Anna Pitto

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Cyclic ADP‐ribose (cADPR) is a universal second messenger that regulates many calcium‐related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD+ (CD38 and BST‐1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 μM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3‐deaza‐cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, M., Zocchi, E., Pitto, A., Usai, C., Franco, L., Bruzzone, S., Guida, L., Bacigalupo, A., Scadden, D. T., Walseth, T. F., De Flora, A., Daga, A. Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB J. 14, 680–690 (2000)

Reduced intensity thiotepa-cyclophosphamide conditioning for allogeneic haemopoietic stem cell transplants (HSCT) in patients up to 60 years of age.

Transplant‐related mortality (TRM) remains a major problem in older patients undergoing allogeneic haemopoietic stem cell transplants (HSCTs). We have therefore explored a less intensive conditioning in 33 patients with a median age of 52 years (range 43–60) transplanted from human leucocyte antigen (HLA)‐identical siblings. The underlying disease was chronic myeloid leukaemia (n = 15), acute myeloid leukaemia (n = 6), myelodysplasia (n = 7) or a chronic lymphoproliferative disorder (n = 5); 15 patients (45%) had advanced disease. The regimen consisted of thiotepa (THIO; 10 mg/kg) on day −5 and cyclophosphamide (CY; 50 mg/kg) on days −3 and −2 (total dose 100 mg/kg). The source was bone marrow (BM) (n = 17) or granulocyte colony‐stimulating factor (G‐CSF)‐mobilized peripheral blood (PB) (n = 16), which were infused without manipulation. Graft‐versus‐host disease (GVHD) prophylaxis consisted of cyclosporin A (CyA) and a short course of methotrexate. Mean time to achieve a neutrophil count of 0·5 × 109/l was 17 d (range 11–23) and full donor chimaerism was detected in 79% of patients by day 100. Acute GVHD grade III or IV occurred in 3% of patients. Chronic GVHD was seen in 45% of patients, with a significant difference for PB (69%) compared with BM transplants (23%) (P = 0·009). For BM grafts, the actuarial 2‐year TRM was 6%, the relapse 56% and survival 87%; for PB grafts, these figures were, respectively, 27%, 33% and 68%. Twenty‐five patients are alive at a median follow‐up of 762 d (range 216–1615) and 20 patients (60%) remain free of disease. Thirteen patients (39%) received donor lymphocyte infusion (DLI) either for persisting or relapsing disease and six patients had complete remission. In conclusion: (i) patients up to the age of 60 years can be allografted with reduced intensity conditioning; (ii) the procedure was associated with a low transplant‐related mortality, particularly for bone marrow grafts, because of a lower risk of chronic GVHD; and (iii) DLI were required after transplant in half the patients for persisting disease or relapse.

Deficient reconstitution of early progenitors after allogeneic bone marrow transplantation

Allogeneic bone marrow transplant recipients maintain normal peripheral blood counts long term, suggesting durable support from engrafted stem cells. In order to investigate late hemopoietic reconstitution at the level of committed and early progenitors (LTC-IC), we studied 64 long-term survivors at a median interval of 6 years (range: 2–20) after allogeneic bone marrow transplant. CFU-GM and BFU-E numbers did not differ from normal controls; CFU-GEMM were found to be significantly decreased (1.2 ± 0.2/105vs 3.1 ± 0.4, P = 0.001). The most remarkable defect was however, the low frequency of LTC-IC (3.2 ± 0.6/106vs 54.2 ± 9.3, P = 0.0001) that did not improve with time and did not correlate with phase of the disease, conditioning regimen, CMV infections or GVHD. Number of infused cells and CFU-GM content of marrow grafts did not seem to influence the number of LTC-IC. This study documents a significantly reduced number of early progenitors in BMT patients despite normal numbers of committed progenitors and normal peripheral blood counts. This finding may suggest a permanent reduction of the stem cell reservoir after allogeneic bone marrow transplantation.

Paracrinally stimulated expansion of early human hemopoietic progenitors by stroma-generated cyclic ADP-ribose.

Both mammalian ectoenzymes CD38 and BST‐1, which convert NAD+ into the potent intracellular calcium regulator cyclic ADP‐ribose (cADPR), are expressed in the bone marrow microenvironment. Therefore, we investigated the effect of extracellular cADPR on human long‐term culture‐initiating cells (LTC‐IC), the most immature hemopoietic progenitors (HP). A threefold expansion of LTC‐IC was elicited by exposure of cord blood mononuclear cells for 24 h either to micromolar concentrations of exogenously added cADPR or to the nanomolar concentrations of cADPR that are produced by CD38‐transfected murine stromal cell lines cocultured with the HP. The latter effect was due to connexin 43‐mediated release of NAD+ from feeder cells and to subsequent CD38‐catalyzed generation of cADPR at their outer surface. The increased [Ca2+]i induced in the feeder cells by autocrinally generated cADPR, however, increased production of interferon γ, a potent hemopoiesis‐inhibiting cytokine. Thus, long‐term culture (5 wk) of HP over undiluted CD38+ feeders decreased LTC‐IC output by 90%, whereas a fivefold increase was observed when HP were cultured over a mixed CD38+/− feeder (1:10), whose ecto‐ADP‐ribosyl cyclase activity was as low as that expressed by native human stroma. These results demonstrate a new paracrine role for cADPR in the regulation of human hemopoiesis.

Transplantation of HLA-mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications.

This trial was designed to test the use of CD34+ selected haemopoietic stem cells (HSC) in HLA‐mismatched donor–recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single‐dose total‐body irradiation (sTBI). 10 patients aged 16–50 with advanced malignancies and a two‐ or three‐antigen mismatched family donor entered this study. Donor marrow and G‐CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34+ cells were 5.66 × 106/kg, with 0.55 × 106/kg CD3+ cells. Nine patients received cyclosporin for graft‐versus‐host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 109/l with a median platelet count of 60 × 109/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0–I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20–144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) (n = 5), renal failure (n = 1), GvHD (n = 1) and Aspergillus meningitis (n = 1). Two patients are alive 365–495 d post transplant, one in remission and one in relapse.

Cyclic ADP-ribose generation by CD38 improves human hemopoietic stem cell engraftment into NOD/SCID mice.

Cyclic ADP‐ribose (cADPR) is a potent and universal intracellular calcium mobilizer, recently shown to behave as a new hemopoietic cytokine stimulating the in vitro proliferation of both committed and uncommitted human hemopoietic progenitors (HP). Here, we investigated the effects of cADPR on engraftment of hemopoietic stem cells (HSC) into irradiated NOD/SCID mice. Two different protocols were used: i) a 24 h in vitro priming of cord blood‐derived mononuclear cells (MNC) with micromolar cADPR, followed by their infusion into irradiated mice (both primary and secondary transplants); and ii) co‐infusion of MNC with CD38‐transfected, cADPR‐generating, irradiated murine 3T3 fibroblasts. We demonstrated a dual effect of cADPR on human HP in vivo: i) enhanced proliferation of committed progenitors, responsible for improvement of short‐term engraftment; ii) expansion of HSC, with increased long‐term human engraftment into secondary recipients and a significantly higher expansion factor of CD34+ progenitors in mice co‐infused with MNC and CD38+ 3T3 fibroblasts. These results hold promise for the possible therapeutic use of cADPR, and of cADPR‐producing stroma, to achieve long‐term expansion of human HSC, that is, those HP capable of self‐renewal and responsible for repopulation of the bone marrow.

Normal primitive haemopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time

We carried out studies to quantify Ph‐negative progenitors both in steady state and during regeneration after chemotherapy and G‐CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady‐state bone marrow, Ph‐negative long‐term culture initiating cells (LTC‐IC) and Ph‐negative colony‐forming‐cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P < 0.001 and P < 0.02, respectively). The absolute number of mobilized Ph‐negative progenitors was markedly higher in group A versus group B (P < 0.02 for LTC‐IC, P < 0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph‐negative LTC‐IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph‐negative LTC‐IC in the bone marrow predicted the yield of Ph‐negative LTC‐IC mobilized into peripheral blood (P < 0.001). The bone marrow frequency of Ph‐positive LTC‐IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph‐negative circulating progenitors would appear to be soon after diagnosis.

Modified in vitro conditions for cord blood-derived long-term culture-initiating cells.

OBJECTIVE The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood. MATERIALS AND METHODS We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4). RESULTS The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p < 10(-4) and p < 0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p < 0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p < 10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p < 10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p < 0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p < 0.02 and p < 0.001 respectively). CONCLUSIONS Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

Progenitor cells trapped in marrow filters can reduce GvHD and transplant mortality

A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filter-discarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001–2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997–2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II–IV graft-versus-host disease (GvHD) (42 vs 15%, P=0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P=0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P=0.15). This study suggests that a significant proportion of LTC-IC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM.