of 4HC showed prolonged survival before death from leukemia and animals given cell suspensions incubated with higher doses of 4HC survival lethal irradiation without the subsequent appearance of leukemia. These studies clearly establish that tumor cells may be eliminated from normal marrow suspensions without completely destroying the pluripotent stem cells.     

Antineoplastic bryostatins are multipotential stimulators of human hematopoietic progenitor cells.

The bryostatins are macrocyclic lactones, extracted from the marine bryozoan Bugula neritina, and have been reported to be potent antineoplastic agents. Results described here demonstrate that the bryostatins may also be useful as stimulators of normal human hematopoietic cells since they can (i) directly stimulate bone marrow progenitor cells to form colonies in vitro and (ii) functionally activate neutrophils. Structure-activity studies with bryostatin congeners indicate that these stimulatory properties may be dependent on the chain length and the unsaturated nature of the acylated group at carbons 20 and 7 of the bryostatin molecule. These stimulatory properties demonstrate that the naturally occurring bryostatins can mimic many of the biological effects of multipotential granulocyte-macrophage colony-stimulating factor. Thus, the coupling of antineoplastic activity with stimulatory growth properties for normal hematopoietic cells makes this agent an excellent probe to dissect the mechanism(s) of normal hematopoiesis. In addition, bryostatin may represent a clinically attractive agent useful for treating bone marrow failure states.     

Regulation of hematopoiesis-IV: The role of interleukin-3 and bryostatin 1 in the growth of erythropoietic progenitors from normal and anemic W/Wv mice

We and others have established a role for T lymphocytes and their products in the regulation of erythropoiesis. Interleukin-3 (IL-3) is a multipotential lymphokine with burst-promoting activity that is produced by activated T lymphocytes. In the anemic, stem cell-defective W/Wv mouse we have described the absence of a functionally active thymocyte population that in normal animals enhances erythroid progenitor growth and stem cell self-renewal. In studies reported here we find that W/Wv mouse marrow responds to exogenous IL-3 by increased erythroid progenitor cell growth. The BFU-E and CFU-E from anemic donors are more sensitive to IL-3 than are those in +/+ marrow. We have recently observed a stimulatory effect of bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate) on normal erythropoiesis in human bone marrow progenitor assays. To test the effects of this molecule on murine normal and anemic W/Wv cells we grew these cells in the presence of increasing doses of bryostatin 1. Bryostatin mimics the stimulatory action of IL-3 on W/Wv bone marrow. Polyclonal antibody directed against murine IL-3 blocks the stimulatory effect of bryostatin on erythropoiesis. Otherwise inactive thymocytes from W/Wv mice in coculture with W/Wv bone marrow showed stimulation of erythropoiesis in the presence of bryostatin. We believe that bryostatin may in part act by stimulating T lymphocytes to release physiologic concentrations of lymphokines.