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.

Hyperviscosity syndrome in hematological diseases and therapeutic apheresis.

The hyperviscosity syndrome classified into pleiocytosic, sclerotic and sieric syndromes according to the blood components involved are characterized by a different degree of clinical signs and symptoms related to rheological modification of blood. Therapeutic management of these syndromes is complex and the choice of apheresis treatment is generally restricted to emergency episodes in order to overcome the acute phase and to reverse the symptoms until definitive therapy is effective.

Randomized trial of sequential administration of G-CSF and GM-CSF vs. G-CSF alone following peripheral blood progenitor cell autograft in solid tumors.

A trial was conducted to investigate whether the sequential administration of recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) could accelerate reconstitution of hematopoiesis, compared with G-CSF alone following high-dose chemotherapy (HDCT). A group of 34 consecutive patients with solid tumors undergoing HDCT and autologous peripheral blood progenitor cell (PBPC) transplantation was studied. Conditioning regimen included carboplatin, etoposide, mitoxantrone, and melphalan for breast cancer and cyclophosphamide or ifosfamide, carboplatin, and etoposide for the other tumors. HDCT was delivered from day -3 to day -1. PBPC were infused on day 0, and on the same day growth factors were administered subcutaneously (s.c.) 5 microg/kg each. Seventeen patients were randomized to receive G-CSF from day 0 to day 13 after HDCT (arm A), and 17 patients received G-CSF from day 0 to day 6 and GM-CSF from day 7 to day 13 (arm B). Patients were stratified, and their characteristics were homogeneous in both arms for age, performance status, and number of previous chemotherapy courses and CD34+ infused. The median time to absolute neutrophil count (ANC) >500/microl was 10 days in arm A and 9 days in arm B (p = 0.96). Days to platelet (PLT) count >20,000 were not different in the two treatment arms (p = 0.1), but patients randomized to arm A had a lower platelet count compared with patients in arm B. One month after PBPC transplantation, a statistically significant difference in PLT count was observed (arm A median 150x10(3)/microl (90-310), arm B median 254x10(3)/microl (117-387),p = 0.0013). The days patients had fever >38 degrees C were 39 in arm A and 26 in arm B (p = 0.18). The difference in the length of hospital stay was not statistically significant between the groups (Mann-Whitney sum rank test). After a median follow-up of 30 months, 21 patients were alive and 20 were disease free. These data show that the two growth factors are associated with different patterns of hematopoietic recovery, and larger randomized trials in groups of more homogeneous patients will be needed to define the effects and benefits of combination growth factor therapies.

Meningitis due to penicillin-resistant Streptococcus pneumoniae in patients with chronic graft-versus-host disease.

Two episodes of meningitis due to penicillin-resistant Streptococcus pneumoniae occurring in two patients with chronic graft-versus-host disease (GVHD) are reported. Both patients were treated with ceftazidime. The first patient died, unresponsive to therapy. The second patient showed clinical improvement, reverting to her baseline mental status. This report draws attention to the fact that in chronic GVHD patients: (1) bacterial prophylaxis does not ensure protection against encapsulated bacteria; (2) rapid microbiological investigation is recommended with any upper respiratory tract infections.

Reconstitution of normal neutrophil function in chronic granulomatous disease by bone marrow transplantation.

Allogeneic bone marrow transplantation was carried out on an 11-year-old boy with chronic granulomatous disease and severe chronic pulmonary insufficiency of restrictive type. After preparative regimen with busulfan (13 mg/kg) and cyclophosphamide (200 mg/kg), the patient received marrow cells from his HLA-identical and MLC-nonreactive sister. Durable sustained engraftment of donor hematopoietic and lymphoid populations occurred, as documented by analysis of genetic markers and complete reversal of the neutrophil function defect. No episode of infection occurred in the post-transplant course and, currently, 40 months after transplantation the patient is in excellent health and growing normally and showing an increasing improvement of his respiratory capacity. The successful outcome in this patient demonstrates that marrow transplantation is at present the only curative approach for this congenital disorder of neutrophil function.

Cytogenetics and bone marrow transplantation.

The authors report hematologic and cytogenetic data on 19 patients treated with allogeneic bone marrow transplantation (BMT) for severe hematologic disorders: 8 patients with chronic myelogenous leukemia, 6 with acute leukemia, 3 with severe aplastic anemia, 1 with refractory anemia, and 1 with beta-thalassemia major. Cytogenetic assays were performed on marrow cells before conditioning, 30 days after BMT, and at subsequent times. The authors discuss the role of cytogenetic studies in the evaluation of bone marrow engraftment, leukemic transformation of the graft, and disease relapse.

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.

Growth factor administration following autologous peripheral blood progenitor cell transplantation.

Hematopoietic growth factor (HGF) administration following autologous peripheral blood progenitor cell transplantation (APBPCT) is a current approach for shortening the duration of high-dose chemotherapy-induced transient peripheral pancytopenia. Several published clinical experiences and a retrospective study reported here show that recombinant human granulocyte colony stimulating factor (rhG-CSF) or recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) administration potentiates polymorphonuclear leukocyte (PMN) and white blood cell (WBC) recovery with some clinical benefits mainly related to the reduction of infectious complications during the shortened period of neutropenia. However, this therapeutic strategy does not produce any enhancement of platelet (PLT) recovery or potentiation of red cell production. Conversely, a recent phase I/II study carried out in our institution showed that the combined administration of rhG-CSF and recombinant human erythropoietin (rhEPO) is able to potentiate trilineage hematopoietic recovery with a reduction of PLT transfusions and to considerably simplify the clinical management of patients as compared to patients treated with APBPCT alone. The post-APBPCT administration of rhEPO with rhGM-CSF decreased the number of days with WBC < 1 x 10(9)/L but failed to produce any appreciable effect on PLT recovery. Both combined treatments significantly reduced the patients' hospital stay and allowed the abrogation of systemic antibiotic administration following APBPCT. A further group of patients were treated with the combined administration of rhEPO, rhG-CSF and rhGM-CSF; they did not show a faster hematopoietic recovery than rhG-CSF plus EPO treated patients and a consistent hyperthermia was observed in most patients as a prominent side effect. Future prospective randomized studies will clarify the efficacy of HGF administration following APBPCT. Moreover, further improvements in the hematopoietic support of transplanted patients may be obtained when stem cell factor, flt3/flk2 tyrosine kinase ligands or megakaryocyte growth and development factor will become clinically available.

Patterns of cytomegalovirus retinitis in immunocompromised patients treated with 9-(2-hydroxy-1-(hydroxymethyl)ethoxymethyl) guanine (ganciclovir).

Five immunocompromised patients, four with AIDS and one who had undergone bone marrow transplantation, showing ocular signs of cytomegalovirus retinitis, were treated with 9-(2-hydroxy-1-(hydroxymethyl)ethoxymethyl) guanine (Ganciclovir), given intravenously at the dose of 5 mg/kg twice daily for a period ranging from 10 to 20 days. At the end of the treatment, in 4 of 5 patients, the ophthalmoscopic picture had improved, with reduced exudation and an arrest in the progression of retinal necrosis, the pattern clearly indicating a trend towards organization and scarring. Complete resolution of the retinitis without subsequent relapse was observed only in the bone marrow transplant patient, who recovered immunologically, whereas improvement of the eye involvement was only transient in the three AIDS patients.

Evaluation of azlocillin-amikacin combination for empirical therapy of infection in febrile neutropenic patients.

We have evaluated the azlocillin-amikacin combination, given at a daily dose of 200 mg/kg and 15 mg/kg respectively, in the treatment of 62 consecutive febrile granulocytopenic patients (less than 500 PMN/microliters) affected by hematological disease. The effectiveness of the treatment was assessed in 60 patients, 44 (73%) of whom responded within 96 hours from the beginning. 36 of the responders showed microbiological and clinical infections, 2 had clinically documented pneumonia and 6 a possible infection. No improvement was obtained in 16 patients; 7 of whom suffered from clinical and microbiological infection, 2 from pulmonary mycosis, 4 from possible infection and 3 from doubtful infection. Seven of these patients subsequently responded to a proven antibiotic treatment, while only one of the remaining responded to a second-line empirical antibiotic schedule. These results suggest that the combination of azlocillin-amikacin was able to overcome about two-thirds of the infections, representing an effective remedy for the empiric treatment of febrile neutropenic patients.

Clinical efficacy and in vitro activity of the ciprofloxacin-azlocillin combination (ratio 1:10) against gram-negative bacteria from non-neutropenic haematologic patients.

The authors report treatment of eighteen haematologic patients (twelve male, six female, age between 21 and 78 years), suffering from upper respiratory tract (ten patients) and/or lower urinary tract (eight patients) infections caused by Gram-negative germs, with a combination of ciprofloxacin-azlocillin in the ratio 1:10. Before treatment, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Fractional Inhibitory Concentration (FIC) index of Gram-negative isolates from patients for the ciprofloxacin-azlocillin combination were evaluated. The in vitro experiments revealed a synergistic activity of the combination for 85% of isolates, while at the same concentration ciprofloxacin alone was 100% effective, and azlocillin alone was 50% effective. The combination was administered to patients as follows: ciprofloxacin: 750 mg "per os" every 12 h; azlocillin 5 g intravenously every 8 h for a therapeutic cycle of 8 days. Seventeen of the eighteen patients that were treated with the combination showed complete eradication of the causative pathogen, sixteen of the eighteen patients recovered fully, whereas the other two showed significant improvements. The tolerability of the combination was excellent in seventeen patients and only one patient developed symptoms of mild gastric intolerance. The results presented here warrant further interest in studies of this antibiotic combination.

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.

Early hematopoietic reconstitution after autologous transplantation with blood-derived stem cells in a patient with advanced lymphoma.

A 30-year-old man with advanced non-Hodgkin lymphoma underwent repeated leukaphereses for harvesting blood-derived hemopoietic stem cells. Collection was started 8-10 days after the end of L-VAMP therapy (3 cycles). Nine procedures were performed and a total of 65.4 x 10(9) mononuclear cells (0.87 x 10(9)/Kg) were collected, processed, cryopreserved and stored in liquid nitrogen. The yields of CFU-GM, BFU-E and CFU-GEMM were respectively 964 x 10(4) (12.4 x 10(4)/Kg), 249 x 10(4) (3.2 x 10(4)/Kg) and 798 x 10(4) (10.4 x 10(4]. The patient received a myeloblative regimen consisting of fractionated total body irradiation (1200 cGy) and cyclophosphamide (120 mg/kg) followed by infusion of his own thawed cells. Early trilineage hematopoietic recovery was first observed on day +8; 1 x 10(9)/l WBC were reached on day +11, 0.5 x 10(9)/l PMN on day +13 and 50 x 10(9)/l platelets on day +11. Course was uneventful and the patient was discharged from hospital on day +21. Eight months after transplant the patient is in continuous unmaintained complete remission with normal blood cell counts. This reports suggests that complete and sustained engraftment can be achieved with peripheral stem cells recruited after "soft" chemotherapy.

Seven-day storage of single donor platelets in polyolefin bags: clinical, biochemical, morphological and microbiological evaluation.

We compared the in vitro and in vivo function of fresh and stored platelet concentrates (PCs) collected by an automated continuous-flow blood cell separator (CS 3000 Fenwal) in a closed-system apheresis kit in order to evaluate the possibility of extending the storage time to seven days with the polyolefin container (PL-732). The initial 220 ml platelet volume (5.14 +/- 1.23 x 10(11) was divided into two parts. Half was transfused and the other half was stored for 7 days. All cultured units were negative for bacterial contamination. Mean counts for fresh and stored platelets were respectively 2.34 +/- 0.59 and 2.17 +/- 0.50 X 10(11)/100 ml of PCs (mean recovery 88.7 +/- 11.9%). The pO2 levels were maintained during storage (179.9 +/- 30.5 mmHg) but pCO2, pH, LDH, osmolality, glucose consumption, bicarbonates, ATP, and osmotic stress values changed significantly after 7 days storage. From a clinical point of view, in 14 patients receiving a total of 38 PC transfusions no statistically significant change in corrected post-transfusional levels was observed between fresh and stored PC. Biochemical and morphological data and clinical results suggest that PCs collected with CS-3000 blood cell separator in a closed system and stored for 7 days in polyolefin bags (PL-732) can be satisfactorily employed in clinical practice.

Enrichment of marrow hemopoietic progenitor cells using a blood cell processor.

A total of 93 bone marrows (BM) from normal donors and patients were processed using the IBM-COBE 2991 blood cell washer to produce a concentrated buffy coat (BC) for either bone marrow transplantation (BMT) or cryopreservation for subsequent autologous BMT. The reduction in volume was 73.3 +/- 8.5% and nucleated blood cells (NBC) recovery was 87.1 +/- 9.1% of original marrow. Red blood cell (RBC) and platelet (PLT) contamination was reduced 64.5 +/- 10.9% and 41.2 +/- 24.1%, respectively. Clonogenic activity indicated that the NBC fraction was highly enriched in hematopoietic progenitor cells (greater than 100%) as assessed in vitro (CFU-GM). Results were not affected by diagnosis, initial marrow volume or cell count of the BM suspension. We conclude that this is a simple and reproducible method using blood bank, facilities and permits BC preparation from BM without significant loss of hematopoietic progenitor cells.

Concentration of human hematopoietic stem cells in bone marrow transplantation: results of a multicenter study using Baxter CS 3000 plus cell separator.

Preliminary BM processing to produce an enriched MNC fraction from large BM volumes improves subsequent pharmacological and/or immunological "ex vivo" treatment and cryopreservation. We detail on a multicenter study (6 Transplant Centers) performed to establish an effective and reliable protocol using a CS 3000 continuous flow separator on a large series of BM processed for autologous (96) and allogeneic (12) transplantation. The reduction in volume was 78.6 + 7.2% while 28.9 + 12.4% of the original nucleated cells were found in the final product. A mean of 84.3 + 13.2% of the staring MNC was yielded in a fraction containing over 81% MNC. Cloning efficiency indicated than the final graft was highly enriched in progenitor cells committed to the granulocyte/macrophage pathway (> 100%) as assessed in vitro (CFU-GM). Removal of RBC and PLT was 98.3 + 1.1 and 37.7 + 14.6%, respectively. The mean dose of MNC and CFU-GM was 0.6 + 0.37 x 10(8) and 0.96 + 1 x 10(5) recipient weight. The entire process was accomplished in 87.5 + 20 min. We concluded that this automated device is a simple and reproducible method for BM processing suitable as first step for further "ex vivo" automated negative and/or positive cell selections.

Rapid diagnosis of bacterial contamination of blood: comparison of three automated microbial detection systems.

Rapid methods of determining microbial contamination are needed in suspecting contaminated banked blood or other blood products. In this study, we experimented contaminated blood units with 122 strains of bacteria and fungi. After innoculation, a comparison between ESP Blood Culture System (Difco Laboratories Inc., Detroit MI), BacT/Alert (Organon Teknica, Durham, NC) and Bactec 9240 System was made for their efficiency in the detection of microbial contamination. Experimental data showed a diagnostic relevance of these automated systems with no significant differences in time detection of microbial contamination between the three methods under comparison.

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.

Comparative analysis of two systems for HCV genotyping.

The HCV genotype can be determined by PCR using nested primers to structural or non-structural HCV regions, followed by hybridization analysis of the amplified products. In this study, two different systems, both based on PCR and hybridization analysis, were used to determine HCV genotype in 32 HCV positive patients at the Clinic of Infectious Diseases, University of Chieti. The main difference between these commercially available systems lies in the different PCR target. Amplification of PCR targets was obtained from all samples. Hybridization analysis gave unequivocal results for all samples with both methods, yielding a 100% rate of genotype determination, with a complete correlation at the genotype level. A lower concordance at subtype level (65% concordance) was found, due only to two types of discrepancies. Both methods proved easy to use in our hands, adding evidence to their potential usefulness and reliability in clinical settings.