Prognostic value of multicenter flow cytometry harmonized assessment of minimal residual disease in acute myeloblastic leukemia.

The assessment of minimal residual disease (MRD) in acute myeloblastic leukemia is of growing interest as a prognostic marker of patients' outcome. Multiparameter flow cytometry (MFC), tracking leukemia-associated immunophenotypic patterns, has been shown in several studies to be a useful tool to investigate MRD. Here, we report a multicenter prospective study which allowed to define a harmonized analysis strategy, as well as the efficacy of MFC MRD to predict outcome. This study included 276 patients, in 10 different MFC centers, of whom 268 had at least 1 MRD check point. The combination of a CD45, CD34, and CD33 backbone, with the addition of CD117, CD13, CD7, and CD15 in 2 five-color tubes allowed to define each patient's multiparameter immunophenotypic characteristics at diagnosis, according to a Boolean combination of gates. The same individual diagnosis gating strategy was then applied at each MRD time point for each patient. MRD levels were stratified according to log by log thresholds, from 5 × 10-2 (the classical morphological threshold to define remission) down to <5 × 10-5 . MRD was found to be constantly negative (<5 × 10-5 ) for 148 patients. Survival analyses significantly associated MRD negativity with a good prognosis and any positive value with poorer outcome. All P values were <0.0001 both for disease-free and overall survival at the earliest time point (post-induction, MRD1) as well as when considering all time points together. Finally, MRD levels were independent of cytogenetics and allowed in fact to further stratify all cytogenetics risk groups. In summary, this multicenter study demonstrates that a simple combination of immunophenotypic markers successfully allows for the detection of MRD in acute myeloblastic leukemia patients, with a strong correlation to outcome.

Erythrocyte density in sickle cell syndromes is associated with specific clinical manifestations and hemolysis.

Dense, dehydrated red blood cells (DRBCs) are a characteristic feature of sickle-cell disease (SCD). DRBCs play a role in the pathophysiology of SCD acute and chronic organ damage because of heightened tendency to undergo polymerization and sickling because of their higher hemoglobin S concentration. Relations between red cell density (assessed with phthalate density-distribution profile method) and several hematologic, biochemical, genetic parameters, and clinical manifestations were studied in a large cohort of homozygous patients. The percentage of DRBCs was significantly higher in patients who experienced skin ulcers, priapism, or renal dysfunction. Presence of α-thalassemia deletions was associated with fewer DRBCs. A multivariable analysis model showed DRBCs to be positively associated with hemolytic parameters such as lactate dehydrogenase and bilirubin and negatively with fetal hemoglobin. The percentage of DRBCs decreased by 34% at 6 months of hydroxycarbamide (xydroxyurea) therapy. Thus, DRBCs are associated with specific clinical manifestations and biologic markers and may be a useful addition to the biologic and clinical evaluation of patients with SCD, because they can easily be measured in a hematocrit tube.

Clinical follow-up of hydroxyurea-treated adults with sickle cell disease.

Hydroxyurea-derived clinical and biological benefits and safety were retrospectively studied for 123 adult patients from 2 sickle cell disease referral centers during a total follow-up of 654 patient-years and total hydroxyurea exposure of 549 patient-years. Fifty-six adverse events occurred (incidence: 12%/patient-year), with leg ulcers being the most frequent. Adverse events could arise at any time and were usually reversible. No malignancy was observed. Clinical and biological benefits of our cohort were similar to those previously reported. Based on this relatively long retrospective study, the risk/benefit ratio for moderate hydroxyurea doses was satisfactory.

Deficiency in calcineurin activity in liver transplantation candidates with alcoholic cirrhosis or hepatocellular carcinoma.

BACKGROUND: Tacrolimus and cyclosporin inhibits the activity of calcineurin, a serine/threonine phosphatase that is involved in many physiological and pathological pathways. However, the baseline calcineurin phosphatase activity (CPA) measured before the transplant is unknown. In this study, we determine baseline CPA in liver transplant (LT) candidates and explore some factors that might modify it. PATIENTS AND METHODS: Thirty-two consecutive LT candidates (25 men, seven women, average age 53.4 years) were included. Seven millilitres of whole blood was collected from each patient. CPA was determined in lymphocytes quantifying a dephosphorylated peptide phosphorylated previously (D-L-D-V-P-I-P-G-R-F-D-R-R-V-S-V-A-A-E) by high-performance liquid chromatography. The relationship between CPA and the quantitative variables was tested according to Pearson's correlation. A two-way analysis of variance was performed to test the independent role of categorical parameters in CPA. RESULTS: The median CPA was significantly lower in LT candidates than in healthy volunteers [179.2 (146.9-226.3) vs 247.8 (220.9-292.5) pmol/min/10(6) peripheral blood mononuclear cell (PBMC), respectively, P=0.0002]. CPA was also significantly lower in alcoholic cirrhosis (152.2 vs 211.1 pmol/min/10(6) PBMC, P=0.04) and in the presence of hepatocellular carcinoma (HCC) (152.0 vs 213.5 pmol/min/10(6) PBMC, P=0.0074) compared with other liver diseases. A two-way analysis of variance showed that these parameters were independently associated with lower CPA (P=0.05 for alcohol and P=0.0056 for HCC respectively). CONCLUSION: This pilot study showed a lower CPA in patients with AC and HCC. This phenomenon may contribute towards lowering the risk of acute rejection in these patients after LT and, on the other hand, may increase the risk of de novo cancers.

h-Goliath, paralog of GRAIL, is a new E3 ligase protein, expressed in human leukocytes.

In Drosophila, the RING finger protein d-Goliath was originally identified as a transcription factor involved in the embryo mesoderm formation [Bouchard, M.L., Cote, S., 1993. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein. Gene 125, 205-209]. In mouse, the m-Goliath mRNA level was shown to be increased in growth factor withdrawal-induced apoptosis of myeloid cells [Baker, S.J., Reddy, E.P., 2000. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1. Gene 248, 33-40]. Due to its putative function of transcription factor in apoptosis, we cloned the human cDNA for h-Goliath and characterized the expression of the protein in blood and bone marrow cells. The human protein of 419 aa (44 kDa) contains a protease-associated domain, a transmembrane domain and a RING-H2 motif. This structure classifies h-Goliath as a new member of a human family of ubiquitin ligases with GRAIL (gene related to anergy in lymphocytes) as founder. This E3 ligase controls the development of T cell clonal anergy by ubiquitination [Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., Soares, L., 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535-547]. In vitro ubiquitination studies support the E3 ubiquitin ligase activity of h-Goliath. In human, the protein is expressed under 3 isoforms, a major one at 28 kDa and two others at 46 and 55 kDa. These proteins come from a common precursor (44 kDa) as we observed using in vitro transcription-translation. Using immunohistochemistry on blood or bone marrow smears, of healthy or leukemia samples, we found that the protein expression was restricted to the cytoplasm of progenitors and fully differentiated leukocyte populations. We did not observe any modification of h-Goliath expression or localization in leukemia. In these cells, this new E3 ubiquitin ligase protein does not seem associated with a differentiation state of the cell or with apoptosis.

Distribution of calcineurin activity in blood cell fractions and impact of tacrolimus inhibition.

The aim of this study was to investigate calcineurin (PP2B) activity in different blood cell fractions and its inhibition by tacrolimus. Basal PP2B activity was measured in each blood cellular fraction collected from healthy volunteers. The inhibition profile of PP2B activity was explored in isolated peripheral blood mononuclear cells (PBMC) and platelets exposed directly to tacrolimus and in PBMC and platelets isolated from whole blood previously exposed to tacrolimus. Contrasting with red blood cells (30%) and platelets (25%), PBMC represented only 8.7% of PP2B activity of unfractionated whole blood. After tacrolimus exposure of isolated PBMC and platelets, the concentration of tacrolimus required to inhibit 50% of PP2B activity (EC(50)) in PBMC was significantly lower than in platelets (0.26 ng/mL vs. 0.83 ng/mL, P < 0.001). EC(50) values were similar in PBMC and platelets isolated from whole blood previously exposed to tacrolimus (7.69 ng/mL vs. 7.42 ng/mL, respectively). These results suggest PBMC is a very suitable matrix for PP2B measurement in monitoring transplant recipients but clinical studies are necessary to solve clearly this issue.

Treatment of cancer cells with methioninase produces DNA hypomethylation and increases DNA synthesis.

Methionine depletion in the human cell line CCRF-CEM through the action of recombinant methioninase (rMETase), a methionine-cleaving enzyme, was previously demonstrated to produce a strong cytotoxic synergistic effect with fluorouracil (FUra) throughout a broad range of concentrations of FUra and rMETase, including subcytotoxic levels of rMETase. Potentiation was associated with a decrease in free thymidylate synthase from preexisting levels. To further investigate the action of rMETase on CCRF-CEM cells, in the present study we explored the effects of rMETase as a single agent on DNA methylation levels and DNA synthesis, which may be changed as a result of deprivation of methionine. Cells treated with rMETase under subcytotoxic conditions contained significantly lower levels of genomic methylated DNA than did control cells, as demonstrated by incorporation of the methyl radical of [methyl-(3)H]S-adenosylmethionine in DNA and by use of methylation-sensitive arbitrarily primed PCR. DNA hypomethylation produced by rMETase was of similar magnitude as that produced with the DNA methyltransferase inhibitor 5-azacytidine. Cells exposed to rMETase synthesized significantly more DNA than did untreated cells. Incorporation of [6-3H]thymidine and [6-3H]2'-deoxyuridine in these cells was augmented over that in control by mean factors of 1.78 and 2.36, respectively. Increased 3H nucleoside incorporation resulted in greater numbers of nuclear grains as demonstrated by autoradiography. The increase in DNA synthesis induced by rMETase is likely to result from enhancement of DNA repair because it was not accompanied by differences in cell cycle phase distribution or in total DNA content as determined by flow cytometry. We hypothesize that potentiation of FUra cytotoxicity by rMETase may result from increased inhibition of thymidylate synthase, together with DNA hypomethylation and enhanced DNA repair that could be involved in cell responses to drug-induced damage.

Effects of the proteasome inhibitor ritonavir on glioma growth in vitro and in vivo.

Glioblastoma is a therapeutic challenge as a highly infiltrative, proliferative, and resistant tumor. Among novel therapeutic approaches, proteasome inhibition is very promising in controlling cell cycle and inducing apoptosis. This study investigated the effect of ritonavir, a protease inhibitor of the HIV and a proteasome modulator, on glioma cells. The hypothesis was that proteasome modulation, mainly by only inhibiting proteasome chymotrypsin-like activity, could be sufficient to control tumor progression. The experiments were done on a human glioblastoma-derived GL15 cell line and a rat nitrosourea-induced gliosarcoma 9L cell line. Culturing conditions included monolayer cultures, transplantations into brain slices, and transplantations into rat striata. The study demonstrates that ritonavir, by inhibiting the chymotrypsin-like activity of the proteasome, has cytostatic and cytotoxic effects on glioma cells, and can induce resistances in vitro. Ritonavir was unable to control tumor growth in vivo, likely because the therapeutic dose was not reached in the tumor in vivo. Nevertheless, ritonavir might also be beneficial, by decreasing tumor infiltration, in the reduction of the deleterious peritumor edema in glioblastoma.

Molecular analysis of an unusual rearrangement between chromosomes 4 and 11 in adult pre-B-cell acute lymphoblastic leukemia.

The reciprocal t(4;11)(q21;q23) is often described in acute lymphoblastic leukemia (ALL). In some cases, variant or complex t(4;11) has been detected in ALL by cytogenetic analysis, but to our knowledge no molecular study has been reported in these variant translocations. We describe a 27-year-old woman suffering from pre-B-cell ALL with an unusual rearrangement between chromosomes 4 and 11. Because this complex rearrangement involved the 11q23 chromosome band known to be associated with poor prognosis, we performed fluorescence in situ hybridization, Southern blot, and reverse transcription polymerase chain reaction analyses. This confirmed myeloid lymphoid leukemia gene rearrangement and showed the presence of MLL/AF4 fusion transcript. These results showed the importance of molecular analysis that allowed minimal residual disease monitoring in this patient.

Hematological reference values for healthy adults in togo.

The hematological reference values are very important for diagnostic orientation and treatment decision. The aim of this study was to establish hematological reference values for healthy adults in Togo. A total of 2571 voluntary blood donors participated to this study. Only 1349 subjects negative for HIV, HBV, HCV, malaria, and without hemoglobin abnormalities in electrophoresis and hypochromia on blood smear, were definitively retained for the study. Median hemoglobin level was higher in males than females (15.1 g/dL versus 13.0 g/dL, p = 0.000). Median total WBC (4.2×10(9)/L) and absolute neutrophil counts (1.6×10(9)/L) were similar by gender. The median lymphocyte counts in males and females were, respectively, 2.1×10(9)/L and 2.2×10(9)/L (p = 0.11). The median platelet count was lower in males than females (236×10(9)/L versus 247×10(9)/L, p = 0.004). Our median values for RBC parameters differ from those of African countries probably because of our inclusion criteria which eliminate most cases with iron deficiency and/or thalassemia.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL.

BACKGROUND: The development of multiparameter flow cytometry (FCM) and increasingly sophisticated analysis software has considerably improved the exploration of hematological disorders. These tools have been widely applied in leukaemias, lymphomas, and myelodysplasias, yet with very heterogeneous approaches. Consequently, there is no extensive reference document reporting on the characteristics of normal human bone marrow (BM) in multiparameter FCM. Here, we report a reference analysis procedure using relevant antibody combinations in normal human BM. METHODS: A first panel of 23 antibodies, constructed after literature review, was tested in four-color combinations (including CD45 in each) on 30 samples of BM. After evaluation of the data, a second set of 22 antibodies was further applied to another 35 BM samples. All list-modes from the 65 bone marrow samples were reviewed collectively. A systematised protocol for data analysis was established including biparametric representations and color codes for the three major lineages and undifferentiated cells. RESULTS: This strategy has allowed to obtain a reference atlas of relevant patterns of differentiation antigens expression in normal human BM that is available within the European LeukemiaNet. This manuscript describes how this atlas was constructed. CONCLUSIONS: Both the strategy and atlas could prove very useful as a reference of normality, for the determination of leukemia-associated immunophenotypic patterns, analysis of myelodysplasia and, ultimately, investigation of minimal residual disease in the BM.

Pharmacokinetic-pharmacodynamic assessment of tacrolimus in liver-transplant recipients during the early post-transplantation period.

During the early post-transplantation period, the limitations of monitoring current tacrolimus dose with classic pharmacokinetics (PK) have been demonstrated in liver-transplant recipients. Evaluation of the pharmacodynamics (PD) using calcineurin activity (CNA) has been proposed to optimize tacrolimus dosing. The aim of the present study was to determine the time of maximal inhibition of CNA, to explore the relation between exposure to tacrolimus and CNA, and to analyze its variability. Blood was drawn from 14 patients 0, 2, 3, 4, 6, and 9 hours after tacrolimus intake on post-transplantation days 8, 21, and 90 to measure blood tacrolimus concentrations using the EMIT 2000 assay and CNA in peripheral blood mononuclear cells. Tacrolimus and CNA data were obtained for 33 blood-sample collection sessions and analyzed using a population approach. Three models were built to describe tacrolimus PK, CNA kinetics, and the relationships between the area under the CNA-time curve (AUC12effCNA) and AUC12Tacrolimus or CminTacrolimus. Coagulation factor V and whole/split liver graft were identified as covariates influencing tacrolimus clearance. Indeed, apparent tacrolimus clearance rose by 14% when factor V increased by 10% and was threefold higher in patients with whole-liver grafts. The median maximal inhibition of CNA was reached 4 hours after tacrolimus intake on days 8, 21, and 90 and represented an 18% drop in CNA compared with activity at drug intake. The variability of the PK-PD relationship was minimal when using AUC12Tacrolimus. The large variability of the PD parameters (coefficient of variation was 89%) that linked AUC12effCNA to AUC12Tacrolimus indicates that monitoring tacrolimus concentrations may not be adequate to control CNA. Measuring CNA in peripheral blood mononuclear cells 4 hours after tacrolimus intake during the first 3 months after liver transplantation could be a means to improve tacrolimus monitoring and thereby avoid acute graft-rejection episodes.

A mouse model provides evidence that genetic background modulates anemia and liver injury in erythropoietic protoporphyria.

Erythropoietic protoporphyria is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin (PP) overproduction by erythrocytes. In humans, it is responsible for painful skin photosensitivity and, occasionally, liver failure due to accumulation of PP in the liver. The ferrochelatase deficiency mouse mutation is the best animal model available for human erythropoietic protoporphyria. The original description, based on mice with a BALB/cByJCrl genetic background, reported a disease resembling the severe form of the human disease, with anemia, jaundice, and liver failure. Using congenic strains, we investigated the effect of genetic background on the severity of the phenotype. Compared with BALB/cByJCrl, C57BL/6JCrl mice developed moderate but increasing anemia and intense liver accumulation of PP with severe hepatocyte damage and loss. Bile excretory function was not affected, and bilirubin remained low. Despite the highest PP concentration in erythrocytes, anemia was mild and there were few PP deposits in the liver in SJL/JOrlCrl homozygotes. Discriminant analysis using six hematologic and biochemical parameters showed that homozygotes of the three genetic backgrounds could be clustered in three well-separated groups. These three congenic strains provide strong evidence for independent genetic control of bone marrow contribution of PP overproduction to development of liver disease and biliary PP excretion. They provide a tool to investigate the physiological mechanisms involved in these phenotypic differences and to identify modifying genes.

Ectopic expression of TAL-1 protein in Ly-6E.1-htal-1 transgenic mice induces defects in B- and T-lymphoid differentiation.

The tal-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor required for primitive and definitive hematopoiesis. Additionally, ectopic activation of the tal-1 gene during T lymphopoiesis occurs in numerous cases of human T-cell acute lymphoblastic leukemia. With the use of transgenic mice, we show that, in adult hematopoiesis, constitutive expression of TAL-1 protein causes disorders in the hematopoietic lineages that normally switch off tal-1 gene expression during their differentiation process. Myelopoiesis was characterized by a moderate increase of myeloid precursors and by Sca-1 antigen persistence. Although no lymphoid leukemia was observed, T lymphopoiesis and B lymphopoiesis were severely impaired. Transgenic mice showed reduced thymic cellularity together with a decrease in double-positive cells and a concurrent increase in the single-positive population. B cells exhibited a differentiation defect characterized by a reduction of the B-cell compartment most likely because of a differentiation block upstream of the intermediate pro-B progenitor. B cells escaping this defect developed normally, but transgenic splenocytes presented a defect in immunoglobulin class switch recombination. Altogether, these results enlighten the fine-tuning of TAL-1 expression during adult hematopoiesis and indicate why TAL-1 expression has to be switched off in the lymphoid lineages.

Exacerbation with granulocyte colony-stimulating factor of prior acute lung injury during neutropenia recovery in rats.

OBJECTIVE: Neutropenia recovery may be associated with an increased risk of respiratory function deterioration. A history of pneumonia complicating neutropenia has been identified as the leading cause of adult respiratory distress syndrome during neutropenia recovery in patients receiving anticancer chemotherapy, suggesting that neutropenia recovery may worsen prior lung injury. DESIGN: Controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: We studied the effect of recovery from cyclophosphamide-induced neutropenia on endotoxin (lipopolysaccharide)- or hydrochloric acid-induced acute lung injury in rats. We also studied the effects of adding granulocyte colony-stimulating factor. MEASUREMENTS AND MAIN RESULTS: Compared with noncyclophosphamide-treated rats, rats undergoing neutropenia recovery had a higher wet/dry lung weight ratio after hydrochloric acid-induced but not lipopolysaccharide-induced acute lung injury. Granulocyte colony-stimulating factor significantly increased both alveolar cell recruitment (bronchoalveolar lavage fluid counts) and pulmonary edema (wet/dry lung ratio) in both acute lung injury models during neutropenia recovery. Furthermore, in an experiment in hydrochloric acid-instilled rats, exacerbation by granulocyte colony-stimulating factor of hydrochloric acid-induced acute lung injury was inhibited by lidocaine, which prevents adhesion of neutrophils to endothelial cells. Tumor necrosis factor-alpha and interleukin-1 beta concentrations in supernatants of lipopolysaccharide-stimulated alveolar macrophages from rats undergoing neutropenia recovery with granulocyte colony-stimulating factor treatment were significantly increased compared with rats undergoing neutropenia recovery without granulocyte colony-stimulating factor. CONCLUSION: Neutropenia recovery can worsen acute lung injury, and this effect is exacerbated by granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor enhances host defenses against bacterial pneumonia following peritonitis in nonneutropenic rats.

OBJECTIVE: Polymorphonuclear cell functions frequently are impaired in critically ill patients, and restoration of normal functions could help to prevent nosocomial infections. The aim of this study was to evaluate the effects of pretreatment with granulocyte colony-stimulating factor (G-CSF) on bacterial pneumonia induced 48 hrs after peritonitis (cecal ligation and puncture [CLP]) in rats. DESIGN: Controlled animal study. SETTING: Research laboratory of an academic institution. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: First, the CLP model was characterized. Second, alveolar endotoxin instillation allowed us to evaluate the ability of neutrophils to migrate to airspaces after CLP was assessed. In the last set of experiments, CLP was followed by G-CSF treatment as a preventive therapy for subsequent bacterial superinfection induced by alveolar instillation. MEASUREMENTS AND MAIN RESULTS: CLP induced a brief increase in proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta) at the 6th hr followed by a longer-lived anti-inflammatory response (interleukin-10 increase from days 1 to 3) in plasma, compared with healthy rats. Impaired neutrophil migration to alveolar spaces denoting immunoparalysis was evidenced after endotracheal endotoxin instillation following CLP, compared with non-CLP rats challenged with endotoxin. No such impairment was found when G-CSF (100 microg/kg: glycosylated recombinant human G-CSF, Lenograstim) was given before endotoxin. G-CSF (100 microg/kg 24 and 48 hrs after CLP) given before endotracheal instillation increased bacterial clearance, as shown by counts in both bronchoalveolar lavage (8.9 x 10 +/- 2.8 x 10 colony-forming units/mL vs. 3.3 x 10 +/- 1.5 x 10 colony-forming units/mL with saline) and lung tissue (4.2 x 10 +/- 1.0 x 10 colony-forming units/g vs. 1.5 x 10 +/- 0.6 x 10 colony-forming units/g with saline). Furthermore, G-CSF pretreatment kept clearance in CLP rats similar to that in non-CLP rats challenged with. CONCLUSION: These results suggest that G-CSF (Lenograstim) may enhance host defenses in rats with peritonitis and immunoparalysis.

Exacerbation by granulocyte colony-stimulating factor of prior acute lung injury: implication of neutrophils.

OBJECTIVE: Granulocyte colony-stimulating factor is widely prescribed to hasten recovery from cancer chemotherapy-induced neutropenia and has been reported to induce pulmonary toxicity. However, circumstances and mechanisms of this toxicity remain poorly known. DESIGN: To reproduce a routine situation in cancer patients receiving chemotherapy, we investigated the mechanisms underlying granulocyte colony-stimulating factor-induced exacerbation of alpha-naphthylthiourea-related pulmonary edema. SETTING: Laboratory research unit. SUBJECTS: Male specific-pathogen-free Sprague-Dawley rats. INTERVENTIONS: The effects of granulocyte colony-stimulating factor given alone or after alpha-naphthylthiourea used to induce acute lung injury were investigated. MEASUREMENTS AND MAIN RESULTS: Lung injury was assessed based on neutrophil sequestration (myeloperoxidase activity in lung tissue) and influx into alveolar spaces (bronchoalveolar lavage fluid cell quantification) and on edema formation (wet/dry lung weight ratio) and alveolar protein concentration into bronchoalveolar lavage fluid. Tumor necrosis factor-alpha and interleukin-1beta were measured in serum, lung homogenates, and isolated alveolar macrophage supernatants. In control rats, granulocyte colony-stimulating factor (25 microg/kg) significantly elevated circulating neutrophil counts without producing alveolar recruitment or pulmonary edema. alpha-Naphthylthiourea significantly increased the wet/dry lung weight ratio (4.68 +/- 0.04 vs. 4.38 +/- 0.07 in controls, p=.04) and induced alveolar protein leakage. Adding granulocyte colony-stimulating factor to alpha-naphthylthiourea exacerbated pulmonary edema, causing neutrophil sequestration in pulmonary vessels, significantly increasing lung myeloperoxidase activity (12.7 +/- 2.0 mOD/min/g vs. 1.1 +/- 0.4 mOD/min/g with alpha-naphthylthiourea alone; p<.0001), and increasing proinflammatory cytokine secretion. alpha-Naphthylthiourea-related pulmonary edema was not exacerbated by granulocyte colony-stimulating factor during cyclophosphamide-induced neutropenia or after lidocaine, which antagonizes neutrophil adhesion to endothelial cells. Tumor necrosis factor-alpha and interleukin-1beta concentrations in alveolar macrophage supernatants and lung homogenates were significantly higher with alpha-naphthylthiourea + granulocyte colony-stimulating factor than with either agent alone, and anti-tumor necrosis factor-alpha antibodies abolished granulocyte colony-stimulating factor-related exacerbation of alpha-naphthylthiourea-induced pulmonary edema. In rats with cyclophosphamide-induced neutropenia, tumor necrosis factor-alpha concentrations in alveolar macrophage supernatants and lung homogenates were significantly decreased compared with rats without neutropenia. CONCLUSION: Granulocyte colony-stimulating factor-related pulmonary toxicity may involve migration of neutrophils to vascular spaces, adhesion of neutrophils to previously injured endothelial cells, and potentiation of proinflammatory cytokine expression.

A closed and single-use system for monocyte enrichment: potential for dendritic cell generation for clinical applications.

BACKGROUND: This study evaluated the ability of a modified cell separator (Cobe Spectra Apheresis) system to isolate monocytes (MOs) by elutriation. The evaluation was performed in two independent international laboratories. The capacity of collected MOs to differentiate into dendritic cells (DCs) was also assessed. STUDY DESIGN AND METHODS: MNCs from platelet apheresis residues were elutriated on a modified cell separator (Cobe Spectra Apheresis system) using a custom disposable set. Cells were separated according to their size and density. Recovery and purity of the collected cell product were evaluated by impedance counting and flow cytometry. DCs were differentiated in culture from the elutriated MOs and characterized by their surface markers and stimulatory capacity in a mixed WBC reaction assay. RESULTS: Six apheresis mononuclear cell products were used by each laboratory. The separation was achieved in less than 1 hour. Collected MOs had the potential to differentiate into DCs. CONCLUSION: The modified cell separator is an easy and fast device to obtain highly enriched MOs with a DC differentiation potential. The system is closed and employs a single-use disposable set and is more amenable to good tissue practice. This method could become a valuable tool for DC-based active immunotherapy.