In vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice.

OBJECTIVE: Intranasal inoculation of the i strain of the parvovirus minute virus of mice (MVMi) into immunodeficient SCID mice induces suppression of myeloid and erythroid progenitors in the bone marrow (BM) and lethal leukopenia. In the present study, we investigated whether the mouse megakaryocytic lineage was susceptible to MVMi. MATERIALS AND METHODS: In vitro and in vivo infections with purified MVMi were conducted and their effects on the megakaryocytic lineage studied. RESULTS: In vitro infection of BM cells showed a multiplicity of infection-dependent inhibition in the colony-forming ability of megakaryocytic progenitors (colony-forming unit megakaryocyte [CFU-MK]). Neutralization or heat inactivation of the virus abrogated this inhibition. Expression of the MVMi nonstructural-1 protein was detected in the in vitro infected and cultured megakaryocytic cells. In vivo, intranasal inoculation of a lethal dose of virus was incapable of producing significant thrombocytopenia, although an increase in mean platelet volume was observed. Significantly, in the BM of these animals, a progressive decrease in CFU-MK was noted from day 14 postinfection, with survival rates less than 1% by day 35 postinfection. At day 35 postinfection, intermediate megakaryocytic differentiation stages showed maintenance of the proportion and ploidy of cells and a moderate decrease in the total number of these cells per femoral BM. CONCLUSIONS: The results demonstrate that MVMi is capable of inhibiting the proliferative capacity of megakaryocytic committed progenitors both in vitro and in vivo. Moreover, the in vivo data show that depletion of BM CFU-MK is compensated by the system, and platelet counts in the peripheral blood are maintained close to normal values.

Conclusions of a national multicenter intercomparative study of in vitro cultures of human hematopoietic progenitors.

With the aim of developing a standardized program of clonogenic cultures, a multicenter intercomparative study of human CFU-GM, BFU-E and CFU-GEMM cultures was conducted. Aliquots of fresh mononuclear cord blood cells, as well as uniform culture materials and instructions for cell culture and for colony scoring were distributed to 28 national laboratories involved in hematopoietic research and transplantation. High interlaboratory coefficients of variation (CV) in the reported number of progenitors were found in our first intercomparative study (range 67-231%). To investigate the relevance of colony scoring in variations of the reported colony numbers, participants were invited to attend a meeting where a single culture dish was scored. In this case, the CVs ranged from 31% to 81%. A subsequent intercomparative assay was then conducted, and significant reductions in the inter-laboratory CVs were obtained with respect to the first study (CVs for colonies grown with two different media: CFU-GMs, 48% and 55%; BFU-Es, 70% and 62%; CFU-GEMMs, 70% and 51%; respectively). In most instances CVs were not significantly different from those obtained in the single plate scoring study, suggesting that the scoring process was the most relevant parameter accounting for variations in the reported colony numbers.

Erythropoietin treatment in allogeneic BMT accelerates erythroid reconstitution: results of a prospective controlled randomized trial.

Twenty-eight allogeneic BMT patients (16 with acute leukemia, 12 with chronic myeloid leukemia) were included in a single center, prospective, randomized, controlled trial to assess the value of recombinant human erythropoietin (rh-Epo) in this setting. rh-Epo was administered through a central venous catheter as a single bolus injection (days 0-7: 100 U/kg/d; days 7-30: 150 U/kg/d). No secondary effects to rh-Epo treatment were detected. An earlier appearance of reticulocytes and a diminished need of red blood cells (RBCs) transfusions were observed in patients who were treated with rh-Epo (4 units vs 12 units; p < 0.05). The time to unsupported platelets above 25 x 10(9)/l was less in patients treated with rh-Epo than in control patients (19 days vs 31; p < 0.05), and they received significantly fewer platelet transfusions (36 units vs 138.5; p < 0.05). Our results show that rh-Epo treatment is capable of accelerating the erythroid reconstitution and decreasing the need for RBC transfusions. A beneficial effect on platelet reconstitution is also suggested, but further studies are necessary to confirm this point.

Fractionated TBI and methotrexate-cyclosporin do not seem to increase relapses in BMT for first chronic phase CML patients: results of a single centre study.

Fractionated total body irradiation (FTBI) and methotrexate-cyclosporin A(MTX-CSA) have been found useful in reducing interstitial pneumonia (IP) and acute graft-versus-host-disease (GVHD) in bone marrow transplantation patients, but an increase in relapse rate has been observed by some authors when these strategies are used. To evaluate this relapse risk, we performed a retrospective analysis in 24 consecutive first chronic phase chronic myeloid leukemia patients who received an HLA-identical non-T cell-depleted graft in a single institution. All were conditioned with cyclophosphamide plus FTBI (12 Gy in six fractions delivered twice daily for 3 days) (CY-FTBI) and received MTX-CSA as GVHD prophylaxis. Serial hematologic and cytogenetic bone marrow analysis were performed at least three times (days +30, +100, +360) and at variable intervals thereafter in long-term survivors. Actuarial probabilities of developing IP and acute GVHD greater than or equal to II were respectively 5.9% and 44.2%, with a GVHD-associated mortality of 33%. Four-year actuarial relapse and disease-free survival rates were 7.7% and 48.2% respectively. No exclusively cytogenetic relapses were observed. Our results suggest that CY-FTBI and MTX-CSA are not associated with an increase in relapse rate in 1CP-CML patients.

Interferon alpha treatment of accelerated-phase chronic myeloid leukemia in relapse after bone marrow transplantation: a case with complete cytogenetic and molecular remission.

We describe here a patient with Philadelphia-positive chronic myeloid leukemia who had a hematologic and cytogenetic relapse after bone marrow transplantation. A diagnosis of accelerated phase was made when an additional malignant clone was detected. This clone was probably derived from the primitive Philadelphia clone, with duplication and rearrangement of the Philadelphia chromosome. The patient was treated with interferon alpha 2a and experienced a complete cytogenetic and molecular remission, with full reconstitution of the donor marrow.

Clinical results in 50 multiply transfused patients with severe aplastic anemia treated with bone marrow transplantation or immunosuppressive therapy.

Fifty patients with aplastic anemia (AA) were treated with BMT or immunosuppressive therapy (IST). Twenty-one patients underwent BMT using cyclophosphamide (CY) and 7 Gy total lymphoid irradiation (TLI) and cyclosporin A (CsA) plus methotrexate (MTX). Actuarial survival is 71% at 5.3 years with an incidence of graft failure of 0% and of acute GVHD of 38.9%. Univariate analysis of variables influencing survival showed a trend for a poorer outcome in patients who received > 30 transfusions prior to BMT and in male recipients from female donors. Twenty-nine patients > 40 years of age or without matched siblings received antithymocyte/antilymphocyte globulin (ATG/ALG). Response rate to the first course of treatment was 46.4%. Subsequent courses of IST rescued 33% of patients who relapsed or had not responded. Actuarial survival is 62% at 8.6 years. In our experience both treatment strategies have given encouraging results although overall morbidity is higher in the IST group because 25% of patients are therapy or transfusion-dependent. The role of irradiation in the conditioning regimen of BMT patients, recently challenged, is discussed.

[Bone marrow autotransplantation in acute myeloblastic leukemia in its first complete remission. The clinical results in 41 patients]. / Autotrasplante de médula ósea en la leucemia aguda mieloblástica en primera remisión completa. Resultados clínicos en 41 pacientes.

BACKGROUND: A single-center experience using autologous bone marrow transplantation (ABMT) as postremission therapy for acute myeloid leukemia (AML) in first complete remission (CR1) in 41 patients is analyzed. PATIENTS AND METHODS: From July 1986 to March 1994, 41 patients with AML in CR1 underwent an ABMT. Source of hematopoietic stem cells was bone marrow in all cases. In eleven patients the marrow was purged with mafosfamide (ASTA-Z 7654). Median age was 31 years (17-59) and median time from CR to ABMT was 7 months (3-27). Busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) was employed as conditioning regimen in 36 patients. The rest 5 patients were prepared with cyclophosphamide (120 mg/kg) and fractioned total body irradiation (12 Gy). Eleven patients received G-CSF after AMBT because of an absolute neutrophil count lower than 0.5 x 10(9) on day +30. Survival analysis was performed according to the methods of Kaplan and Meier and comparison between groups used the log-rank test. RESULTS: With a median follow-up of 26 months (12-75) 21 patients remain alive in CR. Disease-free survival (DFS) at five years was 40% (95% CI: 25-55%). Transplant-related mortality, mainly for infection and hemorrhage, occurred in 6/41 patients (16%). Leukemia relapse was the main cause of treatment failure: 14/35 (40%). Probability of DFS and relapse was similar for those patients with purged ABMT on unpurged ABMT 45 +/- 23% vs 38 +/- 16% and 37 +/- 14% vs 54 +/- 22% respectively. A significantly longer engraftment time for neutrophils (> 0.5 x 10(9)) and platelets (> 20 x 10(9)) was observed in those patients who received a bone marrow treated with mafosfamide compared with the unpurged group (54 vs 29 days for neutrophils and 102 vs 58 for platelets respectively) (p < 0.05). CONCLUSIONS: ABMT is a feasible treatment for AML in CR1. Using bone marrow as hematopoietic stem cell support we observed that delayed engraftment was a common finding among AML patients, specially when the marrow was treatment with mafosfamide. Leukemia relapse remains as the main cause of treatment failure for ABMT.

[A simple method for detecting immature precursors after in vitro bone marrow treatment with ASTA-Z 7654]. / Un método simple para detectar progenitores inmaduros después de un tratamiento in vitro de medula ósea con ASTA-Z 7654.

Cyclophosphamide derivatives for in vitro use, such as mafosphamide L lysine (ASTA-Z 7654) are capable of removing residual leukaemic cells from the bone marrow used for autologous transplantation. ASTA-Z also removes committed stem cells detected in semi-solid cultures (CFU-GM). However, haemopoiesis is preserved by more immature progenitors, undetected in usual cultures but detectable in liquid culture of 21 days (MTC). After treating 15 normal bone marrow specimens with 100 microM ASTA-Z, CFU-GM cultures and medium-term cultures (MTC) are carried out. CFU-GM are cultured on the 21st day of incubation from the cell suspension of the MTC. The CFU-GM made the day of in vitro treatment (0.9 colonies/plate) reappear in the CFU-GM cultures performed on the 21st day of incubation of MTC (42 colonies/plate), although the figures attained in parallel controls without any treatment are not fully reached.

Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells.

BACKGROUND: The transduction of human peripheral blood T cells with retroviral vectors constitutes an attractive approach for the correction of a number of genetic diseases. In this study we have conducted a systematic analysis of the relevance of a large number of parameters currently considered to affect the transduction of, and transgene expression in, human T cells. METHODS: Retroviral vectors encoding the human nerve growth factor receptor (NGFR) were used for transducing human T cells from normal volunteers. The proportion of T cells that expressed the marker transgene was determined by flow cytometry using anti-NGFR antibodies. RESULTS: Spinoculation and static fibronectin (FN)-assisted infections improved to a similar extent the transduction efficiency of PHA/IL-2 stimulated T cells, when compared with samples subjected to standard static infections. When immobilized anti-CD3 (anti-CD3i) or anti-CD3i/28i-stimulated T cells were considered, static infections in FN-coated plates were reproducibly more efficient than spinoculation infections performed on FN-uncoated plates. Under optimized manipulation conditions (three infection cycles of anti-CD3i/28i-stimulated T cells in FN-coated plates) the total number of NGFR+ T cells harvested after 7 days of incubation represented, on average, twice the total number of T cells seeded at Day 0, and up to 95% of the human T cells efficiently expressed the marker transgene. Similar results were obtained regardless of whether samples were manipulated in medium supplemented with fetal bovine serum or with heat-inactivated autologous serum. CONCLUSIONS: Our study offers new experimental conditions for the transduction of human T cells, with obvious implications for the development of gene therapy protocols.

High toxic efficiency of ricin immunotoxins specific for the T-cell antigen receptor of a human leukemia T-cell line.

Immunotoxins (ITs) were prepared by covalently coupling ricin to monoclonal antibodies (MAbs) directed against: (a) 2 different epitopes of the T-cell receptor (TcR) expressed by the Jurkat leukemia T-cell line (JTi2 and JTi4 MAb), (b) 2 epitopes of the CD3 complex (SpV-T3b and 11D8 MAb), (c) the CD2 and the CD8 cell-surface molecules. Conjugates were assayed for their cytotoxic activity by pre-incubating the Jurkat cell line with different concentrations (10-250 ng/ml) of each IT for 2 hr at 37 degrees C in the presence of 0.1 M lactose. After washing, cells were cultured for 24 hr and their protein synthesis and proliferative capacities were assessed. Dose-response experiments indicated that JTi2, JTi4 and anti-CD3 (11D8) ITs inhibited by greater than 90% the cell line proliferation at 50 ng/ml, a 5-fold lower concentration than that required to achieve a similar effect when anti-CD2 and anti-CD3 (SpVT3b) were used. After 4 hr of culture subsequent to treatment with JTi2 or JTi4 ITs (250 ng/ml), protein synthesis was inhibited (greater than 80%). By limiting dilution analysis (LDA) we estimated that the frequency of proliferating Jurkat cells (1/1.5) was reduced to 1/20, 1/460 and 1/300 after treatment with anti-CD3 (SpVT3b), JTi4 and JTi2 ITs, respectively. Phenotypic analysis of 13 clones derived from JTi2 IT-treated Jurkat cells showed that 50% were CD7+ CD3- JTi- variants. When bone-marrow mononuclear cells, previously mixed with low concentrations of Jurkat cells, were treated with anti-JTi ITs, the toxic efficiency estimated by LDA was maintained whereas the growth of CFU-GM remained unaltered.

Functional and phenotypic variations in human T cells subjected to retroviral-mediated gene transfer.

The insertion of suicide genes in donor T lymphocytes constitutes the basis of new approaches aiming at the treatment of the graft-versus-host disease (GVHD), a frequent complication in recipients of allogeneic haematopoietic grafts. In this study we investigated the impact that the ex vivo manipulation required for the retroviral transduction of T cells had on the functionality and differentiation of these cells. Compared to fresh T cells, samples that had been subjected to standard activation (1 microg/ml of both anti-CD3i and anti-CD28i MoAbs) followed by transduction with vectors encoding for the HSV-tk and tNGFR genes maintained the proliferative response to an allogeneic stimulus. These cells, however, had a significantly lower cytotoxic response to allogeneic cells compared to fresh samples. When the concentration of anti-CD3i was reduced to up to 1000-fold (1 ng/ml), similar T-cell transductions were obtained, while the cytotoxicity of the ex vivo manipulated samples was significantly recovered, when assessed either at 7 or 14 days of culture. In all instances, a similar functionality was observed in transduced samples not subjected to immunomagnetic cell sorting, compared to purified fractions enriched in NGFR(+) and NFGR(-) cells. The analysis of CD45RA and CCR7 markers in samples transduced under standard stimulatory conditions showed a differentiation of fresh CD8(+) CD45RA(+)/CCR7(+) naive cells to cells having a predominant central CD45RA(-)/CCR7(+) and effector CD45RA(-)/CCR7(-) memory phenotype. However, when samples were activated with low doses of anti-CD3i, a significant population of naive cells became apparent. Although activation with high doses of anti-CD3i/anti-CD28i resulted in a similar phenotype in both NGFR(+) and NFGR(-) populations, the naive population observed in samples activated with low concentrations of anti-CD3i was almost restricted to the NGFR(-) population. These results show that reducing the stimulation mediated by anti-CD3i in protocols of T-cell retroviral gene transfer significantly helps to preserve the cytotoxic capacity of these cells to allogeneic cells, without affecting the susceptibility of these cells to the retroviral vector. In addition, we observed that modulating the activation of transduced T cells implies the generation of changes in the differentiation of CD8(+) cells, although we could not establish a direct relationship between the CD45RA/CCR7 phenotype of these cells and their cytotoxic reactivity to an allogeneic stimulus.

Collection and transplantation of peripheral blood progenitor cells mobilized by G-CSF alone in children with malignancies.

Results of collection and transplantation of peripheral blood progenitor cells (PBPC) mobilized by G-CSF in 31 children with different malignancies were analysed. A total of 43 aphereses were performed, following administration of granulocyte colony-stimulating factor (G-CSF), using a continuous flow blood cell separator (Cobe Spectra) through a central venous catheter. For patients weighing 0.5 x 10(9)/l and a platelet count of 20 x 10(9)/l without platelet support were 9.5 and 18, respectively. The number of CD34+ cells infused correlated highly with engraftment kinetics. The extra-medullary toxicity was low and manageable.

[Treatment with interferon alfa-2a in the chronic phase of Philadelphia-positive chronic myeloid leukemia]. / Tratamiento con interferón alfa-2a de la leucemia mieloide crónica Ph1 positiva en fase crónica.

PURPOSE: To evaluate the cytologic and cytogenetic response attained with interferon alpha-2a (IFN, Roferon*A) in patients with Ph '-positive chronic myelogenous leukaemia (CML) in the chronic phase. MATERIAL AND METHODS: A prospective study was carried out on 22 CML patients diagnosed in the Haematology Service at the Princesa Hospital in Madrid. The therapeutic regime consisted of two phases: A) Hydroxyurea was given until the white-cell count was reduced to 15-20 x 10(9)/L. B) Roferon*A was then given subcutaneously at a doses of 5 MU/m2 per day. The follow-up was performed weekly, and monthly once the leucocyte count had stabilized. The cytologic and cytogenetic response was assessed by bone marrow aspiration performed after 6, 9, 12 and 18 months. The toxicity was evaluated in accordance with the WHO recommendations. RESULTS: The median follow-up is 263 days (21-930). Thirteen patients (65%) had initial complete haematological response and 3 (15%) had partial response. The mean time to achieve response was 42 days (0-321). In the last evaluation, 69% of the patients were in sustained haematological remission (53% complete and 16% partial) with median follow-up of 232 days (21-930). The cytogenetic response was evaluable in 13 patients (follow up > or = 6 months): three attained complete response (23%) and three others partial response (23%). The commonest untoward effects were hypertriglyceridaemia (100%) and myelosuppression (86%). Grade-III thrombocytopenia was seen in 19% of the patients and grade-III anaemia or leucopenia in 5%. No infectious or haemorrhagic complications have appeared. Therapy was discontinued in 3 patients (14%), two due to severe flu-like syndrome and one for parkinsonism after 809 days of treatment. At the moment of evaluation two patients had died, one in lymphoid blastic crisis on day 217 and the other in the immediate post-BMT period. CONCLUSION: Treatment with interferon-alpha 2A is useful in the chronic phase of CML. An important number of responses can be attained, even in patients in the late chronic phase, and the toxicity seems acceptable.

Erythropoietin in the treatment of delayed immune hemolysis of a major ABO-incompatible bone marrow transplant.

Delayed immune hemolysis can be observed after major ABO-incompatible bone marrow transplants (BMT). The management of these hemolytic episodes includes transfusion of group O red blood cells and increases of immunosuppression. Here we report the case of a 25-year-old patient who developed overt immune hemolysis on day +50 after a HLA-identical ABO-incompatible BMT. To avoid added immunosuppression, erythropoietin was started: an increase in reticulocytes sufficient to maintain hemoglobin despite persistent hemolysis was observed. We conclude that erythropoietin may have a role in the management of delayed-onset hemolysis of major ABO-incompatible BMT, especially when added immunosuppression is undesirable.

Busulfan and melphalan as conditioning regimen for autologous peripheral blood stem cell transplantation in multiple myeloma.

Twenty-four patients with multiple myeloma (MM), three (12.5%) in complete remission (CR) and 21 (87.5%) in partial remission (PR) were treated with high-dose chemotherapy (HDCT) (busulfan 12 mg/kg+melphalan 140 mg/m2) as preparative regimen for autologous peripheral blood stem cell (PBSC) transplantation. These cells were previously collected by leukapheresis after mobilization by high-dose cyclophosphamide (HD Cy)+rhGM-CSF (18 patients) or rhG-CSF alone (six patients). Considering 23 evaluable patients following HDCT, the CR rate was 58% (14 patients) and the PR rate was 38% (nine patients). One transplant-related death occurred following this regimen (4%). With a median follow-up of 20 months (range 4-34) after transplantation, 21 patients are alive (87%). Disease progression after transplantation was observed in four patients. Overall and relapse-free actuarial survival at 24 months was 91% and 74%, respectively. 12 patients (50%) remain in CR 15 months (4-34) post transplant. The major toxicity was mucositis. Busulfan+melphalan is a safe and feasible conditioning regimen for APBSCT in MM with acceptable toxicity and a high objective response rate, which may result in prolonged survival.

Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy.

The immune function of retrovirus-mediated gene modified (GM) T cells is critical for a beneficial effect to follow their adoptive transfer into patients. Recent clinical data show that GM T cells expanded with PHA have reduced function in vivo. However, little functional analysis of PHA stimulation is available. Our results show that expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity and IFN gamma and perforin expression) to allogeneic stimulation or viral antigens in vitro. Conversely, CD3/CD28-based protocols can preserve this immune function. Retroviral transduction did not alter the functional profile induced by polyclonal stimulation. We investigated the mechanisms leading to this functional effect, and identified differential effects of PHA and CD3/CD28 on the distribution of CCR7/CD45RA T cell functional subsets, which may explain the functional differences observed. While CD3/CD28 stimulation parallels the lineage differentiation pattern induced by antigens in physiological conditions, PHA induces a skewed distribution of the CCR7/CD45RA functional T cell subsets, with near disappearance of the subpopulations that display the effector phenotype. Overall, this study demonstrates a functional disadvantage for transduction protocols based on PHA, uncovers mechanisms that may explain this functional effect, and provides us with information to design and select transduction protocols with an improved functional outcome.

Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood cells.

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo-expanded CB cells, purified CD34(+) cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo-expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34(+) cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo-expanded versus the fresh CB cells (CD45(+) repopulation in NOD/SCIDs BM: 3. 7% +/- 1.2% v 26.2% +/- 5.9%, respectively, at 20 days posttransplantation; P <.005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% +/- 1.7% of CD45(+) cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo-expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45(+) and CD45(+)/CD34(+) cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo-expanded samples were found. The analysis of the engrafted CD45(+) cells showed that both the fresh and the in vitro-incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.