Human responses against HER-2-positive cancer cells in human immune system-engrafted mice.

BACKGROUND: Human immune system (HIS)-engrafted mice are new tools to investigate human immune responses. Here, we used HIS mice to study human immune responses against human HER-2-positive cancer cells and their ability to control tumour growth and metastasis. METHODS: BALB/c Rag2(-/-), Il2rg(-/-) mice were engrafted with CD34(+) or CD133(+) human cord blood hematopoietic stem cells (HSC) and vaccinated with human HER-2-positive cancer cells SK-OV-3 combined to human IL-12. RESULTS: Both CD34(+) or CD133(+) human HSC gave long-term engraftment and differentiation, both in peripheral blood and in lymphoid organs, and production of human antibodies. Vaccinated mice produced specific anti-HER-2 human IgG. An s.c. SK-OV-3 challenge was significantly inhibited (but not abolished) in both vaccinated and non-vaccinated HIS mice. Tumours were heavily infiltrated with human and murine cells, mice showed NK cells and production of human interferon-γ, that could contribute to tumour growth inhibition. Vaccinated HIS mice showed significantly inhibited lung metastases when compared with non-vaccinated HIS mice and to non-HIS mice, along with higher levels of tumour-infiltrating human dendritic cells. CONCLUSION: Anti-HER-2 responses were elicited through an adjuvanted allogeneic cancer cell vaccine in HIS mice. Human immune responses elicited in HIS mice effectively inhibited lung metastases.

Effects of granulocyte colony stimulating-factor in a rat model of acute liver injury.

BACKGROUND/AIM: Controversial experimental observations suggest that granulocyte colony stimulating-factor may promote hepatic regeneration after hepatectomy and chemical injury either by directly stimulating adult liver cells or facilitating the mobilization of bone marrow cells and their homing to the liver. We investigated whether different schedules of granulocyte colony stimulating-factor administration protect against experimental acute liver injury. METHODS: Acute liver injury was induced in Sprague-Dawley fed rats by injecting a single intraperitoneal dose of carbon tetrachloride. Recombinant human granulocyte colony stimulating-factor or vehicle was given daily after intoxication (4 days) or before (7 days) and after carbon tetrachloride administration. Liver injury and regeneration were assessed 2 and 4 days after damage. Bone marrow cells mobilization was evaluated by the white blood cell count and the assessment of circulating clonogenic haematopoietic progenitors (colony forming unit-cells). RESULTS: In this experimental model, although granulocyte colony stimulating-factor induced the significant mobilization of colony forming unit-cells, the study cytokine had no effect on liver injury (serum alanine amino transaminase level and necrotic index) and liver regeneration (mitotic index and bromodeoxyuridine incorporation), regardless of the administration schedule. CONCLUSIONS: This study does not support the conclusion that: (1) granulocyte colony stimulating-factor exerts a protective effect against toxic-induced, non-lethal acute liver injury and (2) promotes hepatocyte regeneration.

Italian consensus conference for the outpatient autologous stem cell transplantation management in multiple myeloma.

Multiple myeloma (MM) is the leading indication for autologous stem cell transplantation (ASCT) worldwide. The safety and efficacy of reducing hospital stay for MM patients undergoing ASCT have been widely explored, and different outpatient models have been proposed. However, there is no agreement on the criteria for selecting patients eligible for this strategy as well as the standards for their clinical management. On the basis of this rationale, the Italian Group for Stem Cell Transplantation (GITMO) endorsed a project to develop guidelines for the management of outpatient ASCT in MM, using evidence-based knowledge and consensus-formation techniques. An expert panel convened to discuss the currently available data on the practice of outpatient ASCT management and formulated recommendations according to the supporting evidence. Evidence gaps were filled with consensus-based statements. Three main topics were addressed: (1) the identification of criteria for selecting MM patients eligible for outpatient ASCT management; (2) the definition of standard procedures for performing outpatient ASCT (model, supportive care and monitoring during the aplastic phase); (3) the definition of the standard criteria and procedures for re-hospitalization during the aplastic phase at home. Herein, we report the summary and the results of the discussion and the consensus.

Concomitant and sequential administration of recombinant human granulocyte colony-stimulating factor and recombinant human interleukin-3 to accelerate hematopoietic recovery after autologous bone marrow transplantation for malignant lymphoma.

PURPOSE: To assess the safety, tolerability, and hematopoietic efficacy of sequential and concomitant administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human interleukin-3 (rhIL-3), to accelerate reconstitution of hematopoiesis following myeloablative chemotherapy and autologous bone marrow transplantation (ABMT) for heavily pretreated lymphoma patients. PATIENTS AND METHODS: Fifty-four consecutive patients with refractory or relapsed non-Hodgkin's lymphoma (NHL; n = 30) and Hodgkin's disease (HD; n = 24) were studied. Two different conditioning regimens were used for ABMT: carmustine, cyclophosphamide, etoposide, and cytarabine (BAVC) and carmustine, melphalan, etoposide, and cytarabine (BEAM) for NHL and HD, respectively. Patients were enrolled sequentially onto one of three treatment groups: group 1, G-CSF (5 micrograms/kg/d subcutaneously [SC]) from day +1 after reinfusion of autologous marrow (n = 23); group 2, G-CSF from day +1 combined with IL-3 (10 micrograms/kg/d SC) from day +6 (n = 22, overlapping schedule); and group 3, G-CSF treatment discontinued at day +6 before initiation of IL-3 administration (n = 9, sequential schedule). In the three groups, growth factor(s) was administered until the granulocyte count was greater than 0.5 x 10(9)/L for 3 consecutive days. RESULTS: The study cytokines were generally well tolerated. No side effects were observed when G-CSF was given alone. Four of 31 patients (12.9%) who received SC IL-3 had one severe adverse event defined as World Health Organization (WHO) grade 3 to 4 toxicity (fever, n = 2; pulmonary toxicity, n = 2) and were withdrawn from the study. Groups 2 and 3 did not differ as for treatment tolerability, whereas we observed a trend toward a faster hematopoietic recovery when IL-3 was administered concomitant with G-CSF from day 6 (ie, group 2). Pooled together, patients who received IL-3 showed a median time to achieve a granulocyte count greater than 0.1 and greater than 0.5 x 10(9)/L of 8 and 11 days, respectively. The median time to an unsupported platelet count greater than 20 and 50 x 10(9)/L was 15 and 20 days, respectively, and only one patient did not reach a normal platelet count. The median number of days to hospital discharge was 16 after ABMT (range, 12 to 29). When the hematologic reconstitution of patients in groups 2 and 3 was compared with that of patients in group 1, the addition of IL-3 resulted in a significant improvement of multilineage hematopoietic recovery, lower transfusion requirements, a lower number of documented infections, and shorter hospitalizations. CONCLUSION: We conclude that the combination of G-CSF and IL-3 is safe and well tolerated in intensively pretreated lymphoma patients, undergoing ABMT and results in rapid hematopoietic recovery following myeloablative chemotherapy.

Molecular remission after allogeneic or autologous transplantation of hematopoietic stem cells for multiple myeloma.

PURPOSE: To assess the clinical relevance of minimal residual disease (MRD) in patients with multiple myeloma (MM), 50 patients were monitored while they were in complete clinical remission (CCR) after autologous or allogeneic stem-cell transplantation. PATIENTS AND METHODS: Stringent molecular monitoring using clonal markers based on rearranged immunoglobulin heavy-chain genes was performed in 44 of 50 MM patients in CCR. Molecular clinical remission (MCR) was defined as more than one consecutive negative polymerase chain reaction (PCR) test result. RESULTS: Twelve (27%) of 44 molecularly monitored patients achieved MCR; four of the 12 became PCR-positive, and one of these four relapsed. In comparison with patients who did not achieve MCR, patients who achieved MCR had a significantly lower relapse rate (41% v 16%; P <.05) and longer relapse-free survival (35 v 110 months; P <.005). Fourteen of 26 patients in CCR who had received allografts were evaluated on a molecular basis: seven (50%) of the 14 achieved MCR and did not relapse; one of the seven remaining patients relapsed. Thirty of 47 patients in CCR who received autografts were evaluated on a molecular basis: five (16%) of the 30 achieved MCR; two of these five became PCR-negative, and one of these two relapsed. Ten of the 25 remaining patients later relapsed. For these nonrandomized groups, the higher MCR rate after allograft procedures was statistically significant (P <.01; Fisher's exact test). CONCLUSION: MCR can be obtained in a relatively high proportion of MM patients who have achieved CCR after undergoing allograft procedures and in a smaller fraction of patients after undergoing autograft procedures. In approximately one fourth of MM patients who achieve CCR after transplantation, it may be possible to keep the disease burden constantly below the PCR threshold. Because MCR was associated with prolonged relapse-free survival, these patients could have a relatively favorable clinical outcome.

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)

Combination of hematopoietic growth factors containing IL-3 induce acute myeloid leukemia cell sensitization to cycle specific and cycle non-specific drugs.

Laboratory studies have suggested that hematopoietic growth factors (GF), combined with cytosine-arabinoside (Ara-C) can enhance cytotoxic effects of this agent against acute myeloid leukemia (AML) cells. While clinical trials based on this growth factor/chemotherapy combination (GF/CT) are progressing with discordant results, further information regarding the underlying mechanisms have been reported supporting this rationale and requiring additional investigation. To assess the role of cytokinetic changes in the GF/CT strategy and to evaluate if chemotherapeutic agents regimens other than Ara-C, when combined with GF, can enhance their cytotoxic effects, we have primed AML blasts with two cytokine combinations and then exposed these cells to the S-phase specific agent Ara-C as well as to the phase non-specific drug daunorubicin (DNR) and to the alkylating agent 4-hydroperoxycyclophosphamide (4-HC). The two cytokine combinations used for priming AML blasts were: (i) interleukin-3 (IL-3) + granulocyte-macrophage colony-stimulating factor (GM-CSF) + granulocyte colony-stimulating factor (G-CSF); and (ii) GM + G-CSF. Cytokinetic analysis in ten AML samples and clonogenic growth of leukemic colonies (CFU-L) in methylcellulose were used to detect proliferative and cytotoxic effects on AML samples. We report that in AML clonogenic cell growth can be stimulated by cytokines in 50% of the samples (4/8), and that Ara-C sensitization clearly occurs in two out of these four samples. Among the different cytokine combinations tested, the one containing IL-3 was the most effective through a cytokinetic mechanism consistent with recruitment (averaged G0 decrease p = 0.04; S-phase increase p = 0.005). Furthermore we observed increased cytotoxicity also to the phase non-specific drugs DNR and 4-HC, which may be mediated by other mechanisms recently described. We conclude that GF/CT combinations may also be beneficial in regimens containing drugs other than Ara-C, used for AML treatment, including bone marrow transplantation conditioning regimens.

In vitro cytotoxicity of VP-16-213 and nitrogen mustard: agonistic on tumor cells but not on normal human bone marrow progenitors.

The possible presence of tumor cells in remission bone marrow (BM) is one of the major problems for the success of autologous BM transplantation (ABMT), because the reinfusion of viable malignant cells may result in relapse. In this study we attempted the purging of the malignant cells by the use of VP-16-213 (VP-16) and nitrogen mustard (NM) either alone or in combination. Four cell lines from various hematological malignancies were utilized: SK-DHL-2 was established from a B-cell diffuse histiocytic lymphoma; RAJI was from an Epstein-Barr virus (EBV)-infected B-cell lymphoma cell line; K-562 were from a chronic myelogenous leukemia (CML) blastic crisis; and HL-60, derived from a human promyelocytic leukemia, were used in exponential growth phase. Four logs of tumor cell-elimination were observed after 1-h incubation of RAJI cells with 25 micrograms/ml of VP-16. K-562 and SK-DHL-2 cells showed a greater than 4 logs reduction after 1-h exposure to 75 micrograms/ml of VP-16, and HL-60 cell line growth was inhibited by 3.2 logs. Under the same conditions (i.e., the treatment with 75 micrograms/ml), we observed a mean recovery of 2.7% of BM granulocyte-macrophage colonies (granulocyte-macrophage colony-forming units, CFU-GM), 3.2% of erythroid (erythroid burst-forming units, BFU-E), and 2.5% of pluripotent (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) progenitors, respectively. More than 3 logs reduction of leukemia and lymphoma cell lines were reached following 1-h treatment with 1 micrograms/ml of NM. After exposure to the same concentration of the drug we obtained 2.5% CFU-GM, 1.2% BFU-E, and 2% CFU-GEMM recovery. A drug mixture containing constant doses of VP-16 (10 and 20 micrograms/ml) and NM (1 micrograms/ml) reduced HL-60 and SK-DHL-2 cell growth to undetectable levels (i.e., 4 and 5 logs elimination) in the presence of an excess of irradiated BM cells, whereas it did not further affect the recovery of the BM precursors as compared to the single drugs used alone. These results suggest that the combination of these two drugs at the selected dose level could provide a better therapeutic index (i.e., higher tumor cell killing coupled with no additional cytotoxic effect on normal BM cells) than the same chemotherapeutic agent used alone and that this mixture may be useful for the "ex vivo" treatment of BM grafts.

Interleukin-11 stimulates the proliferation of human hematopoietic CD34+ and CD34+CD33-DR- cells and synergizes with stem cell factor, interleukin-3, and granulocyte-macrophage colony-stimulating factor.

We have studied the effects of recombinant human interleukin-11 (rhIL-11), alone and combined with stem cell factor (SCF or c-kit ligand), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the proliferation of highly enriched human hematopoietic CD34+ and CD34+CD33-DR- progenitor cells. CD34+ cells were purified using the avidin-biotin immunoabsorption technique and CD33+DR+ cells were subsequently removed by immuno-magnetic separation. The colony assays were performed in the presence and absence of exogenous serum. IL-11, as a single agent, induced the growth of a small number of colony-forming units-granulocyte/macrophage (CFU-GM) derived from purified CD34+ cells and failed to support the colony growth of CD34+CD33-DR- cells. The addition of erythropoietin (Epo) to IL-11 induced the growth of erythroid progenitors (BFU-E) derived from CD34+ cells but not from the same population depleted of CD33+DR+ cells. The combination of IL-11 with SCF, IL-3, or GM-CSF, in the presence of Epo, resulted in a synergistic or additive increase in the number of CFU cells (CFU-C) derived from both cell fractions. Moreover, the addition of SCF to IL-11 stimulated the development of macroscopic erythroid and multilineage colonies (CFU-GEMM) containing more than 10(4) cells. A combination of three factors (IL-11, SCF, and IL-3) resulted in the increase of the number of colonies arising from CD34+ and CD34+CD33-DR- cells (but not of their size) compared to the cultures treated with IL-11 plus SCF or IL-11 plus IL-3. The pattern of proliferative response of primitive hematopoietic progenitor cells to IL-11 in serum-free conditions was very similar to the cultures grown in serum-containing medium. It is noteworthy that IL-11 and SCF yielded colony formation that was comparable to that observed in the presence of serum. The effects of IL-11 on CD34+CD33-DR- cells were also studied in a short-term suspension culture system, which was shown to be specific for evaluating the proliferation of pluripotent hematopoietic precursors (Delta assay). In this system, IL-11 had a minimal effect on its own, whereas IL-11 plus SCF acted synergistically and their proliferative activity was improved by the addition of GM-CSF. These experiments indicate that IL-11 may be considered a "permissive" cytokine, capable of initiating the proliferation of very primitive human hematopoietic cells, which are then able to respond to late-acting CSFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Stem cell factor (c-kit ligand) enhances the interleukin-9-dependent proliferation of human CD34+ and CD34+CD33-DR- cells.

We have studied the effects of recombinant human interleukin-9 (IL-9), alone and combined with stem cell factor (SCF, c-kit ligand), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the clonogenic proliferation of highly enriched human hematopoietic CD34+ and CD34+CD33-DR- progenitor cells. Colony assays were performed under serum-containing and serum-free conditions. IL-9, as a single agent, did not support colony formation. The addition of erythropoietin (Epo) to IL-9 induced the growth of erythroid progenitors (BFU-E) derived from both CD34+ and CD34+CD33-DR- cells. The IL-9-dependent growth of BFU-E derived from CD34+ cells was increased in an additive manner by SCF and, to a lesser extent, by IL-3, whereas CD34+CD33-DR- erythroid precursors were also responsive to GM-CSF in combination with IL-9. The addition of SCF to IL-9 did stimulate the development of CD34+ and CD34+CD33-DR- macroscopic, multicentered BFU-E and multilineage colonies (CFU-GEMM). When IL-9 was used in serum-free conditions, the growth of CD34+ and CD34+CD33-DR- BFU-E was observed in the presence of Epo. Moreover, a marked synergy on BFU-E colony formation was evident when IL-9 was combined with SCF, and their activity was enhanced by the addition of IL-3. IL-9 showed a negligible proliferative activity on colony-forming units-granulocyte/macrophage (CFU-GM). However, it increased the number of CD34+CD33-DR- CFU-GM responsive to IL-3 (37% of the colonies generated by phytohemagglutinin-stimulated lymphocyte conditioned medium [PHA-LCM]). The effects of IL-9 on CD34+CD33-DR- cells were also studied in a short-term suspension culture system, which evaluates the proliferation of progenitors earlier than day 14 CFU-C (Delta assay). In this system, IL-9 had a minimal activity on its own. In combination with SCF, however, it induced a nine-fold expansion of CD34+CD33-DR- cells, which generated a greater number of CFU-GM than BFU-E in secondary methylcellulose cultures. These experiments indicate that IL-9 induces the proliferation of very primitive human erythroid cells, and this effect is potentiated by SCF and other cytokines. Furthermore, IL-9 synergizes in vitro with the c-kit ligand in expanding the pool of early pluripotent hematopoietic progenitor cells.

TGF-beta 3 protects normal human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide in vitro.

In this study we have investigated the ability of transforming growth factor-beta 3 (TGF-beta 3, 1000 pM) to protect hematopoietic bone marrow (BM) progenitor cells from the cytotoxic activity of 4-hydroperoxycyclophosphamide (4-HC, 100 microM) in vitro. Hematopoietic progenitors were purified by negative depletion of accessory and maturing cells or enriched by positive (CD 34+ cells) selection. For comparison the same treatment was tested on three different lymphoid cell lines CEM, SK-DHL-2, and LY-16. The experimental protocol was designed to mimic ex vivo purging conditions. Therefore, tumor cells and enriched hematopoietic precursors were mixed with irradiated BM cells. Our results demonstrated that preincubation of enriched progenitor cells with TGF-beta 3 for up to 72 h followed by 4-HC treatment resulted in an increased survival of colonies derived from granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) colony-forming cells, whereas a substantially lower number of colonies was observed in the control group. Similar results were observed when BM cells were first treated with 4-HC followed by TGF-beta 3 incubation for 24 or 48 h. In contrast, TGF-beta 3 provided no protection to the 4-HC cytotoxicity toward the lymphoma and leukemia cell lines. Three to four log of tumor cell killing was induced by 4-HC in the presence or absence of preincubation with TGF-beta 3. These data suggest that TGF-beta 3 is able to protect normal BM progenitors from the cytotoxic activity of an alkylating agent (4-HC) in vitro, whereas it does not offer any protection to lymphoma cell lines. These findings will have important implications for developing better purging conditions for autologous GM transplantation.

Proliferation of human hematopoietic progenitors in long-term bone marrow cultures in gas-permeable plastic bags is enhanced by colony-stimulating factors.

We compared the recovery of human hematopoietic progenitors in long-term bone marrow culture (LTBMC) initiated in tissue culture (TC) flasks to that in "Lifecell" bags, which are gas-permeable plastic bags in which feeder-layer cells cannot adhere. Our results showed that granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) cumulative recovery in cultures from normal donor marrow, expressed as a percent of the initial inoculum, was not statistically different in the two culture systems up to week 8, when the cultures were terminated: 31.5 +/- 19 (flask) vs 30 +/- 14 (bag) and 15.5 +/- 12 (flask) vs 11.5 +/- 8 (bag), respectively. The effects of weekly addition of recombinant human (r-hu)-interleukin 1 (IL1) and r-hu-interleukin 3 (IL3) were then studied, alone and combined, at two different concentrations. Addition of IL1, either alone or combined with IL3, in LTBMC established in flasks induced an increase of hematopoietic progenitors for the first week, but BFU-E and CFU-GM were no longer detectable at weeks 4 and 6, respectively. Analysis of adherent layer cells showed a decreased cellularity, no adipogenesis, and early disappearance of bone marrow (BM) progenitors, whereas the cycling rate of myeloid precursors, by cytosine arabinoside (Ara-C) suicide assay, was similar to that of untreated cultures. Conversely, IL3 alone (5 ng/ml) resulted in 3.6- and 5.4-fold peak increases for CFU-GM and BFU-E, respectively, at week 1 (adherent plus nonadherent cells), and the recovery of BM cells was still higher than that of control flasks at week 8. By comparison, stimulation with colony-stimulating factors (CSFs) of BM cells grown in bags never affected the longevity of the culture. Addition of IL3 (5 ng/ml) induced a higher recovery of total cells, CFU-GM (range: 1.6- to 15-fold peak increase during the culture), and BFU-E (1.2- to 3-fold) compared to the untreated controls. Bags treated with IL1 alone demonstrated only transient beneficial effects, and the number of hematopoietic precursors fell below the level of control bags during the culture. IL1 and IL3 induced 1.8- and 5.3-fold peak increases in BFU-E and CFU-GM at weeks 1 and 4, respectively. Simultaneous flow cytometric analysis of CD34+/CD33+ cells and DNA content showed increased numbers and proliferation of the committed BM progenitors when CSFs were added to the bag.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming growth factor beta3 inhibits chronic myelogenous leukemia hematopoiesis by inducing Fas-independent apoptosis.

OBJECTIVE: Transforming growth factor beta3 (TGF-beta3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-beta3 on highly purified CD34+ cells and more immature CD34-DR(-) cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors. MATERIALS AND METHODS: Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late-acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively. RESULTS: The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-beta3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3-and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-beta3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-beta3 demonstrated a greater inhibitory activity on primitive CD34+DR cells than on more mature CD34+ cells. Again, CML CD34+DR(-) cells were significantly more inhibited by TGF-beta3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-beta induces cell cycle arrest in G(1) phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-beta3 preferentially triggered the programmed cell death of CML CD34-cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion. We demonstrate that TGF-beta3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

Proliferative response of human marrow myeloid progenitor cells to in vivo treatment with granulocyte colony-stimulating factor alone and in combination with interleukin-3 after autologous bone marrow transplantation.

We have recently reported that the hematologic recovery of patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD) undergoing autologous bone marrow transplantation (BMT) is significantly faster when recombinant human interleukin-3 (rhIL-3) is combined with recombinant human granulocyte colony-stimulating factor (rhG-CSF) in comparison with patients receiving G-CSF alone. In this paper, we studied the kinetic response and concentration of BM progenitor cells of 17 patients with lymphoid malignancies submitted to autologous BMT and treated with the G-CSF/IL-3 combination. The results were compared with those of five lymphoma patients receiving the same pretransplant conditioning regimen followed by G-CSF alone. rhG-CSF was administered as a single subcutaneous (sc) injection at the dose of 5 micrograms/kg/d from day 1 after reinfusion of autologous stem cells; rhIL-3 was added from day 6 at the dose of 10 micrograms/kg/d sc (overlapping schedule). In both groups (G-CSF- and G-CSF/IL-3-treated patients), cytokine administration was discontinued when the absolute neutrophil count (ANC) was >0.5 x 10(9)/L of peripheral blood (PB) for 3 consecutive days. After treatment with the CSF combination, the percentage of marrow colony-forming units-granulocyte/macrophage (CFU-GM) and erythroid progenitors (BFU-E) in S phase of the cell cycle increased from 9.3 +/- 2% to 33.3 +/- 12% and from 14.6 +/- 3% to 35 +/- 6%, respectively (p < 0.05). Similarly, we observed an increased number of actively cycling megakaryocyte progenitors (CFU-MK and BFU-MK). Conversely, G-CSF augmented the proliferative rate of CFU-GM (22.6 +/- 0.6% compared to a baseline value of 11.5 +/- 3%; p < 0.05) but not of BFU-E, CFU-MK, or BFU-MK, and the increase of S-phase CFU-GM was significantly lower than that observed in the posttreatment samples of patients receiving IL-3 in addition to G-CSF. The frequency of hematopoietic precursors in the BM, expressed as the number of colonies formed per number of cells plated, was unchanged or slightly decreased in both groups of patients. Because of the increase in marrow cellularity, however, a significant augmentation of the absolute number of both CFU-GM (3605 +/- 712/mL BM vs. 2213 +/- 580/mL; p < 0.05) and BFU-E (4373 +/- 608/mL vs. 3027 +/- 516/mL; p < 0.05) was reported after treatment with G-CSF/IL-3 but not G-CSF alone. Similarly, administration of the cytokine combination resulted in a higher number of CD34+ cells/mL BM, and their concentration was significantly greater than that observed in the posttreatment samples of G-CSF patients. Finally, we investigated the responsiveness to CSFs, in vitro, of highly enriched CD34+ cells, collected after priming with G-CSF in vivo (i.e., after 5 days of G-CSF administration). Our results demonstrated that pretreatment with G-CSF modified the response of BM cells to subsequent stimulation with additional CSFs. The results presented in this paper indicate that in vivo administration of two cytokines increases the proliferative rate and concentration of BM progenitor cells to a greater degree than G-CSF alone. These results support the role of growth factor combinations for accelerating hematopoietic recovery after high-dose chemotherapy.

Pharmacological purging of minimal residual disease from peripheral blood stem cell collections of acute myeloblastic leukemia patients: preclinical studies.

UNLABELLED: In this paper we describe an experimental model for ex vivo purging of contaminating tumor cells from peripheral blood stem cell (PBSC) collections obtained from patients with acute myeloblastic leukemia (AML). We studied the combination of the alkylating agent nitrogen mustard (NM; concentrations ranging from 0.25 to 1.25 microg/mL) and etoposide (VP-16; constant dose of 20 microg/mL), and the conventional cyclophosphamide (Cy)-derivative mafosfamide (concentrations: 20-175 microg/mL). THE AIMS OF OUR STUDY WERE: 1) To compare the toxicity of the purging protocols on bone marrow (BM) and circulating trilineage precursors collected from normal donors after priming with granulocyte colony-stimulating factor (G-CSF) or after complete remission (CR) consolidation chemotherapy and G-CSF (leukemic patients); 2) to demonstrate the survival of very primitive hematopoietic progenitors (LTC-IC) in the peripheral blood (PB) and the BM after pharmacological treatment; and 3) to evaluate the antineoplastic efficacy of purging protocols on PBSC collections using 3 well-established leukemic cell lines. Our results demonstrated that the toxicity on BM and PB progenitor cells could be correlated with the complete killing of committed granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid precursors (BFU-Es), a condition reached at the concentration of 1.5 microg/mL of NM (in addition to 20 microg/mL of VP-16) and 175 microg/mL of mafosfamide. Notably, early and late megakaryocyte progenitor cells (CFU-MKs and BFU-MKs, respectively) showed higher sensitivity to NM/VP-16, but not to mafosfamide, than did CFU-GMs and BFU-Es. The dose of NM capable of inhibiting 95% of CFU-MKs and BFU-MKs (ID95) was 0.75 microg/mL. After incubation with the same dose of NM, the recovery of CFU-GMs and BFU-Es was 20 +/- 8% SD and 25 +/- 10% SD, respectively (p < 0.05). Long-term liquid cultures showed the recovery of primitive hematopoietic cells after incubation with the highest concentrations of NM/VP-16 and mafosfamide, with no significant differences between PB and BM samples. Under the same experimental conditions, we observed a more than 5-log reduction of contaminating leukemic cell lines (i.e., K-562, KG-1, and HL-60). In conclusion, we demonstrated that NM/VP-16 and mafosfamide purging agents are capable of killing leukemic cell lines that contaminate leukapheresis products from patients with AML, whereas an acceptable proportion of primitive LTC-IC is spared. Moreover, despite the different kinetic and functional profile of mobilized and steady-state BM progenitors, we did not observe any difference in toxicity of antineoplastic agents on hematopoietic cells at different levels of differentiation. These data suggest that pharmacological strategies developed for eliminating minimal residual disease (MRD) from BM autografts can be effectively and safely applied to circulating stem cell harvests.

Thrombopoietin and interleukin 11 have different modulatory effects on cell cycle and programmed cell death in primary acute myeloid leukemia cells.

The c-mpl ligand, thrombopoietin (TPO), is a physiologic regulator of platelet and megakaryocytic production, acting synergistically on thrombopoiesis with the growth factors interleukin 11 (IL-11), stem cell factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte-macrophage colony-stimulating factor. Because some of these growth factors, especially TPO and IL-11, are now being evaluated clinically to reduce chemotherapy-associated thrombocytopenia in cancer patients, we evaluated 25 acute myeloid leukemia (AML) samples to test whether TPO, IL-11, and other early-acting megakaryocyte growth factors can affect leukemic cell proliferation, cell cycle activation, and programmed cell death (PCD) protection. TPO induced proliferation in the majority of AML samples from an overall mean proportion of S-phase cells of 7.8% +/-1.5% to 14.5% +/- 2.1% (p = 0.0006). Concurrent G0 cell depletion was found in 47.3% of AML samples. TPO-supported leukemic cell precursor (CFU-L) proliferation was reported in 5 of 17 (29.4%) of the samples with a mean colony number of 21.4 +/- 9.6 x 10(5) cells plated. In 13 of 19 samples, a significant protection from PCD (from an overall mean value of 13% +/-0.7% to 8.8% +/- 1.8%;p = 0.05) was detected after TPO exposure. Conversely, IL-11-induced cell cycle changes (recruitment from G0 to S phase) were detected in only 2 of 14 samples (14.2%). In addition, IL-11 showed little, if any, effect on CFU-L growth (mean colony number = 17.5 9.5) or apoptosis. Combination of TPO with IL-11 resulted in only a slight increase in the number of CFU-L, whereas IL-3 and stem cell factor significantly raised the mean colony numbers up to 119.2 +/- 68.3 and 52.9 +/- 22.1 x 10(5) cells plated, respectively. We conclude that TPO induces cell cycle activation in a significant proportion of cases and generally protects the majority of AML blast cells from PCD. On the other hand, IL-11 has little effect on the cell cycle or PCD. Combination of both TPO and IL-11 is rarely synergistic in stimulating AML clonogenic growth. These findings may be useful for designing clinical studies aimed at reducing chemotherapy-associated thrombocytopenia in AML patients.

In situ staining of bromodeoxyuridine positive cells in normal and neoplastic colony-forming units grown on plasma clots.

Bromodeoxyuridine, an analogue of thymidine, can be detected by means of monoclonal antibodies and utilized as a marker of the S-phase of the cell cycle. In this paper a method for the detection of the labeling index of normal and neoplastic colony-forming units (CFU) growing in plasma clot semisolid medium is described and preliminary results on the cell cycle of 7th and 14th CFU granulocyte-macrophage are discussed.

Evidence that long-term bone marrow culture of patients with multiple myeloma favors normal hemopoietic proliferation.

Long-term bone marrow cultures (LTBMC) were initiated with marrow aspirate cells from 12 patients with multiple myeloma (MM) using the Dexter system. The myeloid and the neoplastic myeloma cell growths were evaluated for up to 6-9 weeks. Our results demonstrate the development of an adherent layer capable of supporting normal granulopoiesis with a concomitant drop in the growth of myeloma cells. The B lymphocyte monoclonal proliferative compartment was also studied with bromodeoxyuridine (Brdurd), an analog of thymidine incorporated during the S-phase, and the labeling index was calculated. The ability to form myeloma stem cell colonies in a modified plasma clot short-term assay was also evaluated. The results confirmed that the neoplastic B lineage compartment was not able to grow in Dexter's system for more than 4 weeks in 11 of 12 cases studied, with the disappearance of Brdurd-positive cells after two weeks, whereas LTBMC were able to sustain the growth of myeloid progenitors. These data indicate the potential applicability of this culture method in selecting normal hematopoietic progenitors from patients with multiple myeloma. This approach can have significant implications for aggressive treatment of patients with multiple myeloma, especially in trials involving autologous bone marrow transplantation (ABMT).

Bone marrow purging for multiple myeloma by avidin-biotin immunoadsorption.

Avidin-biotin immunoadsorption, a technique based on the high affinity between the protein avidin and the vitamin biotin, has been used to remove neoplastic plasma cells from the bone marrow of patients with multiple myeloma. Buffy coat cells obtained from 25 patients were first incubated with monoclonal antibodies (MoAb) capable of recognizing plasma cell-associated antigens (i.e., 8A, 8F6, 62B1, and cocktails of 8A plus 8F6 or 62B1), then with biotinylated goat antimouse immunoglobulin, and passed over a column containing avidin conjugated to Sepharose GMB. Both non-linked and linked cells were analyzed by immunofluorescence and morphological staining. The results showed that over 98% of plasma cells were removed by using 8A or 8F6 alone, while 99.5% +/- 0.4 SD of plasma cell purging was achieved with 2 associated MoAb. In addition, the overall recovery of committed granulocyte-macrophage (CFU-GM),* erythroid (BFU-e), and multilineage (CFU-GEMM) progenitors after column treatment ranged from 39% +/- 15 SD to 50% +/- 6 SD, from 15% +/- 2 SD to 39% +/- 7 SD, and from 16 +/- 4 SD to 64% +/- 10 SD, according to the MoAb employed. On this basis avidin-biotin immunoadsorption appears to be a suitable technique for ex-vivo manipulation of bone marrow infiltrated by neoplastic plasma cells.