Flt3 ligand promotes myeloid dendritic cell differentiation of human hematopoietic progenitor cells: possible application for cancer immunotherapy

Current in vitro culture systems allow the generation of human dendritic progenitor cells (CFU-DCs). The aim of this study was to assess the effect of Flt3 ligand (FL) on the proliferation of human peripheral blood-derived myeloid CFU-DCs and their differentiation into more committed precursor cells (pDCs) using in vitro culture systems. Immunomagnetically separated CD34+ cells were cultured in serum-free, as well as in serum-containing, liquid suspension cultures to investigate the expansion and/or proliferation/differentiation of CFU-DCs, pDCs, and more mature dendritic cells (DCs). FACS-sorted CD34+Flt3+/- cells were cultured in methylcellulose to assay hematopoietic progenitors, including CFU-DCs. In the clonal cell culture supplemented with granulocyte/macrophage (GM) colony-stimulating factor (CSF), interleukin-4, and tumor necrosis factor alpha, the frequency of CFU-DCs was significantly higher in the CD34+Flt3+ fraction than in the CD34+Flt3- population, thus suggesting functional Flt3 expression on CFU-DCs. Serum-free suspension culture of CD34+ cells revealed the potent effect of FL on the expansion of CFU-DCs in synergy with GM-CSF and thrombopoietin (TPO). In addition, FL strongly induced the maturation of CFU-DCs into functional CD1a+ pDCs in serum-containing liquid suspension culture. Moreover, these FL-generated pDCs showed remarkable potential to differentiate into mature DCs with surface CD83/CD86 expression, which induced a distinct allogeneic T-cell response. These results clearly demonstrate that FL supports not only the proliferation of early hematopoietic progenitor cells, but also the maturation process of committed precursor cells along with the DC-lineage differentiation. Therefore, it is possible to develop a more efficient DC-based cancer immunotherapy using this specific cytokine combination, GM-CSF+TPO+FL in vitro in the near future.     

恶性造血细胞表面Flt3受体的表达及其对Flt3配体的反应性研究

目的 :研究恶性造血细胞表面Flt3受体的表达 ,TNFα和地塞米松 (DXM )对Flt3受体表达的作用及重组人Flt3配体 (rhFL)对恶性造血细胞增殖的影响。方法 :用流式细胞仪测定 18株体外培养的恶性造血细胞株细胞表面Flt3受体 ,用MTT法测定rhFL对恶性造血细胞增殖的影响。结果 :5株细胞表面存在Flt3受体。B淋巴瘤细胞株Raji、Daudi及多发性骨髓瘤细胞株 82 6 6表达高水平Flt3受体 ;髓系白血病细胞株HL 6 0和多发性骨髓瘤细胞株XG 6表达低水平的Flt3受体。用 10 -6mol/LDXM培养 2 4h后 ,上述细胞Flt3受体表达降低 ;用2 0ng/mlTNFα培养 2 4h后 ,Raji和 82 6 6细胞Flt3受体表达降低 ,HL 6 0和XG 6细胞Flt3受体表达增高 ,Daudi细胞Flt3受体表达不受影响。rhFL在 10～ 10 0ng/ml浓度时 ,刺激Raji和HL 6 0细胞的增殖 (P <0 0 5 ) ,但对绝大多数的恶性造血细胞体外无刺激作用。结论 :多数恶性造血细胞不表达Flt3受体 ;rhFL也不引起此类细胞的增殖。地塞米松降低Flt3受体的表达 ,有可能用于防止或降低FL对部分恶性造血细胞的刺激作用 ,使FL的应用更为安全。     

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.     

Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.     

Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by co-culture with feeder cells. The feeder cells are important for the growth of hESCs. A novel spontaneously immortalizated mouse fetal liver stromal cell line, named KM3, was isolated from a?13.5?day mouse fetal liver. In this study, we examined whether KM3 cells could be used as feeders to support the growth of hESCs. hESCs cultured on KM3 cells showed a similar proliferation rate and characteristics to mouse embryonic fibroblasts?(MEFs) after prolonged culture, including morphology, unlimited and undifferentiated proliferative ability, maintenance of normal karyotypes, formation of embryoid bodies in?vitro and typically immature teratomas in?vivo. Our results indicate that the immortalized KM3 cell line has the potential to support the growth and maintenance of hESCs. The cell line may be used for the large-scale expansion of hESCs in a low-cost and less labor-intensive manner.     

A phase III study of high-dose intensification without hematopoietic progenitor cells support for patients with high-risk primary breast carcinoma

Patients with more than nine ipsilateral lymph node involvement or inflammatory breast cancer have a 5-year survival rate of approximately 50%. We studied the efficacy of high-dose intensification, comparing it with the standard dose chemotherapy for patients with high-risk primary breast cancer. Patients with inflammatory breast cancer or more than nine ipsilateral lymph node involvement without evidence of distant metastasis were randomized to receive either standard dose 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC) every 3 weeks for nine courses (control) or six courses of FAC followed by two courses of cyclophosphamide (5.25 g/m2), etoposide (1,500 mg/m2), and cisplatin (165 mg/m2) (HDCVP). The study was terminated in 1998 because of slow accrual of patients. Forty-six patients were entered in the study. At 4 years, the overall survival was 72.8% (SE 11.9%) and 61.7% (SE 12.4%), and disease-free survival were 45.5% (SE 12.3%) and 33.7% (SE 11.9%) for the control and HDCVP groups, respectively (p = 0.757 and 0.720). With the small number of patients in our study, a small overall survival benefit of high-dose intensification compared with the standard therapy cannot be excluded. However, any substantial benefit is unlikely.     

Peripheral blood expansion of early progenitor cells after high-dose cyclophosphamide and rhGM-CSF

In 20 patients with non-Hodgkin lymphoma or breast cancer, high-dose cyclophosphamide induced, during the post-nadir period of rapid leucocyte recovery, on median day 19 about a 30-fold increase in the peak concentration of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming cells, and an even higher increase in the more immature pluripotent progenitors (CFU-Mix, 72-fold). After infusion of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), peak concentration was reached earlier (median day 15) and with further enhancements (159, 116 and 283-fold, respectively, in the number of CFU-GM, BFU-E and CFU-Mix). Most CFU-GM were immature, lacking the differentiation antigen CD15, and gave rise to large myeloid colonies, reflecting a high proliferative capacity of the founder cells. Very immature maphosphamide-resistant progenitors were detectable. The marked expansion in the circulating pool was predictable and reliable, allowing harvesting, after two or three leukaphereses, of sufficient haematopoietic progenitors for autologous bone-marrow reconstitution.     

IKAROS isoform 6 enhances BCR-ABL1-mediated proliferation of human CD34+ hematopoietic cells on stromal cells

The BCR-ABL1 induces chronic myelogenous leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL). Recent studies revealed high ratios of loss of the IKZF1 gene which encodes IKAROS in BCR-ABL1+ ALL and lymphoblastic crisis (LBC) of CML. However, little is known about the cooperativity between the aberrant IKAROS and BCR-ABL1 in primary human hematopoietic cells. We investigated the effects of expression of BCR-ABL1 and/or IK6, a natural dominant negative isoform of IKAROS, on proliferation and differentiation of human CD34+ cord blood cells with or without human bone marrow-derived stromal cells which support early B cell differentiation. Cell proliferation was remarkably enhanced by co-expression of BCR-ABL1 and IK6, with reduced expression of glycophorin A and increased expression of CD41, especially on stromal cells, compared with expression of BCR-ABL1 alone that resulted in expansion of erythroid progenitors. Interestingly, p190BCR-ABL1 showed higher dependency on stromal cells to stimulate cell growth with IK6, than p210BCR-ABL1. Furthermore, the cooperation was found to depend on direct cell adhesive interaction of hematopoietic progenitors with stromal cells. These findings suggest that IK6 and BCR-ABL1 synergistically contribute to leukemogenesis in human bone marrow stromal microenvironment, and may provide a clue to elucidate the mechanisms of leukemogenesis of Ph+ ALL and CML-LBC.     

Granulocyte-macrophage colony-stimulating factor modulation of the inhibitory effect of transforming growth factor-beta on normal and leukemic human hematopoietic progenitor cells.

Experiments were undertaken to investigate the molecular basis of primitive hematopoietic progenitor cell regulation in both the long-term culture system and in methylcellulose, particularly with a view to characterizing factors either able or unable to influence the behaviour of primitive leukemic cells from patients with chronic myeloid leukemia (CML). Long-term cultures of CML cells with or without irradiated normal marrow feeder layers were initiated from peripheral blood cells of CML patients with high white blood cell counts. Three weeks later the effect of exogenously added transforming growth factor-beta 1 (TGF-beta 1) on progenitor cycling status was examined. A single addition of 5 ng/ml TGF-beta 1 was able to reversibly arrest the otherwise uninterrupted turnover of primitive leukemic erythroid and granulopoietic progenitors for a period of up to 7 days both in the presence and absence of a normal adherent cell population. When TGF-beta 1 was incorporated into methylcellulose cultures, its ability to inhibit colony formation by CML progenitors showed the same differential activity on primitive cell types exhibited by normal progenitors. Dose-response curves for analogous populations of normal and leukemic cells were indistinguishable. Increasing the concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose colony assays decreased the sensitivity displayed by normal clonogenic cells to TGF-beta 1 and no differences were detectable when CML cells were used in such regulator competition experiments. These findings support a general model of primitive hematopoietic cell regulation in which entry into S-phase is determined at the intracellular level by multiple convergent pathways that may deliver either positive or negative signals from activated cell surface receptors for distinct extracellular factors. The present study shows for the first time that primitive CML progenitors exposed to TGF-beta 1 in vitro can be transiently blocked in a noncycling state for several days without loss of viability and that the mechanisms responsible for the emergence and maintenance of a clonal population of CML cells in vivo do not appear to involve changes in their sensitivity to TGF-beta 1. It is thus unlikely that the heightened proliferative activity exhibited by primitive CML progenitors both in vivo and in long-term culture can be explained by an abnormality in the intracellular mechanisms normally activated by TGF-beta 1 receptor-ligand binding. We suggest that primitive CML cells are either defective in their ability to see (or activate) endogenously produced TGF-beta 1, or are defective in their responsiveness to another, undefined, regulator.     

Immortalization of bone marrow-derived human mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the ability to support expansion of cord blood hematopoietic progenitor cells

Human marrow-derived mesenchymal stem cells (MSC), which have the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose and bone marrow stroma, were transduced with a retroviral vector carrying the simian virus 40 large T antigen, hygromycin-resistant gene and herpes simplex virus thymidine kinase gene, that can be excised by Cre/loxP site-specific recombination. This resulted in establishment of an MSC cell line, HMSC-1, which retained original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than parental cells. HMSC-1 expressed mRNAs of BMP-4, Jagged-1, and SCF that are known to promote hematopoiesis. Human CB CD34+ hematopoietic progenitor cells (HPC) cultured on a layer of HMSC-1 cells showed high expansion of CD34+CD38- immature HPC, capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. This cell line may be of value for developing strategies for ex vivo expansion of human HPC.     

Human Flt3 ligand from Pichia pastoris inhibits growth of lymphoma and colon adenocarcinoma in mice

Potent effects of Flt3 ligand (FL) on the development of the immune system have generated much interest in application of FL in cancer immunotherapy. OBJECTIVE: To evaluate the effects of Pichia pastoris secreted rhFL on the growth of mouse EL-4 lymphoma and C26 colon adenocarcinoma injected in syngeneic mice for the first time. METHODS: Mice were placed into one of two treatment groups. 2 x 10(5) EL-4 or C26 cells were injected subcutaneously (SC.) into mice on day 0. Group 1 received subcutaneous PBS injections from Day -7 to Day 14 and group 2 received subcutaneous rhFL injections at 30 microg/day from Day -7 to Day 14. Serial tumor areas were measured. On Day 22, mice from each group were sacrificed, and weight of tumors and spleens were evaluated. Data analysis used Student t tests. RESULTS: Pichia pastoris secreted rhFL resulted in tumor growth delay for both EL-4 lymphoma and C26 colon adenocarcinoma compared with control (P < 0.01). Tumors from rhFL-treated mice were smaller (P < 0.01) than controls while spleens larger (P < 0.01) than controls. Histological examination of tumor sections revealed an obvious increase in regions composed largely of infiltrating cells in the rhFL-treated tumors. Infiltrating cells could be detected in clusters among tumors from mice treated with rhFL whereas these cells were only occasionally detected in sections of control tumors. CONCLUSION: Treatment of rhFL expressed from Pichia pastoris resulted in an antitumor response against EL-4 and C26 tumors injected in syngeneic mice.     

Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells

The purpose of this study was to evaluate the individual radioprotective effects of 4 distinct purified recombinant human hematopoietic growth factors, namely recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF), recombinant human granulocyte colony stimulating factor (rG-CSF), recombinant human interleukin 1 (rIL-1), and recombinant human interleukin 2 (rIL-2) on human myeloid (CFU-GM) and erythroid (BFU-E) bone marrow progenitor cells. We demonstrate that (a) preconditioning with rGM-CSF, rG-CSF, or rIL-1 enables CFU-GM to repair sublethal radiation damage and renders CFU-GM less radiosensitive, (b) preconditioning with rGM-CSF or rIL-1 enables BFU-E to repair sublethal radiation damage, and (c) preconditioning with rIL-2 does not increase the radiation survival of CFU-GM or BFU-E. The effects of recombinant growth factors, in particular rGM-CSF, on the radiation damage repair, radiosensitivity, and proliferative activity of bone marrow progenitor cells resulted in a substantial increase in the mean numbers of progenitor cell-derived hematopoietic colonies in irradiated marrow samples. The effects of rGM-CSF on the radiation response of CFU-GM and BFU-E, and the effects of rG-CSF as well as rIL-1 on the radiation response of CFU-GM did not appear to require the presence of T-cells/T-cell precursors, NK-cells, B-cells/B-cell precursors, monocytes, macrophages, MY8 antigen positive non-CFU-GM myeloblasts, promyelocytes, myelocytes, metamyelocytes, granulocytes, or glycophorin A positive erythroid cells since virtually identical results were obtained with unsorted marrow samples or highly purified fluorescence activated cell sorter (FACS) isolated progenitor cell suspensions. To our knowledge, this report represents the first study on recombinant human growth factor-induced modulation of the radiation responses of normal human bone marrow progenitor cells.     

The effects of transforming growth factor beta 3 on the growth of highly enriched hematopoietic progenitor cells derived from normal human bone marrow and peripheral blood

The effects of transforming growth factor beta 3 (TGF-beta 3) on growth in semisolid cultures of enriched hematopoietic progenitors derived from normal human marrow and blood were evaluated. Conditioned media from the Mo-T cell line (MoCM) were the source of colony-stimulating factors used to optimally stimulate primitive progenitors. To assess whether a proportion of granulocyte/monocyte (GM) progenitors were prevented from cycling, all sizes of GM aggregates were evaluated from 3 to 20 days. The activity of TGF-beta 3 on the growth of erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) was similar to that observed for TGF-beta 1. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or 72 h after initiation of culture, did not significantly affect the total number of marrow GM aggregates at 3, 7, 14, and 20 days, but TGF-beta 3 (1,000 pmol/liter), added initially, reduced the total number of blood GM aggregates. This suggests that some blood GM progenitors might be blocked from cycling but that the great majority of marrow GM progenitors are not blocked. Whether TGF-beta 3 (10, 100, and 1,000 pmol/liter) was added initially or after 72 h of stimulation by MoCM, there was a dose-dependent reduction of marrow and blood GM colony size even when the total number of colonies was unaffected. TGF-beta 3 (10, 100, and 1,000 pmol/liter), added initially or at 72 h, reduced in a dose-dependent manner the size of marrow and blood-derived BFU-E. TGF-beta 3 (1,000 pmol/liter) was more likely to reduce the total number of marrow and blood BFU-E, and this increased sensitivity of the erythroid lineage may prevent the development of this population in colonies derived from multipotential colony-forming unit-granulocyte/erythroid/monocyte (CFU-GEM). The results suggest that the main effect of TGF-beta 3 and TGF-beta 1 is to slow the rate of proliferation of hematopoietic progenitors rather than to prevent them from beginning proliferation. This results in a reduction in colony size which prevents the identification of primitive versus mature progenitor on the basis of standard criteria of colony size.     

Proliferative response of human acute myeloid leukemia cells and normal marrow enriched progenitor cells to human recombinant growth factors IL-3, GM-CSF and G-CSF alone and in combination.

The effects of human recombinant colony-stimulating factors (r-CSFs), interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on inducing the growth of colonies derived from patients with acute myeloid leukemia (AML) (CFU-L) were investigated and compared to the proliferative response of CFU-GM derived from highly enriched normal blast cell populations. The effects of GM-CSF and IL-3 alone were similar. Both only minimally stimulated normal colonies derived from CFU-GM when compared to stimulation with MoCM (a mean of 28% of the total colonies and 17% of the colonies greater than 100 cells obtained with MoCM). Similarly, the number of leukemic colonies was substantially less than with MoCM (less than 30% of MoCM) in all but 3/10 AML patients and both were only able to significantly stimulate CFU-L derived colonies greater than 50 cells from 2/10 patients. G-CSF alone stimulated some CFU-L derived colony growth in 9/10 patients but the number stimulated was minimal relative to MoCM in five of the patients and significant stimulation of colonies greater than 50 cells occurred in only one patient. The mean number of normal CFU-GM derived colonies stimulated by G-CSF was 41% of the total colonies and 34% of the colonies greater than 100 cells generated by MoCM. The combination of G-CSF with GM-CSF and G-CSF with IL-3 resulted in a synergistic or additive increase in the number of CFU-L in 5/10 and 7/10 patients, respectively, and a synergistic increase in the size of CFU-L in 5/10. The same combinations resulted in a significant synergistic effect on size of normal CFU-GM derived colonies. There was no evidence of a synergistic increase in the number or size of CFU-L and CFU-GM derived colonies stimulated with GM-CSF in combination with IL-3. In addition, a combination of all three (G-CSF + GM-CSF + IL-3) did not enhance the effect of G-CSF + GM-CSF or G-CSF + IL-3. These results suggest that there is significant heterogeneity among AML patients in the pattern of responsiveness of the leukemic cells to the recombinant growth factors. In addition, their responsiveness does not significantly differ from that of normal progenitors. In view of the current clinical trials with r-CSFs and cytotoxic drugs in AML patients, this issue is important and worthy of further investigation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer.

PURPOSE: To evaluate preoperative dendritic cell (DC) mobilization and tumor infiltration after administration of Flt3 ligand (Flt3L) to patients with metastatic colon cancer. PATIENTS AND METHODS: Twelve patients with colon cancer metastatic to the liver or lung received Flt3L (20 microg/kg/d subcutaneously for 14 days for one to three cycles at monthly intervals) before attempted metastasectomy. The number and phenotype of DCs mobilized into peripheral-blood mononuclear cells (PBMCs) were evaluated by flow cytometry. After surgical resection, metastatic tumor tissue was evaluated for DC infiltration. In vivo immune responses to recall antigens were measured. RESULTS: After Flt3L administration, on average, the total number of leukocytes in the peripheral blood increased from 5.9 +/- 1.0 x 10(3)/mm(3) to 11.2 +/- 3.8 x 10(3)/mm(3) (mean +/- SD, P: =. 0001). The percentage of CD11c(+)CD14(-) DCs in PBMCs increased from 2.4% +/- 1.8% to 8.8% +/- 4.7% (P: =.004). Delayed-type hypersensitivity (DTH) responses to recall antigens (CANDIDA:, mumps, and tetanus) showed marginally significant increases in reactivity after Flt3L administration (P: =.06, P: =.03, and P: =.08, respectively). An increase in the number of DCs was observed at the periphery of the tumors of patients who received Flt3L compared with those of patients who had not. CONCLUSION: Flt3L is capable of mobilizing DCs into the peripheral blood of patients with metastatic colon cancer and may be associated with increases in DC infiltration in the peritumoral regions. Flt3L mobilization is associated with a trend toward increased DTH responses to recall antigens in vivo. The use of Flt3L to increase circulating DCs for cancer immunotherapy should be considered.     

Variable effects of tamoxifen on human hematopoietic progenitor cell growth and sensitivity to doxorubicin

To determine the influence of tamoxifen on the drug sensitivity of normal human hematopoietic progenitor cells, T-cell- and adherent-cell depleted human bone marrow mononuclear cells (T^–, Ad^–) were exposed in vitro to 5 M tamoxifen for 24 h. The effects of tamoxifen were highly variable, as exposure to tamoxifen produced an increase (97%±12.3%) in the growth of day-12 committed myeloid progenitors (CFU-GM) in only four of ten experiments utilizing bone marrow from different donors. When T^–, Ad^– myeloid progenitor cells treated with tamoxifen were subsequently exposed to doxorubicin, 7 of 14 experimental samples studied demonstrated a net increase in the number of surviving clonogenic cells as compared with cells exposed to doxorubicin alone. Tamoxifen also stimulated the growth of a more purified (CD34⁺-selected) progenitor cell population in four of four experiments (by 62.5%±4.9%) but did not increase the survival of these cells upon exposure to doxorubicin; in fact, in five of ten experimental samples, tamoxifen enhanced cell sensitivity to doxorubicin. Taken together, these observations indicate that tamoxifen produces variable stimulation of committed myeloid progenitor cell growth in vitro. Furthermore, while under some circumstances, tamoxifen appears to have the capacity to enhance CFU-GM survival in the presence of doxorubicin, this drug combination may also result in enhanced toxicity to normal bone marrow progenitors.Abbreviations CFU-GM granulocyte-macrophage colony-forming units - T^–, Ad^– T-cell- and adherent-cell-depleted bone marrow mono-nuclear cells     

Clinical promise of new hematopoietic growth factors: M-CSF, IL-3, IL-6

Hematopoietic growth factors are reaching maturity in clinical trials. There is a wide spectrum of disorders of bone marrow dysfunction that can be effectively treated by currently available hematopoietic growth factors. Newer growth factors are entering clinical trials. rhM-CSF has a variety of biological activities. It may be useful in hematology/oncology and infectious disease settings. Recombinant human interleukin-3 (rhIL-3) has undergone extensive trials in nonhuman primates that suggest that this hematopoietin is a potent stimulus of bone marrow function following chemotherapy and may be synergistic with other growth factors, such as rhGM-CSF. Other pleotrophic hematopoietic growth factors, such as interleukin-6, are currently being developed and may exert a wide spectrum of activities in disease states.     

The effects of exogenous growth factors on matrix metalloproteinase secretion by human brain tumour cells

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-beta1,2) and vascular endothelial growth factor (VEGF)].on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-beta1 and TGF-beta2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.