Short-term treatment with novel ribonucleotide reductase inhibitors Trimidox and Didox reverses late-stage murine retrovirus-induced lymphoproliferative disease with less bone marrow toxicity than hydroxyurea.

We evaluated the ability of a short course of treatment with the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) and two novel RR inhibitors Trimidox (TX) and Didox (DX) to influence late-stage murine retrovirus-induced lymphoproliferative disease. LPBM5 murine leukaemia virus retrovirus-infected mice were treated daily with HU, TX or DX for 4 weeks, beginning 9 weeks post-infection, after development of immunodeficiency and lymphoproliferative disease. Drug effects on disease progression were determined by evaluating spleen weight and histology. Effects on haematopoiesis were determined by measuring peripheral blood indices (white blood cells and haematocrit) and assay of femur cellularity and femoral and splenic content of colony-forming units granulocyte-macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E). HU, TX and DX partially reversed late-stage retrovirus-induced disease, resulting in spleen weights significantly below pre-treatment values. Spleen histology was also improved by RR inhibitor treatment (DX>TX>HU). However, as expected, HU was significantly myelosuppressive, inducing a reduction in peripheral indices associated with depletion of femoral CFU-GM and BFU-E. In contrast, although TX and DX were moderately myelosuppressive, both drugs were significantly better tolerated than HU. In summary, short-term treatment in late-stage murine retroviral disease with HU, TX or DX induced dramatic reversal of disease pathophysiology. However, the novel RR inhibitors TX and DX had more effective activity and significantly less bone marrow toxicity than HU.

Cerebral excitability and the mechanisms of blood transportation of sodium.

The basic, fundamental property of living structures is excitability. This process defines how an organism responds to both internal and external stimuli. Previous studies have indicated the existence of physical and chemical interactions between cations and anions sites of proteins within the extracellular environment that have a specific functional importance. However, it is not well understood whether specific cations may alter the function of specific proteins. We report here the results of studies that indicate interaction of specific cations such as sodium may alter the physico-chemical action of heparin. The importance of these interactions is discussed.

Low dose fractionated radiation enhances the radiosensitization effect of paclitaxel in colorectal tumor cells with mutant p53.

BACKGROUND: The current study was undertaken to investigate the influence of wild-type or mutant p53 status on the radiosensitizing effect of paclitaxel in colorectal tumor cell lines. METHODS: HCT-116 (contains wild-type p53) and HT-29 (contains mutant p53) established from moderately differentiated colorectal carcinomas were used in this study. Colony-forming assay was performed after exposure to either different radiation doses (0.5-6 gray [Gy]) or paclitaxel (1-10 nM) or in combination. Induction of p53 and p21(waf1/cip1) by these treatments were determined by immunocytochemistry and Western blot analysis. RESULTS: Radiation caused an increase in nuclear p53 and p21(waf1/cip1) proteins in HCT-116 cells, indicating that p53 functionally induced p21(waf1/cip1). However, induction of nuclear p53 and p21(waf1/cip1) protein was not evident in HT-29 cells, suggesting that p53 was not functional in these cells. Survival data showed that the HCT-116 cells (survival fraction of exponentially growing cells that were irradiated at the clinically relevant dose of 2 Gy [SF(2)] = 0.383; dose required to reduce the fraction of cells to 37% [D(0)] = 223 centigray [cGy]) were significantly sensitive to ionizing radiation (P < 0.008) when compared with the HT-29 cells (SF(2) = 0.614; D(0) = 351 cGy). Paclitaxel caused a higher degree of clonogenic inhibition in HCT-116 (D(0) = 0.7 nM) than HT-29 (D(0) = 1.11 nM) cells (P < 0.06). When paclitaxel and radiation were combined, an enhanced radiosensitizing effect (P < 0.05) was observed in HCT-116 cells (SF(2) = 0.138; D(0) = 103 cGy), whereas in HT-29 cells no significant radiosensitization of paclitaxel was observed (SF(2) = 0.608; D(0) = 306 cGy). However, pretreatment with paclitaxel followed by multifractionated low dose radiation (0.5- or 1-Gy fractions for a total dose of 2 Gy) significantly enhanced the radiosensitizing effect in both HCT-116 and HT-29 cells. CONCLUSIONS: The results of the current study suggested that multifractionated radiation given at very low doses after exposure of cells to paclitaxel conferred a potent radiation sensitizing effect irrespective of p53 status.

In vivo and in vitro comparison of the short-term hematopoietic toxicity between hydroxyurea and trimidox or didox, novel ribonucleotide reductase inhibitors with potential anti-HIV-1 activity.

Inhibitors of the cellular enzyme ribonucleotide reductase (hydroxyurea, [HU]) have been proposed as a new therapeutic strategy for the treatment of HIV type-1 (HIV-1) infection. However, HU use may be limited by the frequent development of hematopoietic toxicity. We report here short-term hematopoietic toxicity in mice receiving HU when compared to either of two more potent enzyme inhibitors, didox (DX) and trimidox (TX). High dose HU, DX, and TX monotherapy (500, 460, and 220 mg/kg/day respectively) was administered by daily i.p. injection (Monday-Friday) to C57BL/6 mice for 10 weeks. Effects on hematopoiesis were established by quantitating peripheral blood indices (hematocrit, hemoglobin, mean corpuscular volume, mean cell hemoglobin, mean corpuscular hemoglobin concentration, RBC, and WBC) and numbers of colony-forming units-granulocyte-macrophage (CFU-GM) and BFU-E from bone marrow and spleen. HU produced rapid induction of a macrocytic hypochromic anemia and altered white blood cell kinetics associated with myelosuppression defined as reduced marrow organ cellularity and induction of splenic extramedullary hematopoiesis. Compared to HU, TX and DX induced fewer changes in peripheral blood indices and CFU-GM and BFU-E per hematopoietic organ. In vitro human and murine marrow CFU-GM and BFU-E colony formations were assayed in the presence of dose escalation HU, DX, or TX (0, 1, 10, 50, 100, and 200 microM). HU inhibited colony formation more than either DX or TX. These in vivo and in vitro studies suggest that novel ribonucleotide reductase inhibitors TX and DX may provide an effective alternative to HU in HIV-1 therapy because they demonstrate reduced hematopoietic toxicity.

Influence of lithium on the cellular environment.

In order to study the influence of lithium on the cellular environment, we conducted research in multiple experimental models: groups of rats with normal cerebral excitability and groups susceptible to audiogenic convulsion, rat neuroglia cultures and perfusion of dog isolated head. We assumed blood composition to be a good indicator of cell environment composition. Blood serotonin level differs in the two groups of animals. Lithium induces a decrease of blood serotonin and an increase of amine concentration in some of the cerebral regions of rats susceptible to audiogenic convulsions. Inverse effects occur in rats with normal cerebral excitability. In the perfused, isolated head of a dog, lithium immediately decreases blood serotonin level. Na and water have a diminished metabolization during the first 24 hrs. in both animal groups. Decrease in metabolization is somewhat greater in hyperexcitable animals. Within 48 hrs. after lithium injection, there is an increase of Na metabolization, probably determined by its storage in the interstice. Renal elimination of K decreases under the influence of lithium 48 hrs. after administering one dose of lithium. Lithium induces, immediately after injection, a decrease of blood Na concentration in the efferent flow of the jugular vein of a perfused dog head. When used in cell cultures, lithium (2 mM concentration) stimulates glial cells division (astrocytes, oligodendrocytes), increases their growth and aging rates. The effects of lithium may be due to its toxicity. Therefore, lithium alters the composition of the cellular environment depending on dose and on the state of the body.

Use of an international faculty/student exchange program as a process to establish and improve graduate education and research within an allied health discipline.

It has been recognized in the allied health professions that allied health disciplines must enhance and increase their research and scholarly activity. If faculty/staff are to be judged in the academic environment in which they work, their efforts to conduct research must be supported. Recognition for academic scholarship measured by the performance of research and scholarly activity is often difficult for faculty/staff to attain because of increased demands for scheduled time devoted to classroom instruction and student advising. This inability for faculty/staff to engage in research and scholarly activity often is enhanced by the lack of proper and adequate facilities and equipment. Also important is the role of graduate education, which itself, provides a stimulus for the performance of research and scholarly activity. This article reports outcomes achieved by an international faculty/staff-student program that provides an opportunity for faculty/staff and students within an allied health discipline to conduct research and scholarly activity. This program could serve as a model to identify the strengths and benefits that can be achieved by such programs. This program is capable of improving the research and scholarly activity of all academic units within an allied health discipline.

The effects of lithium in reversing hydroxyurea induced suppression of hematopoietic progenitor cells in vitro using retroviral infected long-term marrow cultures.

Lithium has been known for its ability to induce the production of hematopoietic cells following administration in vivo to minimize the toxic effects on hematopoiesis as a consequence of drug treatment. The drug hydroxyurea (HU), a ribonucleotide reductase inhibitor, has been used in the treatment of a variety of neoplastic and non-neoplastic diseases, such as cancer and sickle cell anaemia. Hydroxyurea has more recently been implicated for use in the treatment of acquired immunodeficiency syndrome (AIDS). However, its major limitations have been due to its toxicity. Hydroxyurea selectively inhibits DNA synthesis and due to its brief duration, the drug is only toxic to those cells which are selectively synthesizing DNA during the period of exposure. The most important of these toxicities, and which serves as a dose limiting factor in treatment, is the induction of bone marrow suppression. In this study we investigated the possible beneficial effects of administering lithium (LiCl) to murine leukemia virus (MuLV) infected and non-infected long term bone marrow cultures (LTBMC). These cultures were then treated with either 0.2 mM hydroxyurea, 1.0 mM LiCl, or a combination of both. Samples were collected from LTBMC supernatants at 1, 2, 3, 4, 5 and 6 weeks post-treatment. Culture supernatants were then monitored to observe their repopulation of hematopoietic progenitors. The results demonstrated the effects of lithium in restoring hydroxyurea suppressed numbers of myeloid (CFU-GM) progenitors to within a normal range and also in re-establishing erythroid (BFU-E) progenitors.

The effects of lithium gamma-linolenic acid in reversing LPBM5 MuLV induced suppression of hematopoietic progenitor cells in vitro.

Lithium gamma linolenic acid (Li-GLA), was evaluated for its possible role as an antiviral agent. Li-GLA 15 micrograms ml-1 was administered to both normal and LP-BM5 MuLV retroviral infected murine bone marrow cultures. After 2 weeks of treatment, numbers of progenitors being produced by infected/treated cultures were reduced to some 10% that of normal cultures. In the remaining 4 weeks, numbers of CFU-GM and BFU-E hematopoietic progenitors returned within normal range. The efficacy of Li-GLA in relieving retroviral hematopoietic bone marrow suppression correlates to a reduction in interleukin-4 (IL-4) secretion, normally elevated in association with LP-BMP5 infection. These data indicate that this reduction in bone marrow suppression of LP-BMP5 infected cells may be due to a killing of infected cells by the Li-GLA, rather than stimulating hematopoiesis as with other lithium compounds. To conclude this may indicate the possible dual effect of administration of LiGLA to virally infected individuals in reducing viral titre and to lower the toxicities associated with long term drug therapy.

Effective use of ribonucleotide reductase inhibitors (Didox and Trimidox) alone or in combination with didanosine (ddI) to suppress disease progression and increase survival in murine acquired immunodeficiency syndrome (MAIDS).

Ribonucleotide reductase inhibitors (RRIs) have been recently shown to inhibit retroviral replication. We examined a new series of RRIs, 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5-trihydroxybenzohydroxamidoxime (Trimidox) for their ability to alter disease progression in murine acquired immunodeficiency syndrome (MAIDS), both alone and in combination with 2',3'-dideoxyinosine (ddI). MAIDS disease was induced by inoculation of female C57BL/6 mice with the LP-BM5 murine leukemia virus (MuLV) and disease progression characterized by extensive peripheral lymphadenopathy and splenomegaly. Efficacy of treatment with these drugs was based upon their ability to influence survival and disease pathophysiology by monitoring the development of splenomegaly. Toxicity was determined by changes in body weight, total peripheral white blood cell count and hematocrit. Didox or trimidox monotherapy was associated with increased survival and decreased disease pathophysiology, with no apparent toxicity. Combined with ddI, their ability to reduce development of viral induced splenomegaly was enhanced compared to trimidox, didox or ddI alone. These results demonstrate RRIs have potent activity in reversing the disease manifestations characteristic of MAIDS. Further studies are warranted to determine human clinical efficacy.

The regulation of phospholipase-A2 (PLA-2) by cytokines expressing hematopoietic growth-stimulating properties.

Various growth factors released by macrophages and other cell types modulate normal hematopoiesis. The physiological mechanisms whereby these molecules interact with specific target cells are ill defined. Eicosanoids, the products of fatty acid metabolism, are known to regulate cell proliferation and differentiation. The release of membrane-bound phospholipid by phospholipase-A2 (PLA-2) is the first critical step in the initiation of membrane remodeling and eventually eicosanoid synthesis. We report here data that demonstrates how various cytokines exhibit a marked hydrolytic activity mediated through PLA-2 against both [1-14C] oleic acid- and [1-14C] arachidonic acid-labeled Escherichia coli (micelle) substrates. PLA-2 extracts were prepared from neutrophils elicited by injecting rats ip with 8% glycogen. The rate of hydrolysis of free fatty acids from the phospholipid substrate was found to be linear, rapid, and pH dependent and was calculated to be 30 nmoles of phospholipid/hr/mg protein lysate. Cytokines (i.e., interleukin-1 [IL-1, human and murine recombinant, alpha], mouse lung cell-derived colony-stimulating factor [L-CSF], granulocyte-macrophage colony-stimulating factor [murine recombinant GM-CSF], tumor necrosis factor [murine recombinant TNF-alpha], and granulocyte colony-stimulating factor [human recombinant, G-CSF] all induced PLA-2 activity with the release of free fatty acids above basal levels. In contrast, lipopolysaccharide (LPS), interleukin-2, (IL-2, human recombinant), and macrophage colony-stimulating factor (M-CSF) did not significantly activate PLA-2 hydrolysis. The activation of this membrane-bound enzyme-substrate complex by these growth factors may serve as a mechanism whereby the appropriate target cells expressing receptors respond through either direct or secondary signals leading to the formation of free fatty acids with the eventual synthesis of prostanoid or lipoxygenase products, resulting in cellular proliferation and differentiation.

Suppression of hematopoietic support function is associated with overexpression of interleukin-4 and transforming growth factor-beta 1 in LP-BM5 murine-leukemia-virus-infected stromal cell lines.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus (MuLV) is a disease with many similarities to human AIDS. Our previous studies demonstrated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures could be attributed to a defective hematopoietic stroma. We report now the generation of permanent stroma cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by the polymerase chain reaction for detecting viral genome expression of the p12 envelope glycoprotein. The ability of these cell lines to support in vitro hematopoiesis was evaluated. The results demonstrated that when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic progenitors, whereas in virus-infected progenitors was suppressed. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for interleukin (IL)-1 beta, IL-2, IL-3, IL-6, IL-7, IL-10, interferon, tumor necrosis factor-alpha and stem cell factor. However, infection was associated with higher expression of IL-4 and transforming growth factor-beta 1. These findings demonstrate that infected stomal cell lines generate a defective hematopoietic microenvironment to produce altered cytokine expression and faulty hematopoiesis. Further characterization of these defective cell lines should assist elucidation of the mechanism(s) whereby retroviruses alter hematopoiesis ultimately leading to the generation of immunodeficiency.

Effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) on hematopoiesis administered to retrovirus-infected immunodeficient mice receiving dose-escalation zidovudine (AZT).

We have previously demonstrated that continuous administration of dose-escalation zidovudine (AZT) in either normal or LP-BM5 MuLV immunodeficient virus-infected mice (MAIDS) was associated with the development of anemia, neutropenia, and thrombocytopenia. Hematopoietic growth factors/cytokines are being evaluated to determine their efficacy in ameliorating the hematopoietic toxicity associated with AZT. In normal mice receiving AZT, an increase in only plasma erythropoietin and not GM-CSF, Meg-CSF or TNF-alpha has been reported. This article describes studies that investigated the effect of combination interleukin-3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) administered in normal non-viral, viral-infected, and viral-infected C57BL6 mice receiving dose-escalation AZT, i.e. 0.1 mg/ml, 1.0 mg/ml, and 2.5 mg/ml placed in the drinking water. Non-viral control mice responded to IL-3/GM-CSF by increasing erythropoiesis, myelopoiesis and platelet production measured by increased bone marrow and spleen derived erythroid, myeloid and platelet precursor stem cells cultured in semi-solid media. Virus-infected control mice not receiving IL-3/GM-CSF developed pancytopenia. Administration of IL-3/GM-CSF to virus-infected mice receiving dose-escalation AZT did not ameliorate the peripheral pancytopenia associated with immunodeficiency disease and AZT treatment, even though erythroid, myeloid and platelet precursor progenitor cells were increased at certain times when compared to either normal or viral-infected mice receiving IL-3/GM-CSF. These results indicate that the combination use of IL-3 and GM-CSF in vivo is only a partially effective growth factor/cytokine treatment to ameliorate the hematopoietic toxicity associated with the use of the anti-viral drug zidovudine.

Effect of lithium in immunodeficiency: improved blood cell formation in mice with decreased hematopoiesis as the result of LP-BM5 MuLV infection.

Lithium salts have been demonstrated to induce the production of hematopoietic cells following administration in vivo and to minimize the reduction of these cells following treatment with either radiation, chemotherapeutic or antiviral drugs. We have previously demonstrated that lithium, when administered in vivo to immunodeficient mice infected with LP-BM5 MuLV (MAIDS) significantly reduced the development of lymphadenopathy, splenomegaly, and the lymphoma associated with late-stage immunodeficiency disease in this model, and increased the survival of these animals compared to virus-infected controls not receiving lithium. We report here the results of in vivo studies in the MAIDS model that determined the effect of lithium on peripheral blood indices and the number of myeloid (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Meg) hematopoietic progenitors from bone marrow and spleen harvested from immunodeficient mice receiving lithium carbonate (1 mM) placed in their drinking water compared to virus-infected controls not receiving lithium. Time-points evaluated were at weeks 1, 5, 9, 13, 17, and 21 postviral infection. Virus-control mice not receiving lithium demonstrated all the signs that are characteristic of MAIDS, i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, reduced hematopoiesis, and death. Infected mice receiving lithium demonstrated diminished presence of splenomegaly, lymphadenopathy, hypergammaglobulinemia, no suppression of hematopoiesis nor mortality. Enhanced hematopoiesis was demonstrated by neutrophilia, lymphocytosis, thrombocytosis, and erythrocytosis that was evident by increased myeloid, erythroid, and megakaryocyte progenitor cells cultured from bone marrow and spleen. These studies further demonstrate that lithium influences the disease process in the MAIDS model and restricts the development of hematopoietic suppression that develops in this retroviral animal model of immunodeficiency.

Increased hematopoietic toxicity following administration of interferon-a with combination dideoxynucleoside therapy (zidovudine plus ddI) administered in normal mice.

Because of the urgency to develop drugs which will effectively combat HIV infection, many combination therapies which have proved effective against HIV in vitro have undergone, or are undergoing clinical trial. Unfortunately many of drugs are being used without rigorous and exhaustive preclinical evaluation to assess their potential to develop hematopoietic toxicity. We report here the results of two in vivo studies performed to analyze the effect of combined zidovudine (AZT) plus didanosine (ddI) therapy, either with or without interferon-a (IFN-a), on murine hematopoiesis. Normal C57BL/6 female mice were administered AZT (1.0 mg/ml) plus dose-escalation ddI (0.1, 1.0 and 2.5 mg/ml) placed in their drinking water. Control mice received IFN-å (100 units/ml) alone. Mice were serially bled and sacrificed over a six-week period for assessment of hematopoietic toxicity measured by peripheral blood indices and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. AZT plus dose-escalation ddI decreased the hematocrit and white blood cell count when administered to normal mice compared to untreated controls during the six-week examination period. Marrow derived BFU-E, CFU-GM, and CFU-Meg were all reduced, however an increase was observed from the spleen for all three progenitor cell types. Use of IFN-a, in addition to combination AZT plus ddI further decreased the hematocrit, white blood cells and platelets. Marrow derived CFU-GM and CFU-Meg were increased slightly and only marginally for BFU-E with a similar response observed from the spleen. These results demonstrate that combination AZT plus ddI when used in vivo may produce synergistic hematopoietic toxicity, and that the addition of IFN-a to this treatment regimen increases this toxicity. These data indicate caution when this therapeutic approach is suggested for patients infected with HIV. If used, these patients will require careful monitoring for blood cell toxicity.

Influence of human granulocyte-macrophage colony stimulating factor/interleukin-3 fusion protein (PIXY321) on the hematopoietic toxicity associated with anti-viral drugs (zidovudine and didanosine) in vitro using normal human marrow cells.

The antiviral drugs didanosine (ddI) and zidovudine (AZT), synthetic nucleoside analogs, have been used in the treatment of acquired immunodeficiency syndrome (AIDS). Although clinical use of zidovudine (AZT) is still widely used, it is associated with the development of virus disease resistance and toxicity to the hematopoietic system. Alternative nucleoside reverse transcriptase derivatives such as didanosine (ddI) have been developed in order to reduce the incidence of virus disease resistance and hematological toxicity. We report here studies designed to ev evaluate the toxicity profile comparing didanosine (ddI) with zidovudine (AZT) when used alone or in combination with normal non-adherent, T-cell depleted human marrow cells plated in the presence or absence of the human cytokine fusion protein of granulocyte-macrophage colony stimulating factor and interleukin-3 (PIXY321). As expected, didanosine (ddI) was less toxic for human hematopoietic progenitor cells, i.e., CFU-GEMM, CFU-GM, CFU-Meg, and BFU-E than zidovudine. Toxicity was additive when didanosine (ddI) and zidovudine (AZT) were combined. In the absence of drugs PIXY321 colony formation was increased for all progenitor cells cultured. In the presence of didanosine (ddI) or zidovudine (AZT), either as single-agents or combined, PIXY321 reduced toxicity significantly. These results demonstrate PIXY321 is an effective cytokine capable of reversing the toxicity associated with anti-viral drugs when used in vitro where didanosine (ddI) is less toxic than zidovudine (AZT); however their suppression of hematopoietic progenitors is additive when combined.

Stromal cell lines derived from LP-BM5 murine leukemia virus-infected long-term bone marrow cultures impair hematopoiesis in vitro.

Murine acquired immunodeficiency syndrome (MAIDS) induced by defective LP-BM5 murine leukemia virus is a disease with many similarities to human AIDS. Previous studies indicated that the depressed hematopoiesis observed in LP-BM5-infected marrow cultures may be attributable to a defect of hematopoietic stroma. We report here the generation of permanent stromal cell lines from noninfected and LP-BM5-infected marrow cultures. Retrovirus infection was confirmed by polymerase chain reaction for viral genome. The ability of these cell lines to support in vitro hematopoiesis was studied. Results indicated that, when cocultured with normal or infected nonadherent mononuclear cells, noninfected cell lines efficiently supported the production of hematopoietic precursors, whereas viral-infected cell lines induced suppression of both normal and viral-infected progenitors. Expression of cytokine genes in stromal cell lines was also examined. All cell lines expressed equivalent levels of transcripts for stem cell factor and tumor necrosis factor alpha. However, infection was associated with higher levels of interleukin-4 and transforming growth factor beta 1 transcript expression. These findings suggest that infected stromal cell lines exhibit a defective hematopoietic microenvironment that produced altered cytokine expression resulting in faulty hematopoiesis. Further characterization of the defective cell lines should prove valuable for studies of the pathogenesis of murine AIDS.

Prevention of hematopoietic myeloid and megakaryocyte toxicity associated with zidovudine in vivo in mice with recombinant GM-CSF.

We studied the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the suppression of hematopoiesis associated with the use of the antiviral drug zidovudine (AZT) administered in vivo to normal mice, as determined by measuring peripheral blood indices, and assays of hematopoietic progenitors, i.e. erythroid (CFU-E/BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) from bone marrow and spleen. Previous studies from this laboratory have established that dose-escalation zidovudine induced a dose-dependent decrease in hematocrit, WBC, and platelets with altered populations of bone marrow and splenic erythroid, myeloid and megakaryocyte progenitors when administered to normal mice. Daily administration of GM-CSF (10 micrograms/kg/bw) was associated with altered peripheral blood indices and progenitor cells. Dose-escalation AZT, i.e. 0.1, 1.0 and 2.5 mg/ml, was associated with a comparable reduction in all indices, i.e. hematocrit, WBC, and platelets during the 6-week examination period. GM-CSF reduced zidovudine-induced myeloid toxicity (concentration < 2.5 mg/ml) which was associated with an increase in bone marrow and splenic CFU-GM. High concentration, i.e. 2.5 mg/ml still produced myelosuppression irreversible with GM-CSF. GM-CSF induced a reduction in circulating platelets following zidovudine treatment at weeks 2 and 4 with the 1.0 mg/ml and 2.5 mg/ml treatment groups respectively, compared to a persistent decrease in platelets in the presence of zidovudine alone. GM-CSF BFU-E were elevated indicating the restriction in erythoid differentiation was still present. These studies demonstrate GM-CSF influences myeloid and megakaryocyte recovery, but not the erythoid suppression associated with the antiviral drug zidovudine.

Influence of interleukin-3 (IL-3) on the hematopoietic toxicity associated with combination anti-viral drugs (zidovudine and DDI) in vitro using retrovirus-infected bone marrow cells.

The drug zidovudine (AZT), a synthetic thymidine analog, has been used in the treatment of acquired immunodeficiency syndrome (AIDS). Clinical use of zidovudine has been associated with the development of hematopoietic toxicity manifested by anemia, neutropenia, and on occasion thrombocytopenia. This toxicity has resulted in the development of alternative dideoxynucleoside drugs capable of exerting anti-viral potency while minimizing the risk for inducing organ toxicities. One such dideoxynucleoside drug is 2',3'-dideoxyinosine (ddI). Clinical trials are currently evaluating the effect of combination anti-viral drug treatment such as zidovudine plus ddI. We report here the results of studies designed to evaluate the effect of interleukin-3 (IL-3) on its ability to influence the hematopoietic toxicity associated with zidovudine and ddI following combination with retroviral-infected murine bone marrow cells. Toxicity was evaluated by quantitating several classes of hematopoietic progenitor stem cells such as granulocyte-macrophage (CFU-GM), erythroid (CFU-E and BFU-E) and megakaryocyte (CFU-Meg). Dose-escalation IL-3 provided protection of anti-viral drug induced suppression of progenitor cells when combined in the presence of the ID50 concentration of either zidovudine or ddI; however, when zidovudine and ddI were combined, IL-3 was less effective in providing protection against drug-induced toxicity at any concentration examined. These results indicate that IL-3 is effective in reducing anti-viral drug-induced hematopoietic toxicity associated with single-agent use; however, IL-3 is less effective when such drugs are used in combination.

Effect of combination interleukin-1 and erythropoietin in ameliorating the hematopoietic toxicity associated with the use of zidovudine administered to normal mice.

Use of the anti-viral drug zidovudine in the treatment of acquired immunodeficiency syndrome (AIDS) has been associated with the development of hematopoietic toxicity. Several hematopoietic growth factors have been investigated in their ability to modulate such toxicity; however, no single factor has been demonstrated to produce restoration of hematopoiesis following use with zidovudine. We report results describing the effect of combination interleukin-1 (IL-1) and erythropoietin (Epo) in their ability to modulate the hematopoietic toxicity associated with dose-escalation zidovudine administered in normal mice. When administered over a six-week period, IL-1 and Epo raised the packed red cell volume, white blood cell and platelet counts in control mice and mice receiving dose-escalation zidovudine. These effects were attributed in part to the ability of combination IL-1 and Epo to increase erythroid, myeloid and megakaryocyte progenitor stem cells from bone marrow and spleen. These results indicate that use of combined IL-1 and Epo may be efficacious in ameliorating the hematopoietic toxicity associated with the use of zidovudine.

Sustained zidovudine treatment on hematopoiesis in immunodeficient mice.

Zidovudine (AZT) has been the drug of choice in the treatment of human AIDS; however, associated with the use of zidovudine has been the development of hematopoietic toxicity, the mechanism of which is not clearly defined. We report here studies designed to evaluate dose-escalation of zidovudine, i.e. 0.1 and 1.0 mg/ml placed in the drinking water on hematopoiesis in C57BL/6 normal and LP-BM5 immunodeficiency virus-infected mice. Over a 6-week evaluation period, compared to normal, non-virus-infected controls, murine immunodeficiency (MAIDS) infection was associated with reduced hematopoietic progenitors, i.e. CFU-E, BFU-E, CFU-GM, and CFU-Meg from bone marrow and spleen. Following zidovudine treatment, further suppression of marrow-derived progenitors was observed, while increased numbers of progenitors were obtained from the spleen. Spleen-derived erythroid progenitors, i.e. CFU-E, were increased by 950% (P < 0.001) from MAIDS-infected animals receiving 1.0 mg/ml of drug following 4-weeks exposure compared to non-drug-treated MAIDS control animals. Splenic BFU-E were increased 654% following 6-weeks exposure compared to non-drug-treated MAIDS-infected mice. This study suggests that the bone marrow is particularly sensitive to zidovudine toxicity which, at least early in exposure, appears to be compensated by splenic-derived hematopoiesis, in particular, erythropoiesis. Overt toxicity develops when, at least in this immunodeficiency model, the spleen is unable to provide progenitors in response to continued zidovudine exposure in vivo.