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Supplementary Figure S1 from Endothelial Differentiation of Hematopoietic Stem and Progenitor Cells from Patients with Multiple Myeloma

Purpose: Vasculogenesis is a physiologic process typical of fetal development in which new blood vessels develop from undifferentiated precursors (or angioblasts). In tumors, near angiogenesis, vasculogenesis contributes to the formation of the microvascular plexus that is important for diffusion. Here, we show that hematopoietic stem and progenitor cells (HSPC) of multiple myeloma (MM) patients are able to differentiate into cells with endothelial phenotype on exposure to angiogenic cytokines.Experimental Design: Circulating HSPCs were purified with an anti-CD133 antibody from patients with newly diagnosed MM before autologous transplantation and exposed to vascular endothelial growth factor (VEGF), fibroblast growth factor-2 and insulin-like growth factor in a 3-week culture.Results: HSPCs gradually lost CD133 expression and acquired VEGF receptor-2, factor VIII–related antigen, and vascular endothelial-cadherin expression. The expression pattern overlapped with paired MM endothelial cells (MMEC). During culture, cells adhered to fibronectin, spread, and acquired an endothelial cell shape. Differentiated HSPCs also became capillarogenic in the Matrigel assay with maximal activity at the third week of culture. Bone marrow biopsies revealed HSPCs inside the neovessel wall in patients with MM but not in those with monoclonal gammopathy of undetermined significance.Conclusions: In patients with MM, but not in those with monoclonal gammopathy of undetermined significance, HSPCs contribute to the neovessel wall building together with MMECs. Therefore, besides angiogenesis, HSPC-linked vasculogenesis contributes to neovascularization in MM patients. Tentatively, we hypothesize that in HSPC cultures a multipotent cell population expressing low VEGF receptor-2 levels corresponds to the endothelial progenitor cell precursor and seems to be the MMEC precursor.

The impact of chronic lymphatic leukemia (CLL) tumor burden on the autologous immune system has already been demonstrated. This study attempted to elucidate the molecular mechanisms underlying T-cell immunologic deficiencies in CLL.Freshly isolated CD3(+) T cells from patients with a diagnosis of CLL and healthy donors were analyzed by gene expression profiling. Activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity against mutated and unmutated autologous B cells and DAUDI, K562 and P815 cell lines. To investigate T-cell mediated cytotoxicity in vivo, we co-transplanted OKT3-activated T lymphocytes and autologous B-cell CLL (B-CLL) cells into NOD/SCID mice.Gene expression profiles of peripheral blood T cells from B-CLL patients showed 25 down-regulated, and 31 up-regulated, genes that were mainly involved in cell differentiation, proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T-cell activation. After culture, the T-cell count remained unchanged, CD8 cells expanded more than CD4 and a cytotoxicity index30% was present in 5/20 patients. Cytotoxicity against B autologous leukemic cells did not correlate with B-cell mutational status. Only activated T cells exerting cytotoxicity against autologous leukemic B cells prevented CLL in a human-mouse chimera.This study indicates that patients with CLL are affected by a partial immunologic defect that might be somewhat susceptible to repair. This study identifies the molecular pathways underlying T-cell deficiencies in CLL and shows that cytotoxic T-cell functions against autologous B-CLL can be rebuilt at least in part in vitro and in vivo.

Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm characterized by progressive anemia, massive splenomegaly, leukoerythroblastosis, extramedullary hematopoiesis and in about 50% of cases the presence of JAK2V617F mutation. Curative therapy in PMF is currently possible only with allogeneic haematopoietic stem cell transplantation which is, unfortunately, associated with relatively high risks of mortality and morbidity which undermine its broad applications. Non-transplant treatment modalities are used for palliative purposes. Recently, anti-angiogenic drugs such as thalidomide have been used to treat these patients on the basis of the prominent bone marrow angiogenesis. Here, we report the case of a patient suffering from JAK2V617F-positive PMF with marked bone marrow neo-angiogenesis. The patient was treated with thalidomide but after 20 days developed life-threatening toxic epidermal necrolysis (TEN). To the best of our knowledge this is the first case of TEN in a patient with PMF under thalidomide therapy.

MtDNA mutation associated with mitochondrial dysfunction in megakaryoblastic leukaemic cells

Purpose: Vasculogenesis is a physiologic process typical of fetal development in which new blood vessels develop from undifferentiated precursors (or angioblasts). In tumors, near angiogenesis, vasculogenesis contributes to the formation of the microvascular plexus that is important for diffusion. Here, we show that hematopoietic stem and progenitor cells (HSPC) of multiple myeloma (MM) patients are able to differentiate into cells with endothelial phenotype on exposure to angiogenic cytokines. Experimental Design: Circulating HSPCs were purified with an anti-CD133 antibody from patients with newly diagnosed MM before autologous transplantation and exposed to vascular endothelial growth factor (VEGF), fibroblast growth factor-2 and insulin-like growth factor in a 3-week culture. Results: HSPCs gradually lost CD133 expression and acquired VEGF receptor-2, factor VIII–related antigen, and vascular endothelial-cadherin expression. The expression pattern overlapped with paired MM endothelial cells (MMEC). During culture, cells adhered to fibronectin, spread, and acquired an endothelial cell shape. Differentiated HSPCs also became capillarogenic in the Matrigel assay with maximal activity at the third week of culture. Bone marrow biopsies revealed HSPCs inside the neovessel wall in patients with MM but not in those with monoclonal gammopathy of undetermined significance. Conclusions: In patients with MM, but not in those with monoclonal gammopathy of undetermined significance, HSPCs contribute to the neovessel wall building together with MMECs. Therefore, besides angiogenesis, HSPC-linked vasculogenesis contributes to neovascularization in MM patients. Tentatively, we hypothesize that in HSPC cultures a multipotent cell population expressing low VEGF receptor-2 levels corresponds to the endothelial progenitor cell precursor and seems to be the MMEC precursor.

We report a noteworthy case of Eubacterium plautii infection after kidney transplantation. Our 33-yr-old transplant recipient received standard care; his post-transplant course was uneventful. However, on day 44 he underwent an emergency laparotomy for perforation of the ileum. He was initially treated with ceftazidime, fluconazole and metronidazole, but his fever persisted, so he was switched to meropenem and vancocin. We could not find any cause for his infection. On day 70, his temperature normalized. On day 75, he developed severe leukopenia (280 cell/mL). His cytomegalovirus-DNA test result was negative, so all immunosuppressants, except for prednisone, were stopped; instead, antibiotic prophylaxis was started, using caspofungin, trimethoprim-sulfamethoxazole and ciprofloxacin. On day 83, he underwent percutaneous drainage of massive left pleural effusion. We repeatedly cultured the pleural liquid, but it was not till three wk later that we were finally able to identify the causative organism. We hypothesize that the microorganism - which normally resides on the surface of the intestinal lumen - entered the bloodstream via bacterial translocation, eventually colonizing the pleurae. This translocation was favored by our patient poor clinical condition, his immunosuppressive treatment and his heavy antibiotherapy. Our experience highlights the need for wiser use of antibiotics in transplant recipients.

Consolidated evidence highlights the importance of redox signalling in poising the balance between self-renewal and differentiation in adult stem cells. The present study shows that human hematopoietic stem/progenitor cells (HSCs) constitutively generate low levels of hydrogen peroxide whose production is inhibited by DPI, apocynin, catalase, and LY294002 and scarcely stimulated by PMA. Moreover, it is shown that HSCs express at the mRNA and protein levels the catalytic subunits of NOX1, NOX2, and NOX4 isoforms of the NADPH oxidase family along with the complete battery of the regulatory subunits p22, p40, p47, p67, rac1, rac2, NOXO1, and NOXA1 as well as the splicing variant NOX2s and that the three NOX isoforms are largely co-expressed in the same HSC. These findings are interpreted in terms of a positive feed-back mechanism of NOXs activation enabling a fine tuning of the ROS level to be possibly used in redox-mediated signalling for growth and differentiation of HSCs.

In this case, originally reported as primary eyelid plasmacytoma, the tumor recurred on the same eyelid within 2 years of surgery. No plasma cell infiltration was observed at bone marrow biopsy. No serum or urinary monoclonal component was detected at immunofixation. Histology and immunohistochemistry confirmed plasma cell infiltration. Tumor cell clonality was determined by immunohistological staining; cells were positive for kappa light chain like the first eyelid tumor. Surgery was followed by radiotherapy. Twenty months later, biopsy of one enlarged right cervical lymph node showed massive diffuse infiltration of atypical plasma cells (CD20(-), CD79a(+), CD138(+), MUM1/IRF4(+)). Given the rapid diffusion to lymph nodes and the appearance of the monoclonal component, the lymph node was removed surgically. No adjuvant chemotherapy was given. Unexpectedly, the serum monoclonal component normalized. No plasma cell infiltration was observed at bone marrow biopsy. As this case might be a particularly slow-progressing extra-medullary plasmacytoma, this study recommends closely monitored follow-ups so that the aggressive form can be treated in time.

This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34+ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34+ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Purpose Establishment of hematopoietic stem-cell (HSC) transplantation from mismatched relatives is feasible for patients with acute leukemia. As our original method of graft processing was unsuitable for large-scale clinical studies, we use automated devices for CD34+ cell purification. Patients and Methods Sixty-seven patients with acute myeloid leukemia (AML; 19 complete remission [CR] 1, 14 CR 2, nine CR > 2, 25 in relapse) and 37 with acute lymphoid leukemia (ALL; 14 CR 1, eight CR 2, two CR > 2, 13 in relapse) were conditioned with total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. Peripheral-blood progenitor cells were mobilized with recombinant human granulocyte colony-stimulating factor and depleted of T-cells using CD34+ cell immunoselection. No post-transplantation graft-versus-host disease (GvHD) prophylaxis was administered. Results Primary engraftment was achieved in 94 of 101 assessable patients. Six of the seven patients who rejected the primary graft, engrafted after a second transplantation. Overall, 100 of 101 patients engrafted. Acute GvHD developed in eight of 100 patients, and chronic GvHD, in five of 70 assessable patients. Thirty-eight patients died of nonleukemic causes. Relapse occurred in nine of 66 patients receiving transplantation in remission and in 17 of 38 receiving transplantation in relapse. Median follow-up of the 40 patients who survived event-free was 22 months (range, 1 to 65 months). Event-free survival (± standard deviation) rate was 48% ± 8% and 46% ± 10%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission. Conclusion Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.

Establishment of cell lines capable of killing leukemia cells, in the absence of alloreactivity against normal host cells, represents a most desirable goal in bone marrow transplantation (BMT) and cancer immunotherapy. By using a human → mouse chimeric model, we demonstrate that allogeneic anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity are endowed with a potent anti-B-cell chronic lymphocytic leukemia (B-CLL) reactivity. Likewise, CTL preparations generated from autologous T cells of the same patients with B-CLL exhibited comparable leukemia eradication, suggesting that the reactivity of allogeneic anti-third-party CTLs is not mediated by residual antihost clones. This specificity was also exhibited in vitro, and annexin staining revealed that B-CLL killing is mediated by apoptosis. While the CTLs killing of third-party cells could be blocked by anti-CD3 antibody, the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a T-cell receptor (TCR)-independent cytotoxicity. The role of cell contact leading to apoptosis of B-CLL cells is shown in transwell plates and by anti-lymphocyte function-associated antigen-1 (LFA-1)-blocking antibody. Up-regulation of CD54 and the subsequent apoptosis of B-CLL cells depend on the initial LFA-1/ICAM-1 (intercellular adhesion molecule 1) interaction. Taken together, these results suggest that allogeneic or autologous host nonreactive anti-third-party CTLs may represent a new therapeutic approach for patients with B-CLL. (Blood. 2005;105:3365-3371)

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFβ1. When FGF2 and/or TGFβ1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFβ1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases. © 2005 Wiley-Liss, Inc.

Haploidentical stem cell transplantation has became a clinical reality in the last 10 years as it provides the chance of transplant for about 50% of patients with hematological malignancies who do not have a matched related or unrelated donor. Proper graft preparation for this type of transplant is crucial and this paper analyses our work over the past decade in the search for the optimal graft processing procedure moving from E-rosetting and soybean agglutination, through a combination of negative or positive selection of hematopoietic stem cells to the current method of one-step positive selection. In preparing a graft for haploidentical transplant, three essential requisites must be met. It must contain (1) a megadose (>10 x 10(6) x kg recipient b.w.) of hematopoietic stem cells to overcome the HLA histocompatibility barrier; (2) very few T-lymphocytes (CD3+ cells < 3 x 10(4)/kg recipient b.w.) to prevent severe acute and chronic graft-versus-host disease (GvHD); (3) very few B-lymphocytes to prevent Epstein-Barr virus-related lymphoproliferative disorders. With current graft processing technologies based on positive selection of hematopoietic stem cells, these requirements can be met. A 70-80% hematopoietic stem cell recovery ensures the target megadose is achieved in over 70% of cases with a T-cell depletion of more than 4 logs and a B-cell depletion of over 3 logs. Progress in graft processing has ensured primary, sustained engraftment rates of over 90% and has significantly reduced the incidence of severe acute GvHD and EBV-related lymphoproliferative disorders. Modern time-saving automated graft processing devices ensure reproducibility, reliability, and biological safety, which make widespread application of the haploidentical transplant currently feasible.

Many patients with high-risk hematological malignancies or with incurable inborn errors do not have an HLA-matched sibling and cannot find an HLA-matched donor for an allogeneic hematopoietic stem cell transplantation (SCT). Transplantation strategies using mismatched haploidentical family donors have been an important development. Although the procedure has saved patients from certain death, it is still beset by major problems like life-threatening infections—due to profound immunodeficiency following T-cell depletion and to disease relapse. At every International Workshop on Haploidentical Transplants, new data are presented, showing how scientists are attempting to improve the outcomes of mismatched transplants while reducing the severity of complications. The fourth Workshop continues in this tradition of presenting ground-breaking research. It opened with presentations of the current results with unrelated volunteer and umbilical cord blood transplants and proceeded to a session with the results of haploidentical transplants in the world with series of patients with high-risk acute leukemia, ranging in number from well over 100 in Perugia, Italy, and 80 in Haifa, Israel, to smaller groups in Europe and the United States. The session on graft engineering presented the latest results in the search for the optimal graft. The graft-vs.-leukemia effect in the haploidentical transplant was discussed in depth. Subsequently, attention focussed on one of the major problems in haploidentical transplant, that is, the delay in immunological recovery. The Workshop closed with presentations on tolerance induction that was followed by the results of ongoing registration studies being performed by the Italian GIMEMA group and the European Bone Marrow Transplant group.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

MAP kinase/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) kinases are frequently activated in acute myelogenous leukemia (AML), and can have prosurvival function. The purpose of this study was to induce downmodulation of MEK-ERK activation in AML primary blasts in order to detect the effect on cell cycle progression and on the apoptosis of leukemic cells. We investigated 14 cases of AML with high ERK 1/2 activity and four cases with undetectable or very low activity. After 24 h incubation of the AML blasts with high ERK activity using PD98059 (New England BioLabs, Beverly, MA, USA), a selective inhibitor of MEK1 phosphorylation, at concentrations of 20 and 40 microM, we observed a strong decrease in the levels of ERK1/2 activity. A significant decrease of blast cell proliferation compared with untreated controls was found. In contrast, the proliferation of blast cells that expressed low or undetectable levels of ERK activity was not inhibited. Time-course analysis demonstrated that the downmodulation of MEK1/2, ERK1 and ERK2 dual-phosphorylation was evident even after 3 h of treatment with 20 and 40 microM. The cleavage of poly(ADP-ribose) polymerase (PARP), an early sign of apoptosis, appeared after 18 h of PD98059 treatment at concentrations of 20 and 40 microM in eight of the 14 cases. After 24 h of treatment, cleaved PARP appeared in all 14 cases. Time-course analysis of cell cycle progression and apoptosis showed that PD98059 induced a G1-phase accumulation with low or undetectable levels of apoptosis after 24 h incubation; after 48 and 72 h incubation, a significant increase of apoptosis was observed. Thus, the primary effect of ERK downmodulation was a cell cycle arrest followed by the apoptosis of a significant percentage of the leukemic blasts. The preclinical model of leukemia treatment reported in this paper makes further comment with regard to MEK1 inhibition as a useful antileukemic target, and encourages the conducting of in vivo studies and clinical investigations.

To develop a trial with lymphocyte suicide gene therapy in patients with hematological malignancies, we transduced human T lymphocytes with a retroviral vector (LSN-tk) encoding the herpes simplex virus thymidine kinase (tk) and the neomycin resistance (NeoR) genes. Precise quantification of gene transfer is crucial for any gene therapy protocol, but methods using semiquantitative PCR are inaccurate and subject to variations. Real-time quantitative PCR could be a valid alternative. A TaqMan probe was designed to hybridize with the NeoR gene. The PCR product is 64 nucleotides long and readily quantified by TaqMan probe binding. The analysis was performed soon after transduction and repeated after the selection procedure. This method was more accurate, reproducible, and sensitive than the semiquantitative PCR assay. Accuracy was the same whether the analysis was performed at the highest rate or at the lowest rate of transduction. Additionally we used real-time PCR to monitor the kinetics of enrichment of the transduced cells over the selection time and showed how 7 days of selection are needed. This study precisely quantified the percentages of cells transduced by the retroviral vector and could have major implications in gene therapy studies.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used (“megadose” transplants) overcomes major genetic barriers without induction of graft-versus-host disease. Therefore, transplantation from a human leukocyte antigen mismatched

The GIMEMA ALL 0288 trial was designed to evaluate the impact of a 7-day prednisone (PDN) pretreatment on complete remission (CR) achievement and length, the influence of the addition of cyclophosphamide (random I) to a conventional 4-drug induction on CR rate and duration, and whether an early post-CR intensification (random II) by an 8-drug consolidation could improve CR duration. Median follow-up of this study was 7.3 years. From January 1988 to April 1994, among 794 adult (> 12 but

In high-risk acute leukemia patients, a 10-fold increase in the dose of extensively T-cell-depleted hematopoietic stem cells ensures sustained full-donor engraftment of one-haplotype-mismatched transplants without graft-vs.-host disease. Since our first successful pilot study, which exploited the principle of a megadose stem cell transplant, our efforts have concentrated on developing new conditioning regimens, optimizing graft processing and improving the post-transplant immunologic recovery. The results so far achieved in more than 100 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype-mismatched family donor, a T-cell-depleted mismatched transplant can be offered with curative intent, thus extending allogeneic transplantation procedures to virtually all candidates.

Aims and Background To investigate the safety and efficacy of a high-dose chemotherapy regimen with etoposide, carboplatin and thiotepa in high-risk stage II-IIIA breast cancer and in responsive metastatic patients. Study Design From April 1992 to December 1998, 24 patients with high-risk stage II-IIIA breast cancer (≥9 positive nodes) and 9 responsive metastatic patients were enrolled in the trial. After induction chemotherapy with an anthracycline-based regimen, peripheral blood stem cells were mobilized with cyclophosphamide (7 g/m2) and G-CSF (5-16 μg/kg/sc/day). The high-dose chemotherapy regimen consisted of etoposide (1000 mg/m2), carboplatin (800 mg/m2) and thiotepa (500 mg/m2). At the end of the high-dose chemotherapy, all stage II-IIIA patients received radiotherapy to the breast or chest wall and draining nodes; stage IV patients were irradiated to sites of disease, if feasible. All ER+ and/or PgR+ patients were treated with hormone therapy. Results For stage II-IIIA high-risk patients, the median follow-up was 4.36 years (range, 1.93-6.94), and the Kaplan-Meier estimate at 5 years of disease-free survival and overall survival was 54.8 ± 11% SE and 76.73 ± 9.4% SE, respectively. For metastatic patients, the median follow-up was 4.93 years (range, 4.15-7.95), and the Kaplan-Meier estimate at 5 years of progression-free survival and overall survival was 22.2 ± 13.9% SE and 76.2 ± 14.8% SE, respectively. No treatment-related deaths were observed. Conclusions Our results are comparable to those obtained in other high-dose chemotherapy trials but do not seem to be superior to conventional-dose therapy given to similar patients.

In human leukocyte antigen haplotype–mismatched transplantation, extensive T-cell depletion prevents graft-versus-host disease (GVHD) but delays immune recovery. Granulocyte colony-stimulating factor (G-CSF) is given to donors to mobilize stem cells and to recipients to ensure engraftment. Studies have shown that G-CSF promotes T-helper (Th)-2 immune deviation which, unlike Th1 responses, does not protect against intracellular pathogens and fungi. The effect of administration of G-CSF to recipients of mismatched hematopoietic transplants with respect to transplantation outcome and functional immune recovery was investigated. In 43 patients with acute leukemia who received G-CSF after transplantation, the engraftment rate was 95%. However, the patients had a long-lasting type 2 immune reactivity, ie, Th2-inducing dendritic cells not producing interleukin 12 (IL-12) and high frequencies of IL-4– and IL-10–producing CD4+ cells not expressing the IL-12 receptor β2 chain. Similar immune reactivity patterns were observed on exposure of donor cells to G-CSF. Elimination of postgrafting administration of G-CSF in a subsequent series of 36 patients with acute leukemia, while not adversely affecting engraftment rate (93%), resulted in the anticipated appearance of IL-12–producing dendritic cells (1-3 months after transplantation versus > 12 months in transplant recipients given G-CSF), of CD4+ cells of a mixed Th0/Th1 phenotype, and of antifungal T-cell reactivity in vitro. Moreover, CD4+ cell counts increased in significantly less time. Finally, elimination of G-CSF–mediated immune suppression did not significantly increase the incidence of GVHD (< 15%). Thus, this study found that administration of G-CSF to recipients of T-cell–depleted hematopoietic transplants was associated with abnormal antigen-presenting cell functions and T-cell reactivity. Elimination of postgrafting administration of G-CSF prevented immune dysregulation and accelerated functional immune recovery.

Polymeric protein-polyamine conjugates: A new class of uremic toxins affecting erythropoiesis. Background Preliminary evidence on the accumulation of polyamine-protein conjugates (PPCs) was obtained in uremic patients. The presence of these substances in the plasma of hemodialysis (HD) patients was evaluated, and their possible contribution to uremic anemia was investigated by testing the effect of PPC synthesized in vitro on erythroid cell proliferation. Methods Plasma PPC was measured by high-performance liquid chromatography. The in vitro synthesis of PPC from human plasma was carried out by means of the enzyme transglutaminase in the presence of either [3H]-labeled or unlabeled spermidine (SPD). After gel filtration chromatography and detection of the fractions containing [3H]SPD. the latter were tested for their effect on monouclear bone marrow cell proliferation. Results In three out of four patients examined, mainly SPD-protein conjugates (SPD-PC) were observed to accumulate during HD. The levels ranged from 0.17 to 4.93 pmol/mg proteins before dialysis, and these values increased at 30 minutes and at the end of the dialysis up to levels 11.90 pmol/mg. SPD-PC levels in healthy controls were 1.46 ± 0.82. SPD-PCs synthesized in vitro were recovered in two main fractions showing a molecular weight of > 100KD (peak 1) and of approximately 30 to 50 KD (peak 3), respectively. The SPD-PC contained in peak 1 showed the greatest inhibitory effect on colony-forming units-erythroid (CFU-E) proliferation without any appreciable effect on burst-forming units-erythroid (BFU-E). Conclusion We demonstrate that SPD-PC can accumulate in HD patients. These substances, which affect CFU-E proliferation, can be considered as an at yet unrevealed class of uremictoxins contributing to the onset of the uremic anemia.

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41.8 +/- 9.3% SD (range 31.1-48.4%) EGFP-positive cells and a mean of 92 +/- 2% SD (range 90-94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 microg/ml x 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity.

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Objective We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). Materials and methods Healthy donors ( n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 μg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. Results Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34 + cells ( r = 0.6, p = 0.004), CFU-GM ( r = 0.79, p = 0.0005) and CFC ( r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells ( r = 0.68, p = 0.001), numbers of day 4 CD341 cells ( r = 0.52, p = 0.005), CFU-GM ( r = 0.63, p = 0.002), and CFC ( r = 0.61, p = 0.003). Conclusion Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (

We evaluated the efficacy of recombinant human interleukin-3 (rhIL-3) in reducing the number of platelet transfusions and major infections after autologous bone marrow transplantation (ABMT) in patients with malignant lymphoma. 198 patients with non-Hodgkin's lymphoma (NHL, n = 111) and Hodgkin's disease (HD, n = 87) were randomized to receive rhIL-3 10 μg/kg/d (n = 130) or placebo (n = 68) for a maximum of 28 d after ABMT. Several well-known conditioning regimens were used. From day 1 after ABMT patients were treated with placebo or rhIL-3 at a dose of 10 μg/kg/d by continuous i.v. infusion for 7 d and then by s.c. administration for 21 d or until platelet (50 × 109/l) and neutrophil (0.5 × 109/l) recovery had occurred. Treatment was completed in 54% of the patients in the rhIL-3 group versus 75% in the placebo group (P

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210BCR/ABLtyrosine phosphorylation. We report here that preincubation of CML or normal CD34+ cells with graded concentration of AG957 (1 to 100 μmol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 μmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 μmol/L;P = .004), colony-forming unit–granulocyte-macrophage (CFU-GM; 34 v 85 μmol/L; P = .004), and LTC-IC (43 v 181 μmol/L; P = .004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 μmol/L significantly reduced the mean (±SD) percentage of BCR/ABL-positive progenitors (92% ± 10% v 33 ± 5%;P = .001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34+ cells with AG957 (1 μmol/L) and CH11 (1 μg/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34+ cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti–Fas-R antibody CH11. These data may have significant therapeutic applications.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Interferon alpha (alpha-IFN) is increasingly used for the treatment of patients affected by polycythemia vera (PV). As prior studies are difficult to interpret in view of the lack of appropriate controls, we undertook a randomized comparison of lymphoblastoid alpha-IFN (alpha n-1 IFN) treatment against venesection treatment alone. In a crossover trial, we treated 22 PV patients alternatively for 5 months each with 3 MU/day sc of alpha n-1 IFN and phlebotomy alone. During IFN treatment, red blood cell count and hematocrit level were well controlled in both trial groups, reducing or eliminating the need for phlebotomy in all patients; furthermore, platelet number and white blood cell count declined during alpha-IFN therapy. In addition, the number of symptomatic patients was greatly reduced, and in six patients a reduction in splenic size was observed. Finally, the only patient with chromosomal abnormalities showed a complete cytogenetic conversion after 5 months of alpha-IFN therapy. Thus, for the first time, our results provide the unequivocal demonstration that alpha-IFN is superior to phlebotomy in controlling the pathologic expansion of erythroid elements and all the clinical aspects of this disease.

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

FLT3, a receptor belonging to the FMS/KIT family and localized to 13q12, could play a role in the biology of early hematopoietic progenitor cells. Because FMS and KIT are expressed in both normal progenitors and myeloid leukemias, we looked for FLT3 expression in fresh human leukemic cells using Northern blot analysis. High levels of FLT3 expression were detected in 92% of the cases of acute myeloid leukemia (AML) tested, ranging from the M1 to the M5 stages of differentiation assessed in the French-American-British classification. Immature (MO) AML cells, biphenotypic leukemias, and AML with megakaryocytic differentiation (M7 subtype) also expressed the FLT3 transcript. FLT3 was also expressed at high levels in acute lymphoid leukemias of T and B origins. Finally, it was not expressed in chronic myeloid leukemias in chronic phase, whereas it was expressed in most blast crisis samples. This pattern of expression of FLT3 contrasts with the expression of FMS and KIT restricted to myeloid leukemias, and suggests that the FLT3 product could play a role in the expansion of the leukemic blasts of both the myeloid and lymphoid lineages.

Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

This mini-review summarizes evidence, provided by our group, relevant to the understanding of how redox signalling may control the fate of adult hematopoietic stem/progenitor cells (HSPCs). In particular it is shown that bone marrow-derived human HSPC are endowed with a composite panel of constitutively active NADPH-oxidases (NOXs) comprising the cell membrane-localized catalytic subunits of the NOX1, NOX2 and NOX4 isoforms. It is proposed that the coordinated activity of the NOX isoforms in HSPCs function as environmental oxygen sensor and generate low level of ROS, which likely serve as second messengers. The pro-oxidant setting, entering into play when HSPCs leave the hypoxic bone marrow niche, would enable them to be more responsive to proliferative/differentiative stimuli. Moreover it is suggested that enhanced ROS elicit mitochondrial "differentiation" in a pre-commitment phase needed to match the bioenergetic request in the oncoming proliferation/differentiation process.

V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these “pre-B-specific” genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu- pseudolight chain complex was actually replaced by the classical mu- kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

255 patients with acute non-lymphoid leukaemia (ANLL), observed between October 1984 and June 1987, entered a chemotherapy regimen consisting of induction therapy with cytarabine in combination with idarubicin (IDA/ARA) or daunorubicin (DNR/ARA), followed by consolidation with four courses of IDA+ARA plus 6-thioguanine (6-TG) or DNR+ARA+6-TG and a 6 month maintenance therapy with 6-TG and ARA. The median age was 62 years (range 55–78 years) and 33 were aged more than 70 years. The treatment groups were comparable for median age, FAB type, performance status and initial blood counts. 249 patients were randomised, 124 to the IDA/ARA arm and 125 to the DNR/ARA arm. Complete remission was achieved in 50 patients (40%) on the IDA/ARA treatment program and 49 patients (39%) on DNR/ARA. No definite differences were found between patients receiving IDA/ARA and those treated with DNR/ARA as far as complete response (CR), overall survival, failure free and relapse free survival are concerned. 74% of the complete responders in the IDA/ARA arm and 51% in the DNR/ARA arm achieved CR after a single course of treatment. Resistant leukaemia was observed in 13.7% of the patients in the IDA/ARA arm and in 31.2% in the DNR/ARA one, whereas hypoplastic death occurred in 29% and 14.4%, respectively. In conclusion, our data failed to show any advantage of idarubicin over daunorubicin even though there is some evidence that IDA, despite the higher toxicity, is more rapid in eradicating leukaemia as proved by the higher CR rate obtained after one course of induction.

Several reports have documented that leukaemic blasts produce a number of cytokines among them the granulocyte-monocyte colony stimulating factor (GM-CSF). We analysed the structure of the gene that codes for GM-CSF in 44 acute myeloid leukaemia (AML) cases in an attempt to establish whether the autocrine production of GM-CSF was due to a structural gene alteration. No structural alteration was detected in the GM-CSF gene in any of the 44 cases studied. We, therefore, conclude that the autocrine production of GM-CSF by leukaemia blasts is not dependent on gene rearrangement.

Notch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

The discovery of the Janus kinase 2 Val617Phe mutation has brought new insights into the development of myeloproliferative disorders; however, the pathogenesis of essential thrombocythemia and its related thrombotic complications has not been completely understood. Although the Janus kinase 2 Val617Phe mutation confirms the initially suspected clonal character of the disease, factors influencing clonal transformation and expansion in the bone marrow have not been fully detected. Furthermore, patients affected by essential thrombocythemia who are carriers of the Janus kinase 2 Val617Phe mutation show a higher incidence of venous thromboembolism both before, and at the time of diagnosis, compared with noncarriers, and recent evidence of splanchnic and cerebral vein thrombosis in carriers of the Janus kinase 2 Val617Phe mutation has been reported. The intake of oral contraceptives is a strong and independent risk factor for venous thromboembolism. In addition, in-vitro tests showed both an altered primary haemostatic plug formation and enhanced platelet aggregation in patients taking such drugs. Little is known, though, about the influence of steroid hormones on both megakaryopoiesis and platelet function in patients with the Janus kinase 2 Val617Phe mutation. Herewith, we report the case of a 30-year-old woman who took a third generation oral contraceptive for 5 months and developed an essential thrombocythemia with splenog-portal axis and superior mesenteric vein thrombosis. She was found to carry the kinase gene Janus kinase 2 mutation. © 2008 Wolters Kluwer Health | Lippincott Williams & Wilkins.

We report a noteworthy case of Eubacterium plautii infection after kidney transplantation. Our 33-yr-old transplant recipient received standard care; his post-transplant course was uneventful. However, on day 44 he underwent an emergency laparotomy for perforation of the ileum. He was initially treated with ceftazidime, fluconazole and metronidazole, but his fever persisted, so he was switched to meropenem and vancocin. We could not find any cause for his infection. On day 70, his temperature normalized. On day 75, he developed severe leukopenia (280 cell/mL). His cytomegalovirus-DNA test result was negative, so all immunosuppressants, except for prednisone, were stopped; instead, antibiotic prophylaxis was started, using caspofungin, trimethoprim-sulfamethoxazole and ciprofloxacin. On day 83, he underwent percutaneous drainage of massive left pleural effusion. We repeatedly cultured the pleural liquid, but it was not till three wk later that we were finally able to identify the causative organism. We hypothesize that the microorganism - which normally resides on the surface of the intestinal lumen - entered the bloodstream via bacterial translocation, eventually colonizing the pleurae. This translocation was favored by our patient poor clinical condition, his immunosuppressive treatment and his heavy antibiotherapy. Our experience highlights the need for wiser use of antibiotics in transplant recipients.

Summary The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O2 sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane ‘rafts’-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms.

Objective Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. Materials and Methods Human MSCs and different T cell populations (CD3 + , CD3 + /CD45RA + , CD3 + /CD45RO + , CD4 + /CD25 + , CD4 + /CD25 + /CD45RO + , CD4 + /CD25 + /CD45RA + ) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. Results We demonstrate MSC recruit Tregs from a fraction of CD3 + and from immunoselected CD3 + /CD45RA + and CD3 + /CD45RO + fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4 + /CD25 bright /FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25 + , CD4/CD25 + /CD45RA + , and CD4/CD25 + /CD45RO + ) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. Conclusions In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.

Retroviral vectors are used in human gene therapy trials to stably introduce therapeutic genes in the genome of patients' cells. Their applicability, however, is frustrated by the limited viability of transformed cells and/or by risks linked to selection of oncogene-mutated clones. The reasons for these drawbacks are not yet completely understood. In this study, we show that LXSN-NeoR gene/interleukin-7-engineered mesenchymal stromal cells exhibited a marked enhancement of reactive oxygen species production compared with untransfected cells. This effect resulted to be independent on the product of the gene carried by the retroviral vehicle as it was reproducible in cells transfected with the empty vector alone. Stable transfection of mesenchymal stromal cells with the different retroviral vectors pBabe-puro and PINCO-puro and the lentiviral vector pSico PGK-puro caused similar redox imbalance, unveiling a phenomenon of more general impact. The enhanced production of reactive oxygen species over the basal level was attributable to mitochondrial dysfunction and brought back to altered activity of the NADH-CoQ oxidoreductase (complex I) of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with a cAMP analog, thus pointing to alteration in the protein kinase A-dependent signaling pathway of the host cell. Transfection of mesenchymal stromal cells with a PINCO-parental vector harboring the green fluorescent protein gene as selection marker in place of the puromycin-resistance gene resulted in no alteration of the redox phenotype. These novel findings provide insights and caveats to the applicability of cell- or gene-based therapies and indicate possible intervention to improve them. Disclosure of potential conflicts of interest is found at the end of this article.