Granulocyte-macrophage Progenitor Cells Research Articles

Molecular cloning of leukotactin-1: a novel human beta-chemokine, a chemoattractant for neutrophils, monocytes, and lymphocytes, and a potent agonist at CC chemokine receptors 1 and 3.

A new member of human beta-chemokine cDNA was isolated and named leukotactin-1 (Lkn-1). Lkn-1, along with murine macrophage inflammatory protein-related protein-1 and -2, defines a subgroup of beta-chemokines based on two conserved cysteines in addition to the four others conserved in all beta-chemokines. The putative mature Lkn-1 is composed of 92 amino acids with a calculated m.w. of 10,162. The Lkn-1 gene was mapped to human chromosome 17, region q12. Recombinant Lkn-1 was a potent chemoattractant for neutrophils, monocytes, and lymphocytes and induced calcium flux in these cells. Lkn-1 specifically induced calcium flux in CCR1- and CCR3-expressing HOS cell lines. Lkn-1 suppressed colony formation by human granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Hence, we have isolated and characterized a human C6 beta-chemokine that is a potent agonist at CCR1 and CCR3 and shows broad biologic activities, including leukocyte chemoattraction.

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Identification of a Serpin Specifically Expressed in Multipotent and Bipotent Hematopoietic Progenitor Cells and in Activated T Cells

We have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.

Identification of a Serpin Specifically Expressed in Multipotent and Bipotent Hematopoietic Progenitor Cells and in Activated T Cells

AbstractWe have identified a gene that has a high level of mRNA expression in undifferentiated, multipotential hematopoietic cells (FDCP-Mix) and that downregulates both transcript and protein, as these cells are induced to differentiate into mature myeloid cells. Sequence analysis of this gene has identified it as a serine protease inhibitor EB22/3 (serpin 2A). Constitutive expression of serpin 2A in FDCP-Mix cells was associated with an increase in the clonogenic potential of the cells and with a delay in the appearance of fully mature cells in cultures undergoing granulocyte macrophage differentiation when compared with control cells. Serpin 2A was also found to be expressed in bone marrow-derived bipotent granulocyte macrophage progenitor cells (GM-colony forming cell [CFC]), but not in erythrocyte progenitor cells from day 15 fetal liver. Expression of serpin 2A also showed a marked up regulation during the activation of cytotoxic suppressor CD8+ T cells, with a clear lag between the appearance of transcript and detection of protein.

Possible involvement of bone marrow stromal cells in agranulocytosis caused by vesnarinone treatment.

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Perturbation in the ability of bone marrow stroma from patients with acute myeloid leukemia but not chronic myeloid leukemia to support normal early hematopoietic progenitor cells

The ability of bone marrow (BM) stroma derived from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) to support normal hematopoiesis was investigated using a two-stage long-term bone marrow culture (LTBMC) procedure. Of particular interest was whether leukemia-derived stroma were capable of supporting the very immature, uncommitted hematopoietic progenitor cells (HPC) which are considered to have a higher dependence and association with the BM stroma than the more mature committed HPC. Confluent stromal layers were recharged with selected populations of normal HPC enriched for the CD34 +CD38 − cells (immature, uncommitted HPC) or the CD34 +CD38 + cells (mature, committed HPC). The weekly output of clonable granulocyte-macrophage progenitor cells (CFU-GM) was used as an indicator of the hematopoietic-supporting ability of the cultures. Stromal layers derived from 5 7 patients newly diagnosed with AML, showed significantly depressed ability to support the CD34 +CD38 − cells compared to stroma derived from normal donors. The supporting function of the AML-derived stroma for the more mature CD34 +CD38 + cells was similar to that of the normal stroma ( 3 3 cases). Stromal layers derived from patients with chronic-phase CML showed normal or enhanced supporting function for the CD34 +CD38 − cells ( 5 6 cases), and likewise for the CD34 +CD38 + cells ( 3 3 cases). This study revealed a selective defect in the ability of BM stroma from patients with AML to support the maturation of normal early uncommitted HPC, represented by the CD34 +CD38 − cells, whilst the ability to support the committed CD34 +CD38 + cells was not affected. This suggests that the BM microenvironment may be implicated in the disease mechanism of AML. It does not, however, appear to be as clearly implicated in chronic-phase CML.

Hydroquinone Stimulates Granulocyte-Macrophage Progenitor Cells in Vitro and in Vivo

Hydroquinone Stimulates Granulocyte-Macrophage Progenitor Cells in Vitro and in Vivo

Hydroquinone stimulates granulocyte-macrophage progenitor cells in vitro and in vivo.

To investigate whether hydroxylated metabolites of benzene may be responsible for the amplification of granulocyte-macrophage progenitor cells (GM-CFC) observed in mice that inhale benzene, groups of six C57BL6 mice were injected with hydroquinone (HQ) (75 mg/kg) or HQ (50 mg/kg) plus phenol (PHE) (50 mg/kg) twice daily for 11 days. Deviations in blood leukocyte and erythrocyte levels by up to one-third were noted in the treated groups; however, the peripheral blood differential counts were unchanged. Although no changes in bone marrow cellularity were observed in mice treated with HQ, cellularity was decreased by a factor of two in the mice that had received HQ plus PHE. The number of GM-CFC per femur was doubled in both treated groups. In vitro experiments using the murine multipotent hematopoietic progenitor cells FDCP mix also showed a duplication of GM-CFC formation in the presence of HQ at concentrations between 10(-6) M and 10(-10) M. When HQ and PHE were present at equimolar concentrations, significantly increased colony formation was still observed with 10(-12) M of metabolites. The effect was independent of the concentration of GM-colony-stimulating factor used. We suggest that HQ is a major mediator of the stimulatory effect of benzene on GM-CFC in mice. In addition, the in vitro data indicate that a direct effect of GM-CFC is involved.

Mechanisms of immune suppression in patients with head and neck cancer: Influence on the immune infiltrate of the cancer

Freshly excised human head and neck cancers (219 primary cancers; 64 metastatic lymph node cancers) were analyzed for the immune inhibitory mediators released from the cancer tissues and the immune infiltrate within the tumor. Significant levels of the immune inhibitory mediators transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2) and interleukin-10 (IL-10) were released from these cancers. Also released was granulocyte-macrophage colony-stimulating factor (GM-CSF), whose secretion was associated with an intratumoral presence of CD34+ cells. We have previously shown that CD34+ cells within human head and neck cancers are immune inhibitory granulocyte-macrophage progenitor cells. The presence of TGF-beta, PGE2 and IL-10 was associated with a reduced content of CD8+ T-cells within the cancers. The CD4+ cell content appeared to be less affected by these immune inhibitory mediators. Instead, parameters indicative of CD4+ cell function (p55 IL-2 receptor expression, release of IL-2 and IFN-gamma) were diminished in cancers that released higher levels of TGF-beta, IL-10 and GM-CSF and had a higher CD34+ cell content. Furthermore, metastatic cancers released higher levels of the soluble immune inhibitory mediators and lower levels of IFN-gamma and IL-2 than did primary cancers, although CD34+ cells were similarly present in both primary and metastatic cancers. Our results show that human head and neck cancers have a multiplicity of non-mutually exclusive mechanisms of immune suppression that are most prominently associated with reduced CD8+ cell influx and reduced influx and altered function of intratumoral CD4+ cells.

Macrophage-stimulating protein, a ligand for the RON receptor protein tyrosine kinase, suppresses myeloid progenitor cell proliferation and synergizes with vascular endothelial cell growth factor and members of the chemokine family.

Macrophage-stimulating protein (MSP), originally identified as an inducer of murine resident macrophage responsiveness to chemoattractants, is a ligand for human RON/murine STK receptor protein tyrosine kinases. Since STK was cloned from populations enriched for hematopoietic stem cells, we initiated studies on the effects of MSP on colony formation by granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) myeloid progenitor cells. MSP alone had no colony stimulating activity. However, MSP caused about a 50% suppression of CFU-GM colony formation induced by synergistic combinations of SLF or Flt-L plus GM-CSF, G-CSF, or IL-3 and of BFU-E and CFU-GEMM colonies induced by SLF or Flt3-L plus Epo or Epo and IL-3. In contrast, MSP had no effect on progenitors stimulated by one growth factor. MSP also suppressed colony formation by stimulated cord blood progenitors, but only after preinduction to a rapidly cycling state. It was previously reported that several members of the chemokine family synergistically suppress myeloid progenitor proliferation. Likewise, synergistic suppression was observed when MSP was paired with VEGF, MIP-1 alpha, IL-8, PF4, MCP-1, IP-10, or ENA-78, or when VEGF was paired with the chemokines; and the required MSP concentration was more than 100-fold less than for MSP alone. Additionally, MSP or VEGF inhibited proliferation of the human myeloid growth factor-dependent cell line, M07e, but a sustained effect required multiple additions over time. At the least, some of the MSP suppressive effects on myeloid progenitors, as assessed on single isolated CD34 marrow cells, appeared to be directly on the progenitors; sustained additions of MSP were required to see this effect. The suppressive action of MSP and its synergism with proteins of the chemokine family may be of relevance to regulation of blood cell production.

Vitamin D3 treatment of tumor bearers can stimulate immune competence and reduce tumor growth when treatment coincides with a heightened presence of natural suppressor cells.

By secreting granulocyte-macrophage colony-stimulating factor (GM-CSF), Lewis lung carcinoma tumors induce immune suppressive granulocyte-macrophage progenitor cells. Treating mice having established tumors and high levels of suppressor activity with vitamin D3 eliminated suppressor activity, increased anti-tumor immunity, induced an immune stimulatory cell population, and reduced tumor growth. When instead, the vitamin D3 treatment was initiated earlier, when implanted tumors first became detectable and when natural suppressor activity was less prominent, the treatment had no effect. Thus, vitamin D3 treatment can stimulate the immune competence of tumor bearers when treatment is targeted to coincide with a heightened presence of GM-CSF-induced suppressor cells.

Flt3 Ligand prolongs survival of CD34 +++ human umbilical cord blood myeloid progenitors in serum-depleted culture medium

Flt3 Ligand (L), a newly identified stimulating/co-stimulating cytokine for hematopoietic stem and progenitor cells, was assessed for its capacity, in comparison to that of steel factor, to prolong the survival in culture of hematopoietic stem and progenitor cells from CD34++ + human cord blood. The addition of Flt3-L to serum-depleted cultures significantly prolonged the survival of high-proliferative-potential colony-forming cells (HPP-CFC) as well as that of multipotential (CFU-GEMM) and granulocyte-macrophage (CFU-GM) progenitor cells. The concentrations of Flt3-L necessary for these effects were similar to those of SLF, and low concentrations of Flt3-L and SLF which were inactive when used alone were active in these effects when used together. These results may be of practical value for short-term storage, treatment and/or transport of purified populations of cord blood stem and progenitor cells.

Selective protection of toxicity of 2′,3′-dideoxypyrimidine nucleoside analogs by β- d-uridine in human granulocyte-macrophage progenitor cells

β- d-Uridine protected human granulocyte-macrophage lineage cells in both semi-solid (granulocyte-macrophage colony-forming units, CFU-GM) and liquid cultures against the toxic effects of 3′-azido-3′-deoxythymidine (AZT), 3′-fluoro-3′-deoxythymidine (FLT) and a combination of AZT and FLT, without impairment of the activities of these respective drugs against human immunodeficiency virus (HIV) replication. In addition, β- d-uridine also protected human CFU-GM against toxicity of the in vivo AZT metabolite, 3′-amino-3′-deoxythymidine (AMT). β- l-uridine and α- d-uridine, two stereoisomers of the natural form, and the base uracil, were unable to protect cells against either AZT or FLT toxicity, whereas β- d-uridine-5′-bis(SATE)phosphotriester, a prodrug of β- d-uridine-5′-monophosphate, successfully protected cells against AZT toxic effects, suggesting that β- d-uridine needs to be metabolized to its nucleotides to exert a pharmacological effect. These data suggest in addition that AZT, FLT and AMT share a common target site(s) of toxicity involved in myelosuppression.

Suppression of T cell proliferation by tumor-induced granulocyte-macrophage progenitor cells producing transforming growth factor-beta and nitric oxide.

Production of high levels of granulocyte-macrophage CSF (GM-CSF) by LLC-LN7 tumors results in myelopoietic stimulation and an increase in cells having natural suppressor (NS) activity. Prior studies showed these NS cells could be isolated from the bone marrow of tumor-bearing mice with an Ab (ER-MP12) that recognized GM-progenitor cells. The present study showed these cells to also be in the spleen, lymph node, and tumor, and that treatment of tumor-bearing mice with low doses of IFN-gamma plus TNF-alpha reduced the frequency of E-MP12+ cells. Studies focused on characterizing the intratumoral ER-MP12+ cells and the mechanism by which they suppress T cell proliferation. When isolated and seeded in soft agar with CSF-containing LLC-LN7 supernatants, the ER-MP12+ cells grew into colonies, most of which contained both granulocytic and monocytic cells. Tumor-derived ER-MP12+ cells and their culture supernatants were suppressive to T cell proliferation. Among the factors produced by ER-MP12+ cells were TGF-beta, nitric oxide (NO), IL-10, and prostaglandin E2 (PGE2). However, it was TGF-beta and NO that mediated the suppression of T cell proliferation by ER-MP12+ cells. Intratumoral ER-MP12+ cells could be maintained as suppressive blastlike cells for at least 4 days in cultures containing CSFs, but adding IFN-gamma plus TNF-alpha to these cultures caused their differentiation mainly into nonsuppressive TNF-alpha-secreting monocytic cells. These results show that intratumoral ER-MP12+ cells having homology to GM-progenitor cells suppress T cell function by producing TGF-beta and NO. IFN-gamma/TNF-alpha treatment stimulates their differentiation and shift from production of TGF-beta and NO to production of TNF-alpha.

Retroviral Transfer of the Recombinant Human Erythropoietin Receptor Gene Into Single Hematopoietic Stem/Progenitor Cells From Human Cord Blood Increases the Number of Erythropoietin-Dependent Erythroid Colonies

To test whether an enforced expression of a lineage-specific cytokine receptor would influence the proliferation/differentiation of hematopoietic stem/progenitor cells, retroviral vectors containing the human erythropoietin receptor (hEpoR) gene were used to transduce the hEpoR gene into phenotypically sorted subsets of cells. CD34 , CD34++CD33-, and CD34++CD33+ populations of human cord blood, highly enriched for hematopoietic stem/progenitor cells, were sorted and plated as single cells per well in methylcellulose culture medium containing early acting growth factors in the presence or absence of Epo. The hEpoR gene was efficiently transduced into single high proliferative potential colony-forming cells (HPP-CFC) and multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]), erythroid (burst-forming unit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming unit-granulocyte-macrophage [CFU-GM]) progenitor cells. As expected in cultures grown in the absence of Epo, no BFU-E or CFU-GEMM colonies grew. In the presence of Epo, the hEpoR-gene transduced cells formed significantly more CFU-GEMM and BFU-E colonies than did the controls. A significant decrease in HPP-CFC colonies was also observed under these conditions. Little or no effect of hEpoR gene transduction was apparent in the numbers of CFU-GM colonies formed in the presence or absence of Epo. All of the above results were similar whether the cell populations assessed were CD34 or their CD33- or CD33+ subsets plated in the presence of growth factors at 200 cells/mL or after limiting dilution at 2 cells/well. These results suggest that the profile of detectable stem/progenitors can be altered by retrovirus-mediated expression of the hEpoR gene.

Recombinant human granulocyte colony-stimulating factor treatment improves macrophage suppression of granulocyte and macrophage growth after burn and burn wound infection.

Granulocyte and macrophage production after burn injury or burn wound infection is significantly reduced and further compromised by endotoxin (ET). Moreover, the macrophage seems to be the major source of this bone marrow suppression. We sought to determine if recombinant human granulocyte colony-stimulating factor (rhG-CSF), a hematopoietic growth factor that is capable of improving survival after experimental burn wound sepsis, altered postburn macrophage-mediated marrow suppression. Groups of male BDF1 mice (n = 6 to 10) receiving a 15% total body surface area burn +/- infection (B or B + I) with Pseudomonas aeruginosa were injected with 100 ng rhG-CSF twice daily. On day 3, peritoneal-elicited macrophages (5 x 10(6) cells/mL) from either rhG-CSF-treated or control (5% dextrose in water) mice were incubated +/- ET (300 ng/mL). The resultant macrophage supernatant was added to cultures of target marrow granulocyte-macrophage progenitor cells (GM-CFC) at a volume of 1:10. The GM-CFC growth as a percentage of cultures not containing macrophage supernatant were compared and reductions in the number of GM-CFC taken as an index of marrow suppression. Macrophages obtained from B and B + I animals reduced target GM-CFC growth, compared with macrophages from normal animals (B vs. normal animals p < 0.05). In addition, ET-stimulated macrophages induced further bone marrow suppression for all three groups (p < 0.01). Macrophages from granulocyte colony-stimulating factor-treated animals caused significantly less bone marrow suppression, compared with untreated animals for all groups (p < 0.05 to 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic Effect of Granulocyte Colony-Stimulating Factor and Drugs Elevating Extracellular Adenosine on Neutrophil Production in Mice

Experimental evidence suggests that the activation of purinoceptors by extracellular adenosine can modulate proliferation and/or differentiation of hematopoietic cells. The present study was undertaken to investigate the potential interactions of this system of intercellular signaling with the effects of granulocyte colony-stimulating factor (G-CSF) on granulopoiesis in vivo. Elevation of extracellular adenosine in normal mice was induced by the joined administration of dipyridamole, a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The effects of dipyridamole, AMP, and G-CSF, administered either alone or in combinations, were evaluated. The agents were injected to mice in a 4-day regimen, and the hematologic endpoints were determined 24 hours after the completion of the treatment. It was shown that the effects of G-CSF, ie, increases in peripheral blood neutrophils, granulocyte-macrophage progenitor cells (GM-CFC), and morphologically determined granulocytic cells in femoral marrow and a decrease in the marrow erythroid cells, can be enhanced by the combination of dipyridamole plus AMP administered 30 minutes before G-CSF. Furthermore, it was ascertained that the stimulatory action of dipyridamole plus AMP was expressed particularly at lower doses of G-CSF (1.5, 3, and 4.5 micrograms/d). At higher doses of G-CSF (6 and 9 micrograms/d), the interactions were no more evident. When combining dipyridamole, AMP, and 3 micrograms of G-CSF, peripheral neutrophils increased approximately 3.9- to 4.5-fold compared with an approximate 2.2-fold increase induced by G-CSF alone. The results indicate the possible therapeutic potential of combination therapy with G-CSF and drugs increasing extracellular adenosine.

Human chemokines: enhancement of specific activity and effects in vitro on normal and leukemic progenitors and a factor-dependent cell line and in vivo in mice.

The myelosuppressive effects of human chemokines were evaluated in vitro on normal myeloid progenitors obtained from bone marrow and cord blood, on bone marrow progenitors from patients with acute or chronic leukemia, on proliferation of human factor-dependent cell line M07e, and in vivo on myelopoiesis in mice. Preincubation of human MIP-1 alpha, MIP-2 alpha, interleukin (IL)-8, platelet factor (PF) 4, monocyte chemotactic and activating factor (MCAF), and interferon-inducible protein-10 (IP-10) in an acetonitrile (ACN) solution significantly enhanced the specific activity of these chemokines for in vitro suppression of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells stimulated to proliferate with a colony stimulating factor plus steel factor (SLF). Combinations of any two of these ACN-treated chemokines synergized to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM at chemokine concentrations below that at which combinations of non-ACN treated chemokines are active. Cord blood progenitors, as previously reported, were in a slow or noncycling state and nonresponsive to inhibition by chemokines. However, after suspension culture with GM-CSF, IL-3, and SLF, they were placed into rapid cell cycle and were responsive to inhibition by ACN-treated chemokines. Low doses of these ACN-pretreated chemokines were active in vivo in suppressing absolute numbers and cycling status of femoral marrow CFU-GM, BFU-E, and CFU-GEMM in C3H/HeJ mice. Other chemokines, alone and in combination, including MIP-1 beta, MIP-2 beta, GRO-alpha NAP-2, and RANTES, were inactive in vitro and in vivo whether or not they were pretreated with ACN. While heterogeneity in responsiveness of CFU-GM from different patients with leukemia to suppression by ACN-treated chemokines was apparent, if the patients had CFU-GM responsive to one of the active chemokines these cells were responsive to the other active chemokines; if patient CFU-GM were not responsive to one of the chemokines, they were not responsive to the other active chemokines. M07e colony-forming cells were responsive to the growth-inhibiting effects of the active ACN-treated chemokines, alone and in combination, but these effects were rapidly reversible and sustained only by multiple daily additions of chemokines. These results should be of value in considering these chemokines for potential clinical use and for assessment of their mechanisms of action, alone and in combination.

A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors.

A new member of the beta-chemokine family, macrophage inflammatory protein (MIP)-related protein-2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. MRP-2 is composed of 122 amino acids of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. MRP-2 is structurally similar to MIP-related protein-1 (MRP-1) (C10) and MIP-1 alpha. MRP-2 shows a 50.8% sequence identity at the protein level to MRP-1 and 46.3% identity to MIP-1 alpha. MRP-2 detects approximately 1.3 kilobase mRNA from monocyte and macrophage cell lines but does not detect the mRNA from T and B cells. The MRP-2 gene termed Scya9 was mapped to the central region of mouse chromosome 11 near the Scya1 and Scya2 genes, which are also members of the beta-chemokine superfamily. The Scya gene cluster was located between neurofibromatosis type 1 (Nf1) and myeloperoxidase (Mpo). rMRP-2 significantly suppressed colony formation by murine and human bone marrow granulocyte-macrophage (CFU-granulocyte-macrophage), erythroid (burst-forming unit-E), and multipotential (CFU-granulocyte-erythroid-macrophage-megakaryocyte) progenitor cells stimulated by combinations of growth factors.

Antiviral, metabolic, and pharmacokinetic properties of the isomeric dideoxynucleoside 4(S)-(6-amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol

4(S)-(6-Amino-9H-purin-9-yl)tetrahydro-2(S)-furanmethanol (IsoddA) is the most antivirally active member of a novel class of optically active isomeric dideoxynucleosides in which the base has been transposed from the natural 1' position to the 2' position and the absolute configuration is (S,S). IsoddA was active against human immunodeficiency virus type 1 (HIV-1) (strain IIIB), HIV-2 (strain ZY), and HIV-1 clinical isolates. Combinations of the compound with zidovudine (3'-azido-3'-deoxythymidine), 2',3'-dideoxyinosine, or 5-fluoro-2'-deoxy-3'-thiacytidine showed synergistic inhibition of HIV. A moderate reduction of activity was observed with clinical isolates resistant to zidovudine. An IsoddA-resistant virus (eightfold-increased 50% inhibitory concentration) was selected in vitro by repeated passage of HIV-1 (HXB2) in the presence of increasing concentrations of IsoddA. The reverse transcriptase-coding region of the mutant virus contained a single base change resulting in a change at codon 184 from Met to Val. IsoddA was also active against hepatitis B virus (HBV) in vitro; however, it lacked substantial selective activity in an in vivo HBV model. IsoddA was inefficiently phosphorylated in CEM cells; however, the half-life of the triphosphate was 9.4 h, and IsoddATP was a potent inhibitor of HIV-1 reverse transcriptase, with a Ki of 16 nM. The cytotoxicity 50% inhibitory concentrations of IsoddA were greater than 100 microM for CEM, MOLT-4, IM9, and the HepG2-derived HBV-infected 2.2.15 (subclone P5A) cell lines but were 12 and 11 microM for human granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)