Cytotoxic effects of high concentrations of sodium ascorbate on human myeloid cell lines.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

A multiple regression analysis on factors influencing haematopoietic progenitor cell collection for autologous transplantation.

Autologous hematopoietic stem cell (HSC) transplantation today is the standard treatment for a wide variety of haematological and oncological diseases. HSC are collected from peripheral blood by leukapheresis (HPC-A) following chemotherapy and/or growth factor-mediated mobilization. The ideal HPC-A collection allows to reach the CD34(+) target dose through a single, tailored leukapheresis. The aim of this paper was to find out which collection parameter might play a key role in obtaining a CD34 dose >4×10(6)/kg with a reduced number of leukapheresis. To address this issue, a multivariate logistic regression was carried out on several operational and laboratory parameters from 943 HPC-A collections performed in 600 hematological and oncological patients. We observed a CD34(+) cells collection efficiency (CE) >50% when patient's pre-apheresis total WBC count was lower than 12.5×10(6)/mL. At the same time, the likelihood of reaching the CD34(+) cells target dose/kg increased from 6 to 3 times when the pre-apheresis WBC count ×10(6)/mL t was below 4.3 (OR=6.1; 2.6-14.1) and between 4.3 and 7 (OR=2.8; 1.4-5.7) respectively when compared to a pre-apheresis WBC count >36×10(6)/mL.

Survey of current practice for monitoring and management of platelet refractoriness in Italy.

Platelet transfusion failure is a common phenomenon affecting from 7% to 34% of haematology-oncology patients. Monitoring the efficacy of platelet transfusion through the evaluation of a post-transfusion platelet count and clinical response represent an important guide for subsequent transfusions and for the detection of refractoriness. The aim of this survey was to investigate physicians' attitudes and practices regarding the monitoring of platelet response and the management of platelet refractoriness. An e-mail based survey was conducted among the heads of blood banks with a hemapheresis ward in Italy. Heads of 64 centers out of the 122 initially identified (52%) completed the entire survey. Apheresis, buffy-coat pool, and platelet rich plasma represented an average of 46%, 38% and 17% of the total number of transfusions, respectively. In the prophylaxis of hemorrhagic episodes, most of the centers utilized as standard dose one unit of apheresis platelets (55.7%) and/or one unit of buffy-coat pool platelets (42.6%), while 11.4% of respondents used an average of 6 units of platelet rich plasma. In only 27.9% of the centers was the platelet dose established based on the body weight of the recipient. Only one-third of the centers evaluated the response to platelet transfusion in all patients, while the rate increased to 60% in onco-hematological patients. Among patients transfused on an outpatient basis, the rate dropped to 20%, and a platelet sample taken 10 min after transfusion was generally used. The survey documented a substantial lack of interaction between the clinician requesting the transfusion and the one responsible for the preparation and delivery of the product, with both figures involved in the diagnosis of refractoriness in only one-third of the centers. In conclusion, despite being a frequent condition, platelet refractoriness is still managed with a high degree of heterogeneity and often overlooked. Better adherence to existing guidelines and standard operating procedures, as well as the involvement of transfusion centers in prospective evaluations can help reduce this variability and improve the outcome of transfused patients.

A novel flow cytometric approach to distinguish circulating endothelial cells from endothelial microparticles: relevance for the evaluation of endothelial dysfunction.

Circulating endothelial cells (CEC) and endothelial microparticles (EMP) are emerging as markers of endothelial repair and activation/apoptosis. Although significant changes in the number of CEC and EMP in pathological conditions have been reported, their reliable identification and quantification still remain a technical challenge. Here, we present a novel methodology for the identification and quantitation of CEC and EMP based on multicolor flow cytometry. Using a lyse/no wash protocol, we observed that in 50 µl of peripheral blood, the large majority of events expressing an endothelial phenotype (CD45-/CD146+/CD34+) are due to non-nucleated particles (DRAQ5-) carrying mitochondrial activity (MitoTracker+) and, therefore, classified as EMP. We enumerated circulating EMP by single platform absolute count in a lyse/no wash four-color flow-cytometric procedure, which allowed the distinction, within the whole endothelial compartment, of EMP derived from endothelial progenitors (CD45-/CD146+/CD34+/CD117+) and from mature endothelial cells (CD45-/CD146+/CD34+/CD117-). A significant increase in both subsets was observed in patients with diabetes mellitus. Thus, this simple and highly reproducible method may be useful for monitoring endothelial dysfunction in clinical settings.

Amyloid-specific T-cells differentiate Alzheimer's disease from Lewy body dementia.

Alzheimer's disease and dementia with Lewy bodies are the most common neurodegenerative dementias in old age. Accurate diagnosis of these conditions has important clinical implications because they tend to be confounded. In the brain of Alzheimer's disease patients amyloid-beta is produced in excess and deposited as plaques, forming the hallmark of this condition. Lymphocytes have been implicated in the process of amyloid-beta removal and inflammation occurrence. Here we investigated peripheral amyloid-beta1-42-specific T-cells by multicolor flow cytometry to simultaneously detect and characterize activation markers and cell signaling proteins (phospho-protein kinase C) in patients with Alzheimer's disease or Lewy body dementia and in healthy controls. Results indicate that only Alzheimer's disease patients display small subsets of peripheral amyloid-beta1-42-specific T-cells, characterized by bright expression of phosphorylated-protein kinase C-delta or -zeta whose significance although discussed, is far from being understood. The identification of such subsets, anyhow, may strongly contribute to distinguish Alzheimer's disease from dementia with Lewy bodies, opening possible new routes to early therapeutic strategies.

Cell therapy: cGMP facilities and manufacturing.

Advanced therapies constitute one of the most complex, organizational, and regulatory areas currently approached by clinical researchers in order to explore new therapeutic applications. Basic scientists and clinicians trying to implement cell therapies into clinical practice, may feel overwhelmed by the apparently endless regulatory requirements that apply. However, regulatory agencies have primary responsibility on patient safety and law enforcement are, and should be, their main considerations. Cell- and tissue-based therapies have the potential to treat many conditions, where present conventional treatments are inadequate. The current approach to cell- and tissue-based therapy development requires using good manufacturing production facilities through master and working cell banks. Facilities need to be purpose-designed and accredited by their national medicinal regulatory body and production scientists need to work in close tandem with quality assurances and ethics committees to absolutely ensure the safety of this cellular products.

Bacterial contamination of platelets and septic transfusions: review of the literature and discussion on recent patents about biofilm treatment.

In spite of the improvements in transfusion safety occurred in the last decades, platelet septic transfusions still represent a cause for concern. Microbial screening of blood products cannot ensure transfusion sterility, so that pathogen inactivation methods and a timely management of infectious events actually play the most relevant role. Biofilm production has been associated to several human illnesses; also, it promotes bacterial adherence to platelet bags and colonization of recipient's catheter after transfusion. Therefore, facing biofilm communities is required to reduce the contamination risk and the occurrence of post-infusion events. In this context, the use of tigecycline as a wide-spectrum antibiofilm drug is discussed, along with recent patents about biofilm treatment by quorum-sensing blockers, bacteriophage-based therapy and antibiofilm oral compounds.

Management of poor peripheral blood stem cell mobilization: incidence, predictive factors, alternative strategies and outcome. A retrospective analysis on 2177 patients from three major Italian institutions.

CD34+ peripheral blood hematopoietic stem cells (HSC) are usually collected following mobilization therapy accomplished by using growth factors (GF) such as rHuG-CSF or rHuGM-CSF with or without chemotherapy. A target dose of yielded CD34+ is usually prescribed by the attending physician depending on different protocols, which may include single or double transplantation. HSC collection usually is performed when at least 20 CD34+ HSC/microL are detected by means of flow cytometry. A cumulative dose of at least 2 x 10(6)/Kg/bw CD34+ HSC has been considered as the threshold to allow a prompt and persistent hematopoietic recovery. Unfortunately, this goal is not achieved by the totality of patients undergoing mobilization regimen. In fact, 5-46% of patients who underwent mobilization therapy fail HSC collection due to very low peripheral blood HSC CD34+ count. Patients' characteristics, including age, sex, stage of the underlying disease (complete or partial remission), diagnosis, previously administered radio/chemotherapy regimens, time-lapse from last chemotherapy before mobilization and mobilization schedule (including dose of GF) were considered as possibly predictive of poor or failed mobilization. We performed a retrospective analysis in 2177 patients from three large Italian academic institutions to assess the incidence of poor mobilizers within our patients' series. Therefore, a patient who fails a first mobilization (and when an HLA-compatible related on unrelated donor is not available) could undergo a second attempt either with different mobilization schedule or by using different GF, such as stem cell factor, growth hormone (GH), or more recently newly introduced drugs such as AMD3100, alone or in combination with rHuG- or -rHuGM-CSF. Thus, we investigated the fate of those who failed a first mobilization and subsequently underwent a second attempt or alternative therapeutic approaches.

Comparison between the BACTEC 9240 and the Pall eBDS system for detection of bacterial platelet concentrate contamination.

BACKGROUND: Only two commercially available automated systems have been cleared by the FDA for screening of bacterial contamination in platelet (PLT) products. These are the Pall eBDS (Pall Corp.), based on measurement of oxygen consumption by contaminant organisms, and the BacT/ALERT (bioMérieux), revealing increasing carbon dioxide concentration due to bacterial growth. STUDY DESIGN AND METHODS: The authors compared the performance of the Pall eBDS with the BACTEC 9240 (bioMérieux) in detecting PLT contamination. Serial dilutions of 10 bacterial species frequently associated with PLT contamination were prepared in an apheresis PLT unit per organism. Units were from single donors. After 30 minutes from seeding PLT units, a volume of suspension achieving a final bacterial concentration of 1 to 10 colony-forming units/mL for each unit was inoculated in two Pall bags and a BACTEC bottle, and the same was done after 24 hours from seeding. Measurements were performed at 24 and 30 hours. RESULTS: Significant differences between the two instruments were only found when screening PLT units after 24 hours from seeding. The Pall system showed a higher sensitivity than BACTEC 9240, because it revealed 97 and 98% of positive samples at 24 and 30 hours of incubation, respectively, whereas the second detected 86 and 90% of contaminated products. Significance was lost after 35-hour incubation with the BACTEC 9240. CONCLUSIONS: By comparing the two instruments, their performances were found to be comparable; the Pall system appeared as a more suitable method when using 24 to 30 hours as times for readings, but the significant difference was lost after 35-hour incubation.

Immunotherapy with cytokine induced killer cells in solid and hematopoietic tumours: a pilot clinical trial.

BACKGROUND AND OBJECTIVES: CIK cells are a novel population of efficient immune effector cells with high antitumour activity mainly due to the high proliferation of CD3(+)CD56(+) cells, so may play a role in the development of new forms of adoptive cellular immunotherapy. We started a pilot clinical trial with autologous CIK cells in patients with refractory lymphoma and metastatic solid tumours. This study was aimed at determining the feasibility of generating a sufficient number of CIK cells in heavily pretreated patients and at assessing treatment toxicity. DESIGN AND METHODS: CIK cells were generated from peripheral blood mononuclear cells (MNC) and incubated in the presence of IFN-gamma followed by OKT3 and IL-2. Treatment schedule consisted of three cycles of CIK cells infusions at an interval of 3 weeks. RESULTS: At present 12 patients were enrolled: 6 advanced lymphomas, 5 metastatic kidney carcinoma and 1 hepatocellular carcinoma (HCC). The median number of transferred cells per patient was 28 x 10(9) (range, 6-61). Protocol adherence was excellent and the toxicity profile was favourable. After CIK cells infusion, the absolute median count of lymphocytes, CD3(+), CD8(+) and CD3(+)CD56(+) cells significantly increased in patient's peripheral blood. Clinical outcome appeared promising: three patients had complete response (CR) and two patients had stabilization of disease with a median follow-up of 33 months (range, 9-44). INTERPRETATIONS AND CONCLUSIONS: These preliminary data showed that adoptive immunotherapy with CIK cells is a safe therapy with some suggestion of efficacy that significantly enhances immune functions increasing absolute numbers of effector cells without side effects. If confirmed in larger scale studies, these promising results may have a favourable impact on conventional treatment strategy of malignancies.

Long-term results of survival in patients with thalassemia major treated with bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) is the only available curative approach for thalassemia major, although long-term morbidity and mortality are not established. The aim of this study was to assess the long-term clinical and hematological results in children and adults with thalassemia major treated with BMT. We analyzed the outcome of 115 patients (median age 9 years, range 11 months to 28 years) with thalassemia major undergoing BMT from a related donor between 1983 and 2006. All patients received the same protocol, consisting of busulfan and cyclophoshamide as conditioning therapy and cyclosporin (CSA) alone or CSA and methotrexate for graft-versus-host disease (GvHD) prophylaxis. The cumulative probability of graft rejection was 6.7%. The transplant-related mortality at 1 year was 8.7%. The 20-year Kaplan-Meier estimate of overall survival and disease-free survival was 89.2% and 85.7%, respectively. Ninety-nine patients out of 103 survivors were in excellent clinical and hematological conditions at last visit following a median follow-up of 15 years (range, 1-24 years) with the exception of two patients who had invalidating chronic GvHD. This study conducted with a large cohort of patients and covering a long period of observation time, showed BMT to be curative for the majority of patients with thalassemia major. The impact of long-term transplant-related sequelae was very limited.

Evaluation of an automated closed fluid management device for processing expanded cytokine-induced killer cells to use in immunotherapy programs for cancer.

BACKGROUND: Cytokine-induced killer (CIK) cells are a heterogeneous population of immune cells derived from peripheral blood lymphocytes with a high proliferative potential ex vivo. This study shows a rapid and reproducible protocol for adoptive immunotherapy with CIK cells in patients with hematologic malignancies. For this purpose a new automatic cell processing device (CytoMate, Baxter Oncology) was tested to improve extensive manipulations of these cells. STUDY DESIGN AND METHODS: Twenty CIK expansions obtained from healthy donors and patients with hematologic malignancies were washed and refilled with fresh medium during culture with the CytoMate. Recovery, viability, and cytotoxic activity were evaluated. Six cryopreserved CIK procedures were thawed and processed for washing out dimethyl sulfoxide automatically. Recovery of cells, viability, and early apoptosis were measured immediately after washing, and cytotoxic activity against target cell lines K562 and Daudi was tested after short culture. RESULTS: Prewash volume of CIK cultures was 3600 mL (range, 1970-6000 mL). After automatic wash, the total CIK cell recovery was 85.3 percent (range, 78.5%-97.5%), and living cells were greater than 95 percent. After thawing, the median recoveries of total nucleated cells and natural killer T (NKT) cells were, respectively, 80.7 percent (range, 65%-95.5%) and 90.5 percent (range, 70.5%-98.5%). Thawed cells preserved their cytotoxic activity after cryopreservation (approx. 50% lysis at effector:target ratio of 40:1). CONCLUSION: The automatic wash with the CytoMate showed a good recovery of viable CIK cells during expansion and allowed an efficient manipulation of thawed cells. The use of this simple and efficient washing technique is suitable for clinical-grade processing in cellular therapy protocols.

Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching.

PKCalpha was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

Long-term remission in BCR/ABL-positive AML-M6 patient treated with Imatinib Mesylate.

BCR/ABL-positive acute myeloid leukemia (AML) is a rare disease, characterized by a poor prognosis, with resistance to induction chemotherapy and frequent relapses in responsive patients. Here we report a case of BCR/ABL-positive AML-M6 who, after relapse, was treated with Imatinib Mesylate (600 mg/die) and within 4 months achieved a cytogenetic and molecular complete response. After more than 4 years of continuous Imatinib therapy, nested RT-PCR for BCR/ABL is persistently negative. The case reported shows that the response obtained with Imatinib Mesylate in BCR/ABL-positive AML may be long lasting, offering a chance of successful treatment for this poor prognosis group of patients.

TRAIL regulates normal erythroid maturation through an ERK-dependent pathway.

In order to investigate the biologic activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human erythropoiesis, glycophorin A (GPA)+ erythroid cells were generated in serum-free liquid phase from human cord blood (CB) CD34+ progenitor cells. The surface expression of TRAIL-R1 was weakly detectable in the early-intermediate phase of erythroid differentiation (days 4-6; dim-intermediate GPA expression), whereas a clear-cut expression of TRAIL-R2 was observed through the entire course of erythroid differentiation (up to days 12-14; bright GPA expression). On the other hand, surface TRAIL-R3 and -R4 were not detected at any culture time. Besides inducing a rapid but small increase of apoptotic cell death, which was abrogated by the pan-caspase inhibitor z-VAD-fmk, the addition of recombinant TRAIL at day 6 of culture inhibited the generation of morphologically mature erythroblasts. Among the intracellular pathways investigated, TRAIL significantly stimulated the extracellular signal-regulated kinase 1/2 (ERK1/2) but not the p38/mitogen-activated protein kinase (MAPK) or the c-Jun NH2-terminal kinase (JNK) pathway. Consistently with a key role of ERK1/2 in mediating the negative effects of TRAIL on erythroid maturation, PD98059, a pharmacologic inhibitor of the ERK pathway, but not z-VAD-fmk or SB203580, a pharmacologic inhibitor of p38/MAPK, reverted the antidifferentiative effect of TRAIL on CB-derived erythroblasts.

Matrix metalloproteinase-9 (gelatinase B) is elevated during mobilization of peripheral blood progenitor cells by G-CSF.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9 or gelatinase B) has recently been implicated in the IL-8-induced mobilization of HPCs in rhesus monkeys and mice. It is not known whether administration of G-CSF causes expression of MMP-9 during HPC mobilization. STUDY DESIGN AND METHODS: Blood samples from 15 allogeneic progenitor cell donors were collected before and during G-CSF-induced HPC mobilization. The expression of the gelatinases MMP-2 and MMP-9 in the plasma of the donors was analyzed by ELISA and zymographic analysis. Gelatinolytic activity was measured with a fluorometric assay that was specific for gelatinases. Expression of IL-6, IL-8, and soluble vascular cell adhesion molecule (VCAM) was measured by ELISA. RESULTS: Highly elevated latent gelatinolytic activity was found on Days 4 and 5 of G-CSF treatment in comparison to pretreatment activity. ELISA and zymographic analyses revealed pro-MMP-9 as the major source of the latent gelatinolytic plasma activity during mobilization. Pro-MMP-2 was not elevated compared with pretreatment levels. As IL-8 has been implicated in the expression of MMP-9, IL-8 concentrations were measured in plasma samples from donors and patients immediately before the start of HPC apheresis, but no significantly elevated IL-8 concentrations were noted. In contrast, pro-MMP-9 and latent gelatinolytic activity was highly correlated with IL-6, which was strongly elevated during mobilization therapy. Finally, soluble VCAM was equally significantly elevated on the days of apheresis. CONCLUSIONS: G-CSF mobilization treatment induces MMP-9, IL-6, and soluble VCAM. Expression of MMP-9 might be involved in the mobilization of human HPCs and might be a final common pathway of different mobilization therapies. Our data do not support a role of IL-8 in G-CSF-induced mobilization. In contrast, IL-6 might be involved in the G-CSF-induced expression of MMP-9.