TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway.

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.

Comparison of ciprofloxacin, ofloxacin and pefloxacin for the prevention of the bacterial infection in neutropenic patients with haematological malignancies.

The efficacy of oral prophylaxis with ciprofloxacin, ofloxacin or pefloxacin was assessed in preventing bacterial infection in neutropenic patients with treatment being allocated randomly before beginning chemotherapy. Bacteraemia developed in six of 78 episodes (8%) treated with ciprofloxacin, in eight of 80 (10%) allocated to ofloxacin and in 12 of 77 (16%) when pefloxacin was given. However, there were no episodes involving Gram-negative bacilli among those given ciprofloxacin whereas three and seven episodes occurred in patients given ofloxacin or pefloxacin respectively (P = 0.013). With the exception of Pseudomonas aeruginosa, all potential pathogens isolated were resistant to all three fluoroquinolones. Faecal anaerobes were not affected by treatment with pefloxacin whereas their total numbers were reduced in 12 cases who had received ofloxacin and in nine cases who had been given ciprofloxacin (P = 0.002). Fourteen patients (18%) were colonized with pefloxacin resistant P. aeruginosa at the end of treatment with this agent compared with only two and five of those given ciprofloxacin or ofloxacin respectively. A similar trend was seen with other resistant Gram-negative bacilli colonizing 14%, 20% and 23% of patients for ciprofloxacin, ofloxacin and pefloxacin, respectively. Ciprofloxacin was therefore superior to the other two fluoroquinolones in preventing infections due to Gram-negative bacteria in this population of neutropenic patients.

Haemopoietic reconstitution after autologous blood stem cell transplantation in patients with malignancies: a multicentre retrospective study.

A retrospective study was undertaken to evaluate the efficacy of autologous blood stem cell transplantation (ABSCT) in terms of haemopoietic reconstitution after ablative chemotherapy or chemo-radiotherapy. 55 patients with malignancies, observed in four Italian institutions from January 1987 to June 1991, were eligible for evaluation. This series included 19 non-Hodgkin's lymphoma, 11 multiple myeloma, nine ovarian cancer, seven Hodgkin's disease, seven non-lymphocytic leukaemia, one acute lymphoblastic leukaemia, one neuroblastoma. 522 PBSC collections were performed on 55 patients. Following ABSCT, the rate of engraftment was positively related to the dose of CFU-GM infused and negatively to the presence of bone marrow involvement at conditioning. 48 patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant-related complications. Considering that 60% of the patients in this series were in partial remission or in progressive disease at the time of ABSCT, we conclude that ABSCT is a safe approach for the use of ablative conditioning therapy in patients with a wide scope of malignancies, provided that a large number of CFU-GM have been collected after mobilizing treatment.

The role of hemapheresis technology in allogeneic bone marrow transplantation.

In this paper the impact of hemapheresis technology on 238 allogeneic bone marrow transplants performed in Pescara from 1982 through 1993 is described. Granulocyte transfusions were limited to patients with neutrophil level < 0.2 x 10(9)/L. An average of 4 units of packed red blood cells were required to maintain adequate hemoglobin levels. Patients with major ABO incompatibility showed an increased requirement of red blood cell support as compared to patients ABO-matched and ABO minor mismatched. For platelet support single-donor platelets collected on a blood-cell separator were given. A total of 1548 platelet transfusions were examined. The median number of platelet transfusions for each patient was 5. Platelet refractoriness occurred in 44% of patients. The hemorrhage related mortality was 0.9%. The advancement made in the field of hemapheresis technology, as well as the improved transplant technique, have contributed to increase the post-transplant survival from 17% in the early experience (1976-1982) to 88% in the recent years (1992-1993).

Autologous blood stem cell collection after chemotherapy in patients with sensitive and refractory malignancies: a multicenter retrospective study.

A retrospective study was undertaken to assess the factors affecting the yield of peripheral blood stem cell (PBSC) collections after chemotherapy. Fifty-five patients with malignancies, observed in 4 Italian Institutions from January 1987 to June 1991 were eligible for evaluation. This series included 19 non-Hodgkin lymphoma, 11 multiple myeloma, 9 ovarian cancer, 7 Hodgkin disease, 7 acute non-lymphocytic leukemia, 1 acute lymphoblastic leukemia, 1 neuroblastoma. Five hundred and twenty two PBSC collections were performed on 55 patients after a median of 18 days after the start of chemotherapy. The yields of PBSC collections were related to the dose of cytoreductive chemotherapy exploited for PBSC mobilization and to the number of circulating white blood cells, colony forming unit granulocyte/macrophage (CFU-GM) and the percentage of monocytes at the time of collection. Forty-eight patients out of 55 transplanted (87%) had rapid, complete and sustained engraftment. Three patients (5%) died of transplant related complications.

Survival after PBSC transplantation and comparison of engraftment speed with autologous and allogeneic marrow transplantation: results of a multicenter study.

We have analyzed the results of a multicenter study on peripheral blood stem cell transplantation (PBSCT) performed on 55 patients suffering from various neoplastic diseases. After myeloablative therapy, they received a median of 6.8 x 10(8)/kg MNC and 11.4 x 10(4)/kg CFU-GM harvested by a median of 9 apheresis after mobilization with chemotherapy alone. As of date, 34 of the 55 patients are alive and 28 of them are in continuous complete remission after a follow-up of 30 months. The probability of survival was related to the disease status at transplant, CR/PR vs. PD (p = 0.0001) and the bone marrow involvement, BM-vs. BM+ (P = 0.009). Furthermore, a comparative study on speed of engraftment and clinical management was conducted on the 55 PBSCT patients as well as on 41 autoBMT and 52 alloBMT patients. Days to reach WBC > 1.0 x 10(9)/L, PMN > 0.5 x 10(9)/L and PLT > 50 x 10(9)/L was 12/14/33 for PBSCT, 17/20/23 for ABMT and 15/16.5/18 for BMT, respectively. Days with fever > 38 degrees C, systemic antibiotic therapy and length of hospitalization was 3/12/36 for PBSCT, 5/18.5/42 for ABMT and 9/25/46 for BMT respectively.

Autologous blood stem cell transplantation in malignant lymphomas: an Italian Cooperative Study.

Twenty-three patients with malignant lymphoma, (7 Hodgkin's, and 16 non-Hodgkin's) in different phases of disease were autografted in 4 Italian Haematology institutions using only chemotherapy-mobilized blood stem cells (BSC) collected by apheresis. Clinical and laboratory data were analysed centrally and showed mean collection yields of 8.1 x 10(8) kg mononuclear cells (MNC) (SE 0.5; range 2.6-13.8) and 24.1 x 10(4) kg CFU-GM (SE 7.4; range 1.4-162.9). The mean times required to attain 0.5 x 10(9)/l neutrophils and 50 x 10(9)/l platelets after marrow-ablative high-dose chemo+radiotherapy and BSC reinfusion were 14.9 days (SE 1.5; range 7-38) and 18.6 days (SE 2.6; range 6-49) respectively. The incidence of early deaths was < 5% and the requirement for support with blood product transfusion was moderate. The progression free survival (PFS) is > 50% at 3 years with a median follow-up of 17.3 months. Results were significantly better for patients autografted in remission. These results suggest that autologous blood stem cell transplantation (ABSCT) may be proposed for the primary treatment of poor prognosis malignant lymphomas. However, ABSCT needs to be compared with autologous bone marrow transplantation (ABMT) followed by infusion of growth factors to accelerate recovery.

Comparison of norfloxacin and pefloxacin in the prophylaxis of bacterial infection in neutropenic cancer patients.

In this study the efficacy of norfloxacin and pefloxacin for the antibacterial prophylaxis of granulocytopenia was compared in cancer patients following cytostatic treatment. A total of 136 patients was randomly selected to receive either norfloxacin or pefloxacin. Nineteen patients remained afebrile in the norfloxacin group compared with thirty one in the pefloxacin group (p = 0.045). Twenty four microbiologically documented infections (twelve with and twelve without bacteraemia) occurred in sixty seven patients taking norfloxacin, and twelve in sixty nine patients taking pefloxacin (five with and seven without bacteraemia) (p = 0.015). Only one infection caused by Gram-negative bacilli was observed in the pefloxacin group compared with seven in the norfloxacin group (p = 0.019). In conclusion, both microbiological and clinical results showed pefloxacin to be a better antibacterial agent than norfloxacin for these patients.

Detection of bacterial contamination in bone marrow graft.

A rapid method of determining bacterial contamination was used in a series of fifty-five bone marrow harvests (20 allogeneic and 35 autologous transplantations). The microbiological culture assays executed on bone marrow samples soon after the harvest and before the manipulation, showed a positivity only in the autologous bone marrow group. Another positivity was revealed after the freezing process of bone marrows for autologous transplantation. Two samples in the bone marrow group submitted to purging were positive after treatment and freezing process. In this study, the increase of bacterial contamination frequency seemed to be related to extensive handlings, such as pharmacological in vitro purging and freezing procedures.

Density gradient separation of hematopoietic stem cells in autologous bone marrow transplantation.

Recently, the principles of density gradient cell separation have been transferred to the marrow fractionation, and the Ficoll technique by using a COBE 2991 blood cell processor has been developed and widely employed as well. This method is particularly useful in view of a chemical antineoplastic purging intended for autologous marrow transplantation. Forty marrows, which derived from patients suffering with leukemia and lymphoma, were fractionated with Ficoll on a COBE machine and in vitro purged with Mafosfamide at a dose of 50 micrograms/ml/1 x 10e7 MN cells. The density gradient separation enables to reduce the initial volume to 10%, the contaminating RBC to less than 1%, the total nucleated cells to 25% (greater than 80% of MNC) sparing about 80% of the CFU-GM. After purging, the surviving hemopoietic progenitor cells were 2.5%. The clinical effects of the fractionated purged cells were studied in 11 autotransplanted patients and compared with 14 transplants performed with untreated buffy-coat marrow derived cells. Ficoll cells produced less adverse effects at the time of reinfusion, while, as expected, the time of hematopoietic recovery was delayed in these patients (mafosfamide treated cells). These results confirm the usefulness of the gradient density cell separation to reduce the side effects of the DMSO and to make reliable the Mafosfamide purging manoeuvre, preventing the interference of contaminating RBC aldehyde dehydrogenase.

Collection of peripheral blood stem cells in normal donors.

Although committed progenitor cells have been harvested during the standard plateletpheresis, only few experiences were specifically carried out in normal donors. The major drawback is the very low number of circulating haemopoietic stem cells and, besides a donor manipulation, a good harvesting technology is a fundamental prerequisite. To verify the effectiveness of an automate system, twenty normal volunteer donors were randomly assigned to four protocols of simultaneous PBSC and platelet collection. A Baxter separator CS 3000 was employed and we compared the yields and the efficiency of the modified Program 1 plus isoradial chamber (Protocol A), the modified Program 1 plus granulo chamber (Protocol B), the standard Program 3 plus isoradial chamber (Protocol C) and the standard Program 3 plus granulo chamber (Protocol D). The absolute yields for WBC (x10(9)), MNC (x10(9)), platelets (x10(11)) and CFU-GM (x10(4)) were respectively: 10.4, 10.1, 6.5 and 29.5 for Protocol A; 10.1, 9.9, 5.2 and 14.7 for Protocol B; 10.0, 9.9, 5.2, 14.7 for Protocol C, and 12.7, 12.4, 6.8 and 57.4 for Protocol D. The best CFU-GM efficiency was achieved with Protocol D (33%) vs. Protocol A (27.5%), Protocol B (14.5%) and Protocol C (27%). The above differences, although remarkable, were not statistically significant. Though preliminary, this study demonstrates the feasibility of a combined platelet and PBSC collection in normal donors.

Pharmacological mobilization of haemopoietic progenitor cells in human peripheral blood.

We investigated Prednisone (PRD) and Folinic Acid (FA) ability to recruit stem cells (CFU-S) into the circulating compartment, evaluating CFU-GM, BFU-E and CFU-GEMM count increases in peripheral blood (PB). Fifteen healthy donors were administered 75 mg PRD in one trial, and 15 mg FA p.o. through 5 consecutive days in another trial. We observed a significant expansion for BFU-E and CFU-GEMM compartments, though not parallelled by CFU-GM expansion. FA determined a significant increase in CFU-GM on the 5th day of administration, as well as in BFU-E on day 7. It fails to raise CFU-GEMM. The authors suggest a less empiric persuing for such attempts to optimize the use of non-chemotherapic drugs to recruit hemopoietic precursors into PB, so that these can suit to meet stem cell needs for autologous and allogeneic transplantations.

Human bone marrow processing using Cobe 2991 and CS 3000 blood cell separators for further ex vivo manipulation.

Several automated procedures are now available to enrich stem cells from large bone marrow (BM) volumes prior to ex vivo treatment or cryopreservation. This report details our experience using a ficoll-hypaque (F/H) gradient on Cobe 2991 cell washer and a CS 3000 continuous flow separator, on 90 BM processed for allogeneic and autologous transplantation. In the Cobe series, from 70 BM aspirates, 89 +/- 5% of the original mononuclear cells (MNC) was found in the light density fraction with a erythrocytes (RBC) and granulocytes (PMN) removal of 98 +/- 1 and 97 +/- 4.5%, respectively. Over 80% of the initial myeloid precursors (CFU-GM) were recovered in a small final volume. Twenty BM processing were performed with a CS 3000 separator using program "3" and granulo chamber. This technique yielded 86 +/- 9% of the initial MNC while 85 +/- 10% of RBC and 90 +/- 1.1% of PMN was removed. Over 75% of the original CFU-GM was recovered in the final product. Both techniques are effective to large-scale purification of progenitor cells and readily available as routine procedures for marrow processing.

Autologous blood stem cell transplantation as intensive consolidation therapy in hematological malignancies.

Ten consecutive patients (pts), suffering from hematological malignancies (5 NHL, 3 ANLL and 2 HD), received high doses of radio-chemotherapy and autologous peripheral blood stem cell (PBSC) transplantation. Seven of them were in 1st CR, two in 3rd CR and one in refractory status. The recruitment of PBSC was performed during the conventional non-intensive schedule of therapy. The median interval between the remission and the transplant was 6 months (3-15). The pts received a median of 0.96 x10(9) MNC/Kg b.w. and of 3.8 x10(4) CFU-GM/Kg b.w. Hemopoietic recovery occurred promptly and the median number of days to reach WBC greater than 1.0 x10(9)/L was 10 (7-15), PMN greater than 0.5 x10(9)/L was 12 (10-34), platelets greater than 50 x10(9)/L was 13 (10-41) and reticulocytes greater than 20 x10(9)/L was 12 (10-15). Three pts relapsed after 14, 12 and 6 months, respectively. Two of them achieved a further remission and one died. The remaining seven, are alive and disease-free with a follow-up of 11 months (range 2-27).

Infection after autologous blood-derived stem cell transplantation.

Ten patients who underwent autologous blood-derived stem cell transplantation (APBSCT) were studied retrospectively to determine the frequency and outcome of transplant associated infections during the hemopoietic reconstitution. Six patients developed an infection after transplant. Severe bacterial infections were unfrequent and mostly caused by gram-positive cocci. In no case pneumonia was observed and this scarce morbility of infection could be the result of early engraftment. None of them died for infection. The frequency of infections in APBSCT patients appears lower than the one observed in autologous bone marrow transplant patients.