Corneal explantation activity in 2002: an analysis of the modified operational schedule.

After data have been gathered about corneal explants performed within the AOP health-care web throughout 2001, the resulting findings were used to update the selection system for donation fitness and operational procedures. The rejection of anti-HBc-positive grafts and tissues coming from subjects more than 79 years old resulted in decreased donations (256 donations, that is 492 corneal explants in 2001 vs 140, that is 273 in 2002), although the number of deaths was unchanged (1298 in 2001 vs 1294 in 2002). Corneas fit for transplantation did not change in number-126 (25.6% of the total available) in 2001 and 113 (41.4%) in 2002-while the instances of rejected corneas occurred 56.3% less frequently, allowing a savings of great deal of human and money resources. After activity schedules were modified, the results analysis confirmed the expected improvement in 2001.

Searching for the magic bullet against cancer: the butyrate saga.

n-Butyric acid and its "polymorphic" derivatives have been largely but somehow "blindly" studied in oncology and in red cell diseases with consistent results through decades indicating a strong maturative effect determined by enhancement of gene transcription. Although these effects have been observed mainly in vitro, the relative absence of systemic toxicity of butyrates render these compounds appealing as specific therapeutic agents. More interestingly, their specific mechanism of action, i.e. inhibition of histone deacetylase and de-repression of transcription represents at present an unique tool for diseases such as acute leukemias which are characterised by a disregulation of co-repressors and co-activators of gene transcription. More insight into specificity and modalities of action of different butyrate derivatives may be a guarantee for excellent tailored antileukemic therapy in the future.

Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide.

Adoptive transfer of ex vivo-generated cytomegalovirus (CMV)-specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV-specific T lymphocytes based on the antigen-presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201-restricted pp65 nonamer peptide. CMV-specific T lymphocytes were identified following induction of interferon gamma (IFN-gamma) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV-specific T lymphocytes were selectively produced in these cultures and showed CMV-restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV-specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV-specific lymphocytes.

Treatment of large cell lymphoma with the Fi2 regimen (doxorubicin, vincristine, cyclophosphamide, bleomycin and prednisone): 25-year experience.

The CHOP protocol is the reference treatment for large cell lymphomas, but several other schemes of different intensity have recently been studied with controversial clinical findings. We report here the results obtained at our institution with a CHOP-like regimen called Firenze 2 (Fi2), which is characterised by an original scheduling of chemotherapy administration. A total of 225 patients, who were diagnosed from 1974 to 1996, were included in this retrospective study. All patients received the Fi2 regimen as a first-line intervention. One-hundred and sixty-two (72%) achieved complete remission; the overall survival at 120 months was 51% with a disease-free survival of 67% (median follow-up = 78 months). The survival curve showed a stable plateau of 42% after 16 years, which remained stable for further 4 years. In a multivariate survival analysis, achievement of complete remission (p<0.001) and IPI index of 0 or 1 (p=0.05) were significantly associated with a better survival. Overall, the outcome of our patients was similar to that reported by others, but the distinguishing feature of our study is the very long follow-up of the patients. Our study confirms that first generation regimens are effective and can cure a substantial proportion of patients. The long-term results of our study are helpful to retrospectively identify high-risk patients whose prognosis is poor and who can be candidates for more aggressive schemes of chemotherapy.

Amifostine in the treatment of low-risk myelodysplastic syndromes.

BACKGROUND AND OBJECTIVE: The phosphorylated aminothiol agent amifostine (Ethyol) protects bone marrow and other tissues from toxicity due to ionizing radiation and antineoplastic drugs, and stimulates progenitors from normal and myelodysplastic bone marrow. Contrasting results have been published so far on the effectiveness of amifostine in correcting cytopenia in patients with myelodysplastic syndromes (MDS). DESIGN AND METHODS: In a pilot phase II study we treated 26 patients with low risk MDS (13 RA, 2 RARS, 2 CMML, 9 RAEB with blasts < 10%) with amifostine (200 mg/m(2 )x 3/week for 4 weeks). RESULTS: Hemoglobin concentration, reticulocyte, neutrophil and platelet counts increased respectively in 6 (23%), 11 (42%), 13 (50%) and 9 (34%) of patients. Red cell transfusions were reduced (> 50%) in 4/26 patients and abolished in 1/26. Unexpectedly a significant decrease in soluble transferrin receptor level at week 4 of therapy, compared to the basal level (p<0.04), was observed in the whole population of patients. INTERPRETATION AND CONCLUSIONS: Amifostine can ameliorate cytopenia in some patients with MDS, with few and mild side effects. Neutropenia is more likely to be corrected than anemia or thrombocytopenia. Mechanisms underlying this biological effect remain to be clarified.

Immunohistochemistry applied to the study of bone marrow Gaucher's cells: a case report.

Gaucher's disease is frequently associated with immunologic abnormalities, e.g. hypergammaglobulinemia, polyclonal gammopathy and benign monoclonal gammopathy. A patient with Gaucher's disease and a selective accumulation of IgM k in Gaucher's cells without serum monoclonal gammopathies is described. The selective accumulation is detected by immunohistochemistry analysis performed on cryostat bone marrow biopsies.

An open evaluation of triple antibiotic therapy including vancomycin for febrile bone marrow transplant recipients with severe neutropenia.

Infectious complications still represent a major problem in patients submitted to bone marrow transplant (BMT); approximately 40% of febrile episodes are associated with infection and one-third of these are bacteremias. Opinions about the best appropriate empiric regimens are based on evaluation of cost, potential for adverse side-effects, development of bacterial resistance, prevalent nosocomial infections. In order to assess the clinical and microbiological effectiveness of an aggressive approach, we performed a prospective open study in 72 neutropenic febrile BMT patients, employing a triple antibiotic association including amikacin 500 mg x 8h, ceftazidime 2 g x 8 h, vancomycin 500 mg x 8 h as first-line empiric treatment. For the purpose of this study, a lasting return of temperature to normal and complete disappearance of either clinical or bacteriological signs of infection without any modification of therapy was considered as success; the persistence of fever after 72 hours or a protocol change was considered as failure. Eighty episodes were enrolled during the course of the study; bacteriological evidence of infection was obtained in 23 (28.7%) febrile episodes. Median duration of antibiotic administration and of febrile episodes were 5 and 2 days respectively. Overall response rate based on clinical responses was 87% and 91% in microbiological documented infections. Death due to sepsis nor toxicity were observed. This triple antibiotic combination appears to be a very effective regimen for the empiric treatment of febrile episodes in severely neutropenic BMT recipients.

Maturation and apoptosis of primary human acute myeloblastic leukemia cells are determined by TNF-alpha exclusively through CD120A stimulation.

BACKGROUND AND OBJECTIVE: Tumor necrosis factor-a plays an important role in hematopoiesis. Its effects are mediated through two membrane-bound receptors: TNF-R I (p55; CD 120a) and TNF-R II (p75; CD 120b). The aim of our study was to investigate the relative roles of these receptors. DESIGN AND METHODS: We analyzed in 16 acute myeloid leukemia cases whether TNF-alpha could induce in vitro maturation and apoptosis. We then investigated which of the two receptors was provoking monocytic maturation and which was responsible for apoptosis by using the agonistic MoAb HTR-9, directed at CD120a, and the CD120b antagonistic MoAb UTR-1. RESULTS: Monocytic maturation (morphologic and immunologic) was induced in all cases studied, although to different rates, by TNF-alpha and by HTR-9 incubation. The addition of UTR-1 to TNF-alpha did not abolish maturation, nor did it affect apoptosis, which was present in primary AML cultures after 4 and 10 days. INTERPRETATION AND CONCLUSIONS: We present here evidence that the sole stimulation of CD 120a, but not of CD120b, by TNF-alpha is responsible for bot monocytic maturation and apoptosis of primary AML blasts.

Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA.

There is considerable interest in an autologous transplantation (AT) programme for patients with high-risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a 'nested' reverse transcriptase-polymerase chain reaction (RT-PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT-PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte-colony stimulating factor (rhG-CSF). K19 transcripts in one or both BM samples were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR- and IHC-positive BM samples were diagnosed as 'normal' on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19-negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19-negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high-risk non-metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.

The effectiveness and tolerability of epoetin alfa in patients with multiple myeloma refractory to chemotherapy.

Anemia is a frequent complication of multiple myeloma, becoming chronic in patients who are resistant to chemotherapy. This randomized, parallel, controlled multicenter study (71 patients receiving concomitant chemotherapy) evaluated the efficacy and safety of epoetin alfa in improving anemia and eliminating the need for transfusions in multiple myeloma patients refractory to conventional first- or second-line chemotherapy. Forty patients were treated with subcutaneous epoetin alfa (150 IU/kg per dose, increasing to 300 IU/kg per dose, every 3 weeks) for 6 months, and 31 entered a control group. The epoetin alfa group had a significantly (P < or = 0.001) greater percentage of patients (75% vs. 21%) with increases in hemoglobin levels and/or reduced transfusion requirements. In 44 non pre-transfused patients (20 controls, 24 in the epoetin alfa group), the mean increase in hemoglobin was significantly (P < or = 0.0001) greater in the epoetin alfa group (+2.1 vs. -0.2 g/dl). Increases in hematocrit and red blood cells were also significantly (P < or = 0.0001) greater in epoetin alfa-treated patients, with corresponding reductions in transfusion requirement. In the 27 pre-transfused patients (11 controls, 16 in the epoetin alfa group), there was a trend towards reduced transfusional need in epoetin alfa-treated patients. Thus, in patients with multiple myeloma refractory to chemotherapy epoetin alfa is a well-tolerated treatment which improves anemia in non pre-transfused patients and appears to reduce transfusion need in those previously transfused.

Fatal herpesvirus 6 encephalitis after unrelated bone marrow transplant.

A complex pattern of neurological dysfunctions with generalized seizures and visual allucinations, but without focal signs, suddenly arose 20 days after an unrelated bone marrow transplant for chronic myelogenous leukemia (CML) in a 13-year-old girl, accompanied by signs of acute skin graft-versus-host disease (GVHD). Magnetic resonance imaging (MRI) revealed multiple bilateral foci of signal abnormalities, which were exclusively localized in the grey matter, sparing the white. Extensive microbiological and virological assays of cerebrospinal fluid (CSF) allowed the identification of HHV-6, variant A, DNA. Further progression of both neurological alterations and of skin and gut GVHD led to a fatal outcome 2 weeks later. A retrospective analysis of both the recipient and donor mononuclear cell suspensions supported the hypothesis that HHV-6 had been acquired from the donor with the bone marrow graft. This report suggests a pathogenetic role of HHV-6 in viral encephalitis in immunocompromised bone marrow transplant (BMT) recipients, and its possible association with GVHD.

Butyrate-stable monosaccharide derivatives induce maturation and apoptosis in human acute myeloid leukaemia cells.

The rapid degradation and subsequent lack of efficacy of n-butyric acid in vivo has been improved by the synthesis of monosaccharide stable pro-drugs of butyric acid. We studied the effects of D1 (O-n-butanoyl-2,3-O-isopropylidene-alpha-D-mannofuranoside), G1 (1-O-n-butanoyl-D,L-xylitol), and F1 (1-O-n-butanoyl 2,3-O-isopropylidene-D,L-xylitol) on the maturation and proliferation of AML cell lines HL 60 and FLG 29.1 and of purified blast cells from 10 cases of de novo acute myeloid leukaemia (AML). AML cell maturation was measured by surface antigen expression, morphology and cytochemistry. Toxicology in mice was also evaluated (DL50 1000 mg/kg). In HL 60 cells G1 and D1 increased the expression of CD15 and CD11a (presenting 62% of promyelo-metamyelocytes), and in 7/10 cases of primary AMLs that of CD11a, CD11b, CD15, and myeloperoxidase. D1, G1 and F1 induced a dose-dependent inhibition of tritiated thymidine uptake. Apoptosis (evaluated by flow cytometry and agarose gel electrophoresis) was induced in AML blasts by D1 and F1 (79% and 94% respectively for HL 60 cells) and, with less effect, by G1 (27%). The persistence of maturative and apoptotic activity in these new pro-drugs of butyric acid, hydrolysed only inside the tumour cell, suggests a possible use in differentiation therapy of myelodysplastic syndromes and AMLs.

Severe hypoxia enhances the formation of erythroid bursts from human cord blood cells and the maintenance of BFU-E in vitro.

Incubation in severe hypoxia (1% oxygen) increased the number of erythroid bursts generated from full-term CD34+, or premature mononucleated, human cord blood (CB) cells, in semisolid cultures containing stem cell factor (SCF), interleukin (IL)-3 and erythropoietin (EPO). Severe hypoxia also enhanced the maintenance of erythroid burst-forming units (BFU-E) in CB cell liquid cultures. These positive effects of hypoxia on the maintenance and cloning efficiency of BFU-E did not extend to the other progenitors assayed. Hypoxia, on the other hand, markedly reduced the size and level of hemoglobinization of bursts and, in liquid cultures, suppressed the growth factor-stimulated numerical increase in BFU-E and inhibited the expression of CD36, a marker of erythroid colony-forming units and maturing erythroid precursors. However, when transferred to clonal assays incubated in air, cells from liquid cultures incubated in hypoxia or in air generated fully expanded and hemoglobinized bursts, suggesting that in hypoxia the clonogenic potential of BFU-E was maintained and the development of erythroid clones reversibly inhibited. These results indicate that hypoxia inversely regulates two subsequent phases of erythropoiesis, i.e., it enhances the maintenance of BFU-E and the early development of erythroid clones but inhibits the terminal expansion and maturation of these clones. The cloning of CB cells selected for CD34 positivity, when compared with that of the total population of mononucleated CB cells, revealed that the early development of erythroid bursts was either hypoxia-enhanced or hypoxia-insensitive, reflecting the existence of two different types of BFU-E. Hypoxia-enhanced BFU-E are relatively immature, are maintained in hypoxia but not in air, and account for a large part of CD34+ BFU-E and for a high percentage of the BFU-E in premature CB. Hypoxia-insensitive BFU-E are mostly CD34- and are largely predominant in full-term CB, and most probably correspond to a more mature type of BFU-E.

Randomized trial comparing netilmicin plus imipenem-cilastatin versus netilmicin plus ceftazidime as empiric therapy for febrile neutropenic bone marrow transplant recipients.

The aim of this study was to compare the clinical and microbiological efficacy of netilmicin plus imipenem-cilastatin (Net + Imi) vs netilmicin plus ceftazidime (Net + Cef) as empiric antimicrobial therapy in bone marrow transplant (BMT) febrile neutropenic patients (pts). Sixty-six pts undergoing BMT for hematological malignancies and solid tumors were randomized to receive Net + Imi or Net + Cef as first-line antibiotic therapy. A lasting return of temperature to normal and complete disappearance of either clinical or cultural signs of infection without any modification of therapy was considered as improvement; the persistence of fever after 72 hours, the addition of a third antibiotic or a protocol change was considered as failure. Sixty-nine episodes were randomized during the course of the trial; bacteriological evidence of infection was obtained in 17 (25%) febrile episodes. Overall outcome based on clinical responses was as follows: 80% of pts on Net + Imi responded compared to 73% of those on Net + Cef. For microbiologically documented infections response rates were 70% in Net + Imi group and 43% in the Net + Cef group (p = ns). Neither septic death nor toxicity were observed. Both empiric regimens were shown to be effective; Net + Imi appeared to be more effective in microbiologically documented infections but there was no statistical significance. In conclusion, both Net + Imi and Net + Cef are active and safe as empirical treatment of febrile episodes in neutropenic BMT pts.

High doses of recombinant human erythropoietin fail to accelerate platelet reconstitution in allogeneic bone marrow transplantation. Results of a pilot study.

BACKGROUND AND OBJECTIVE: The effectiveness of recombinant human erythropoietin (rhEpo) in accelerating erythroid engraftment in patients undergoing allogeneic bone marrow transplantation (BMT) has been demonstrated in previous studies. On the other hand, there are experimental data suggesting that high doses of rhEpo might also exert a stimulatory effect on thrombopoiesis. METHODS: We carried out a pilot study on the use of high doses of rhEpo (500 U/kg/day for 30 days after transplant) in ten patients (HD-Epo group) receiving BMT to evaluate the effects on both erythroid and platelet (Plt) engrafment. This group was compared to ten BMT patients who had not received the hormone (Placebo group). RESULTS: The HD-Epo group patients showed signs of accelerated erythropoietic recovery; in fact, the time required to reach a reticulocyte count higher than 30 x 10(9)/L was significantly shorter than in the Placebo group, while the number of high RNA content reticulocytes (HFR) was about three times greater. Circulating transferrin receptor (TfR) levels 30 days after BMT were also significantly higher in the HD-Epo group than in the other. Finally, the number of red blood cell (RBC) transfusions in the first 30 days following BMT was about twofold lower in the HD-Epo group; moreover, 4/10 patients who were treated with HD-Epo did not require any RBC units. No significant effects on the engraftment of platelets or on the number of Plt transfusions were observed in the HD-Epo as compared to the Placebo group. No adverse effect was noted on granulocytopoiesis, nor were any adverse clinical experiences found in patients who had been treated with erythropoietin at high dosages. INTERPRETATION AND CONCLUSIONS: These data confirm that rhEpo may stimulate erythroid reconstitution after BMT, while its effects on Plt engraftment and on Plt transfusion requirements are minimal.

Combination therapy with G-CSF and erythropoietin after autologous bone marrow transplantation for lymphoid malignancies: a randomized trial.

Previous studies have shown that, unlike in patients submitted to allogeneic BMT, administration of recombinant erythropoietin (Epo) after autologous BMT (ABMT) had no significant effect on erythroid recovery and transfusional requirements. On the other hand, it has also been shown that combining Epo with recombinant granulocyte colony-stimulating factor (G-CSF) in patients with the acquired immunodeficiency syndrome (AIDS) and with myelodysplastic syndromes resulted in additive effects on erythropoiesis. To test the effects of combined G-CSF + Epo therapy on erythroid recovery after autologous bone marrow transplantation a pilot randomized, three-arm trial was designed. Thirty patients suffering from lymphoid malignancies submitted to ABMT were randomly assigned to receive G-CSF alone (5 micrograms/kg, from day + 1 up to reaching an ANC > or = 10(9)/1), G-CSF + Epo (150 U/kg, from day +1 to +21), or neither of these (controls). Patients receiving G-CSF + Epo had significantly more reticulocytes on day +21 and reached 30 x 10(9)/1 reticulocytes earlier when compared to both G-CSF and control patients; however, the number of red blood cell (RBC) transfusions was not modified by the addition of Epo to G-CSF, although both groups had significantly fewer units transfused than controls. No effect on platelet recovery or platelet transfusional requirements was observed. Myeloid recovery was comparable in the G-CSF and G-CSF+Epo groups, and significantly accelerated as compared to controls. We conclude that the addition of Epo to G-CSF causes a slight acceleration of erythroid recovery after ABMT, but is not associated with transfusional benefits. Therefore, the present data do not support the use of Epo in association with G-CSF to hasten erythroid recovery after ABMT.

Effects of fludarabine and gemcitabine on human acute myeloid leukemia cell line HL 60: direct comparison of cytotoxicity and cellular Ara-C uptake enhancement.

This study was designed to compare the effects of fludarabine and gemcitabine on cytosine arabinoside (Ara-C) uptake and retention, and their specific cytotoxicity on HL 60 human acute myeloid leukemia cells. The leukemic blasts were exposed to either drug at equimolar concentrations (10 microM) for 3 h and further incubated with Ara-C (5 microM), added immediately (day 0) or after an interval of 24 h in cells were kept in a drug free medium (day 1). On day 0, leukemic cells exposed to fludarabine 10 microM had a significant (P<0.01) increase in Ara-C uptake (297 +/- 11 pmol/10(7) cells) with respect to control cells (not pre-treated: 195 +/- 10 pmol/10 (7) cells). After treatment of leukemic cells with fludarabine, cytoplasmic Ara-C peaked after 180 min of exposure, as well as nuclear bound Ara-C. At the same time, a significant decrease in the number of S-phase leukemic cells, consistent with depressed [3 H]TdR uptake was observed. Although on day 0 gemcitabine 10 microM did not have potentiating effects on Ara-C uptake, it showed a high degree of intrinsic cytotoxicity as a single agent(clear from cell cycle distribution, [3H]TdR uptake, plating efficiency (PE) data and percentage of apoptotic cells). Cells exposed to gemcitabine, on the other hand, showed on day 1 a significant increase in Ara-C uptake (2.4 x control values in the cytoplasmic and 3x in the nuclear fractions) and a reduced number of S-phase blasts, [3H]TdR uptake and PEs, as well as an increased apoptotic cell death. Evidently, it is possible to modulate Ara-C uptake by leukemic cells with gemcitabine. Although this effect is similar to that demonstrated with fludarabine, its kinetics and time of efficacy are different and also, because of its intrinsic higher cytoxicity and lack of important side effects, gemcitabine could be considered a suitable candidate for Ara-C association therapy in acute leukemia.