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Background: Epithelial cytokines, including IL-33 and Thymic stromal lymphopoietin (TSLP), have attracted interest because of their roles in chronic allergic inflammation-related conditions such as asthma. Mast cells are one of the major targets of IL-33, to which they respond by secreting cytokines. Most studies performed thus far have investigated the acute effects of IL-33 on mast cells. In the current study, we investigated how acute vs. prolonged exposure of mast cells to IL-33 and TSLP affects mediator synthesis and IgE-mediated activation. Methods: Human lung mast cells (HLMCs), cord blood-derived mast cells (CBMCs), and the ROSA mast cell line were used for this study. Receptor expression and the levels of mediators were measured after treatment with IL-33 and/or TSLP. Results: IL-33 induced the release of cytokines. Prolonged exposure to IL-33 increased while TSLP reduced intracellular levels of tryptase. Acute IL-33 treatment strongly potentiated IgE-mediated activation. In contrast, 4 days of exposure to IL-33 decreased IgE-mediated activation, an effect that was accompanied by a reduction in FcεRI expression. Conclusion: We show that IL-33 plays dual roles in mast cells, in which its acute effects include cytokine release and the potentiation of IgE-mediated degranulation, whereas prolonged exposure to IL-33 reduces IgE-mediated activation. We conclude that mast cells act quickly in response to the alarmin IL-33 to initiate an acute inflammatory response, whereas extended exposure to IL-33 during prolonged inflammation reduces IgE-mediated responses. This negative feedback effect suggests the presence of a novel regulatory pathway that modulates IgE-mediated human mast cell responses.

Clonal chromosome abnormalities in Philadelphia-negative cells could concern chronic myeloid leukemia patients treated by tyrosine kinase inhibitors. The European LeukemiaNet distinguishes -7/del(7q) abnormalities as a "warning". However, the impact of clonal chromosome abnormalities, and specifically those of -7/del(7q), in Philadelphia-negative cells on clinical outcomes is unclear and based on case-reports showing morphological dysplasia and increased risk of acute myeloid leukemia, suggesting the coexistence of chronic myeloid leukemia and high-risk myelodysplastic syndrome. The aim of this study was to determine whether the impact of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells on the clinical outcome is different from that of other types of abnormalities, and we argue for an underlying associated high-risk myelodysplastic syndrome. Among 102 chronic myeloid leukemia patients with clonal chromosome abnormalities in Philadelphia-negative cells with more than a median of 6 years of follow up, patients with -7/del(7q) more frequently had signs of dysplasia, a lower cumulative incidence of deep molecular response and often needed further treatment lines, with the consequent impact on event-free and progression-free survival. Morphological features of dysplasia are associated with myelodysplastic syndrome/acute myeloid leukemia mutations and compromise the optimal response to tyrosine kinase inhibitors, irrespectively of the type of clonal chromosome abnormalities in Philadelphia-negative cells. However, mutation patterns determined by next-generation sequencing could not clearly explain the underlying high-risk disease. We hereby confirm the pejorative prognostic value of -7/del(7q) clonal chromosome abnormalities in Philadelphia-negative cells and suggest that myelodysplastic features constitute a warning signal that response to tyrosine kinase inhibitors may be less than optimal., (Copyright© 2019 Ferrata Storti Foundation.)

The French Working Group on Perioperative Haemostasis (GIHP) and the French Study Group on Haemostasis and Thrombosis (GFHT), in collaboration with the French Society for Anaesthesia and Intensive Care (SFAR), drafted up-to-date proposals on the management of antiplatelet therapy for non-elective invasive procedures or bleeding complications. The proposals were discussed and validated by a vote; all proposals could be assigned with a high strength. Management of oral antiplatelet agents in emergency settings requires knowledge of their pharmacokinetic and pharmacodynamic parameters, evaluation of the degree of alteration of haemostatic competence and the associated bleeding risk. Platelet function testing may be considered. When antiplatelet agent-induced bleeding risk may worsen the prognosis, measures should be taken to neutralize antiplatelet therapy, by considering not only the efficacy of available means (which can be limited for prasugrel and even more for ticagrelor), but also the risks that these means expose the patient to. The measures include platelet transfusion at the appropriate dose and haemostatic agents (tranexamic acid; recombinant activated factor VII for ticagrelor). When possible, postponing non-elective invasive procedures at least for a few hours until the elimination of the active compound (which could compromise the effect of transfused platelets) or, if possible, for a few days (reduction of the effect of antiplatelet agents) should be considered., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Severe postpartum hemorrhage (sPPH) is an obstetric emergency that needs prompt and effective therapy to reduce the risk of complications. In this study, women who developed sPPH (study cohort, n = 27) were treated according to a standardized management protocol prescribing sequential administration of uterotonic drugs, crystalloids, tranexamic acid, labile blood products, low-dose fibrinogen, and recombinant activated factor VII (rFVIIa). This group was compared to patients treated with different strategies during 2 preceding periods: an in-house guideline regulating the administration of rFVIIa (historical cohort 1, n = 20) and no specific guideline (historical cohort 2, n = 27). The management protocol was used over 33 months. The study cohort had a lower estimated blood loss ( P = .004) and required less red blood cell concentrates ( P = .007), fresh frozen plasma units ( P = .004), and platelet concentrates ( P = .020) compared to historical cohort 1 and historical cohort 2, respectively. The necessity of emergency postpartum hysterectomy was lower in the study group ( P = .012). In conclusion, in patients with sPPH treated with this standardized management protocol, we observed a decreased requirement of labile blood products and lower need to proceed to emergency postpartum hysterectomy.

Hemoglobinopathies are congenital disorders resultimg from hemoglobin abnormalities. Major forms are often severe, their management is difficult and associated with a great psychosocial impact on patients and their families. They are classified as rare diseases and are still insufficiently known by health professionals. This lack of knowledge is at the origin of diagnostic errors, delay in their management and therefore high morbidity and mortality rate for these patients. In 2008, the World Health Organization (WHO) has published data on hemoglobinopathies epidemiology: more than 330.000 cases of hemoglobinopathy occur each year (83% of cases of sickle cell anemia, 17 % of cases of thalassemia). Hemoglobin disorders are responsible for approximately 3.4% of deaths among people under the age of 5. At the global level, approximately 7% of pregnant women would be carriers of a form of thalassemia and 1% of couples are at risk. However, they are relatively frequent in some regions of the globe where consanguineous marriages are common. We conducted a descriptive cross-sectional study based on two surveys, the first in May 2015 and the second in June of the same year. It was performed in the immunization days to deliver pneumococcal vaccine to the index cases and it was aimed to describe the epidemiological features of families at risk of hemoglobinopathies (index case study), whose index cases were treated in the Department of Pediatrics at the Provincial Hospital El Idrisi, Kenitra, Morocco. After having collected the epidemiological data from patients, laboratory tests were performed including: blood count with red blood cells morphological assessment using the MGG assay and automatic numbering of reticulocytes; hemoglobin electrophoresis at alkaline pH (8.8) and then at acid pH (5.4) on agarose gel and densitometric integration. 275 patients had laboratory profiles compatible with hemoglobinopathy. The majority of these patients were born to consanguineous marriages (83.1%) and came from the north regions of Morocco. This family survey allowed to identify families at risk with a high frequency of sickle cell anemia. Our results confirm the existence of hemoglobinopathies variants among Moroccan population.

Systemic mastocytosis (SM) is a clonal bone marrow disorder, where therapeutical options are limited. Over 90% of the patients carry the D816V point mutation in the KIT receptor that renders this receptor constitutively active. We assessed the sensitivity of primary mast cells (MC) and mast cell lines HMC1.2 (D816V mutated), ROSA (KIT WT) and ROSA (KIT D816V) cells to histone deacetylase inhibitor (HDACi) treatment. We found that of four HDACi, suberoyl anilide hydroxamic acid (SAHA) was the most effective in killing mutated MC. SAHA downregulated KIT, followed by major MC apoptosis. Primary SM patient MC cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas primary MC from healthy subjects were less affected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive SM, with almost 100% cell death among MC from the mast cell leukemia patient. Additionally, ROSA (KIT D816V) was more affected by HDACi than ROSA (KIT WT) cells. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/H3 decreased significantly in the KIT region. This epigenetic silencing was seen only in the KIT region and not in control genes upstream and downstream of KIT, indicating that the downregulation of KIT is exerted by specific epigenetic silencing. In conclusion, KIT D816V mutation sensitized MC to HDACi mediated killing, and SAHA may be of value as specific treatment for SM, although the specific mechanism of action requires further investigation.

Homozygous sickle cell disease is one of the most frequent haemoglobinopathies in Morocco. Sickle cell disease is characterized by a large clinical and biological expression variability which depends on modulating genetic and environmental factors. Clinical manifestation includes regenerative anemia whose severity may vary among individuals. In the absence of treatment, it results in premature death. Sickle cell disease is characterized by a large clinical and biological expression variability which depends on genetic and environmental factors. A severe clinical picture marked by high early transfusion frequency, severe infectious complications and early mortality. A constant inflammatory condition characterized by elevated inflammatory proteins and compromised nutritional status. The objective of this study is to determine the hematological parameters profile in moroccan patients with homozygous sickle cell (SS) disease during stationary phases. We conducted a cross-sectional descriptive study of 87 patients with sickle cell (SS) disease. We performed a biological study based on: Hemogram with morphological assessment of red blood cells stained with MGG and automated reticulocyte counting; Hemoglobin electrophoresis test performed on alkaline agarose gel (pH 8.8) and densitometric integration. The average age is 13.22 years ± 16.36, ranging betrween 0.6 and 36 years, with a sex ratio (M/F) of 1.175. Biological effects of anemia were intense in 88.5% of patients; 67.8% of patients had normocytic anemia compared with 29.9% with microcytosis, and 2.3% with macrocytosis. The degree of anisocytosis was related to the degree of anemia, very evocative in patients with homozygous S/S (95.4%). Reticulocytosis was observed in 81.6% of patients; 52.9% of patients had thrombocytosis. Leukocytosis was observed in 64.4% of patients; 80.5% of patients had neutropenia. The parameters of the hemogram will serve as a basis for comparison during crises and will make it possible to evaluate the effectiveness of patient management. High white blood cell count, platelets and MCHC seem to be determinant of sickle cell anemia severity in Morocco. The haematological profile of moroccan patients with sickle cell disease exhibits data similar to those reported in literature relating to patients with leucocytosis from Central Africa. The results of our study suggest that sickle cell anemia is the most common health problem in Morocco and they are similar to those for major sickle cell syndrome., Competing Interests: Les auteurs ne déclarent aucun conflit d'intérêt.

Systemic mastocytosis are rare neoplasms characterized by accumulation of mast cells in at least one internal organ. The majority of systemic mastocytosis patients carry KIT D816V mutation, which activates constitutively the KIT receptor. Patient with advanced forms of systemic mastocytosis, such as aggressive systemic mastocytosis or mast cell leukemia, are poorly treated to date. Unfortunately, the lack of in vivo models reflecting KIT D816V+ advanced disease hampers pathophysiological studies and preclinical development of new therapies for such patients. Here, we describe a new in vivo model of KIT D816V+ advanced systemic mastocytosis developed by transplantation of the human ROSAKIT D816V-Gluc mast cell line in NOD-SCID IL-2R γ-/- mice, using Gaussia princeps luciferase as a reporter. Intravenous injection of ROSAKIT D816V-Gluc cells led, in 4 weeks, to engraftment in all injected primary recipient mice. Engrafted cells were found at high levels in bone marrow, and at lower levels in spleen, liver and peripheral blood. Disease progression was easily monitored by repeated quantification of Gaussia princeps luciferase activity in peripheral blood. This quantification evidenced a linear relationship between the number of cells injected and the neoplastic mast cell burden in mice. Interestingly, the secondary transplantation of ROSAKIT D816V-Gluc cells increased their engraftment capability. To conclude, this new in vivo model mimics at the best the features of human KIT D816V+ advanced systemic mastocytosis. In addition, it is a unique and convenient tool to study the kinetics of the disease and the potential in vivo activity of new drugs targeting neoplastic mast cells.

Little is known about inherited factors associated with the risk of developing chronic myelogenous leukemia (CML). We used a dedicated DNA chip containing 16 561 single nucleotide polymorphisms (SNPs) covering 1 916 candidate genes to analyze 437 CML patients and 1 144 healthy control individuals. Single SNP association analysis identified 139 SNPs that passed multiple comparisons (1% false discovery rate). The HDAC9, AVEN, SEMA3C, IKBKB, GSTA3, RIPK1 and FGF2 genes were each represented by three SNPs, the PSM family by four SNPs and the SLC15A1 gene by six. Haplotype analysis showed that certain combinations of rare alleles of these genes increased the risk of developing CML by more than two or three-fold. A classification tree model identified five SNPs belonging to the genes PSMB10, TNFRSF10D, PSMB2, PPARD and CYP26B1, which were associated with CML predisposition. A CML-risk-allele score was created using these five SNPs. This score was accurate for discriminating CML status (AUC: 0.61, 95%CI: 0.58-0.64). Interestingly, the score was associated with age at diagnosis and the average number of risk alleles was significantly higher in younger patients. The risk-allele score showed the same distribution in the general population (HapMap CEU samples) as in our control individuals and was associated with differential gene expression patterns of two genes (VAPA and TDRKH). In conclusion, we describe haplotypes and a genetic score that are significantly associated with a predisposition to develop CML. The SNPs identified will also serve to drive fundamental research on the putative role of these genes in CML development.

Core-binding factor acute myeloid leukemia is a favorable acute myeloid leukemia subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, disrupting RUNX1 (previously CBFA/AML1) or CBFB transcription factor functions. The receptor tyrosine kinase KIT is expressed in the vast majority of these acute myeloid leukemias and frequent activating KIT gene mutations have been associated with a higher risk of relapse. This phase II study aimed to evaluate dasatinib as maintenance therapy in patients with core-binding factor acute myeloid leukemia in first hematologic complete remission, but at higher risk of relapse due to molecular disease persistence or recurrence. A total of 26 patients aged 18-60 years old previously included in the CBF-2006 trial were eligible to receive dasatinib 140 mg daily if they had a poor initial molecular response (n=18) or a molecular recurrence (n=8). The tolerance of dasatinib as maintenance therapy was satisfactory. The 2-year disease-free survival in this high-risk population of patients was 25.7%. All but one patient with molecular recurrence presented subsequent hematologic relapse. Patients with slow initial molecular response had a similar disease-free survival when treated with dasatinib (40.2% at 2 years) or without any maintenance (50.0% at 2 years). The disappearance of KIT gene mutations at relapse suggests that clonal devolution may in part explain the absence of efficacy observed with single-agent dasatinib in these patients (n. EudraCT: 2006-006555-12)., (Copyright© Ferrata Storti Foundation.)

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3'untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199., (© 2014 by The American Society of Hematology.)

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients., (© 2014 by The American Society of Hematology.)

Systemic mastocytosis with associated hematologic clonal non-mast cell disease (SM-AHNMD) is a rare and heterogeneous subtype of SM and few studies on this specific entity have been reported. Sixty two patients with Systemic mastocytosis with associated hematologic clonal non-mast cell disease (SM-AHNMD) were presented. Myeloid AHNMD was the most frequent (82%) cases. This subset of patients were older, had more cutaneous lesions, splenomegaly, liver enlargement, ascites; lower bone mineral density and hemoglobin levels and higher tryptase level than lymphoid AHNMD. Defects in KIT, TET2, ASXL1 and CBL were positive in 87%, 27%, 14%, and 11% of cases respectively. The overall survival of patients with SM-AHNMD was 85.2 months. Within the myeloid group, SM-MPN fared better than SM-MDS or SM-AML (p = 0.044,). In univariate analysis, the presence of C-findings, the AHNMD subtypes (SM-MDS/CMML/AML versus SM-MPN/hypereosinophilia) (p = 0.044), Neutropenia (p = 0.015), high monocyte level (p = 0.015) and the presence of ASXL1 mutation had detrimental effects on OS (p = 0.007). In multivariate analysis and penalized Cox model, only the presence of ASXL1 mutation remained an independent prognostic factor that negatively affected OS (p = 0.035). SM-AHNMD is heterogeneous with variable prognosis according to the type of the AHNMD. ASXL1 is mutated in a subset of myeloid AHNMD and adversely impact on OS.

Background: Molecular monitoring of chronic myeloid leukemia patients treated with tyrosine kinase inhibitors is essential for therapeutic stratification. Inter-laboratory reproducibility is, therefore, a crucial issue which requires standardization and strict alignment of BCR-ABL1 values to the international scale. An automated cartridge-based assay (Xpert BCR-ABL Monitor(™), Cepheid) had been proposed as a robust alternative to non-automated assays. This study aimed to compare inter-laboratory reproducibility of automated and non-automated quantification, the possibility of converting automated results to the international scale, and the potential economic impact of automation., Design and Methods: One hundred and eighteen blood samples from chronic myeloid leukemia patients treated with tyrosine kinase inhibitors were prospectively analyzed in two laboratories using both automated and non-automated assays. The economic evaluation involved a micro-costing study and average costs were assessed as a function of sample throughput., Results: Automated assays achieved similar inter-laboratory reproducibility to highly standardized non-automated assays and a short delay (≤6 h) between sampling and blood lysis had a positive impact on inter-laboratory reproducibility. Reporting automated BCR-ABL1 ratios on the international scale was possible using a specific conversion factor which may vary with batches. Cost assessment showed that automated assays could be relevant for annual activity levels below 300 since average costs were lower than those of the non-automated assays., Conclusions: The Xpert BCR-ABL Monitor(™) assay could be appropriately used in a near-patient setting for routine quantification of e13/e14-a2 transcripts, preferably in partnership with a regional reference laboratory. However, its prognostic impact relative to non-automated quantification remains to be tested prospectively within appropriate clinical trials.

Interleukin 10 (IL10) is associated with maternal immunotolerance. IL10 also down-regulates decidual cell tissue factor expression, the main molecule triggering coagulation activation: this antithrombotic effect may protect the umbilicoplacental vasculature from the 10th wk of gestation onward. IL10 down-regulation may thus dispose to early pregnancy loss (PL) due to maternal immunotolerance defect or late pregnancy failure due to placental vascular insufficiency. IL10 gene promoter polymorphisms associated with cytokine down-regulation may help to identify the actual and probable mechanisms of IL10 modulation in pregnancy outcomes. We investigated the following four IL10 promoter polymorphisms associated with IL10 down-regulation: two single-nucleotide polymorphisms rs1800871 and rs1800872 and two polymorphic CA repeat microsatellites IL10 X78437.2:g8134CA(14_29) and IL10 X78437.2:g.5325CA(11_15). Each microsatellite was analyzed as a biallelic polymorphism. Based on a review of the literature, we define a short allele and a long allele for each microsatellite. We compared their frequencies in early PL occurring before 10 wk of amenorrhea (n = 342) and in PL occurring later on (n = 123). The mutated alleles rs1800871T (odds ratio, 3.083; 95% confidence interval, 1.984-4.792) and rs1800872A (odds ratio, 3.013; 95% confidence interval, 1.924-4.719) were associated with early PL. The haplotype rs1800872A/rs1800871T/X78437.2:g.8134CA[14_25]/X78437.2:g.5325CA[11_13], which includes the two mutated alleles, was significantly associated with the risk of early PL in a dose-dependent manner. Positivity for one haplotype was significantly associated with a 5.6-fold increase in the risk of early pregnancy failure, and positivity for two haplotypes was associated with an 8-fold increase in risk. In women with PL, some polymorphisms of the IL10 gene promoter seem to be constitutional risk factors for early (embryonic) pregnancy failure.

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, chromosome fragility, and cancer susceptibility. Eight FA-associated genes have been identified so far, the products of which function in the FA/BRCA pathway. A key event in the pathway is the monoubiquitination of the FANCD2 protein, which depends on a multiprotein FA core complex. In a number of patients, spontaneous genetic reversion can correct FA mutations, leading to somatic mosaicism. We analyzed the FA/BRCA pathway in 53 FA patients by FANCD2 immunoblots and chromosome breakage tests. Strikingly, FANCD2 monoubiquitination was detected in peripheral blood lymphocytes (PBLs) in 8 (15%) patients. FA reversion was further shown in these patients by comparison of primary fibro-blasts and PBLs. Reversion was associated with higher blood counts and clinical stability or improvement. Once constitutional FANCD2 patterns were determined, patients could be classified based on the level of FA/BRCA pathway disruption, as "FA core" (upstream inactivation; n = 47, 89%), FA-D2 (n = 4, 8%), and an unidentified downstream group (n = 2, 4%). FA-D2 and unidentified group patients were therefore relatively common, and they had more severe congenital phenotypes. These results show that specific analysis of the FA/BRCA pathway, combined with clinical and chromosome breakage data, allows a comprehensive characterization of FA patients.

The Sysmex XE-2100 (Sysmex Corp. Kobe, Japan) is a latest-generation hematology analyzer. Its optical and electrical measuring technology is improved by the addition of flux cytometry, fluorescence, and differential lysis. Its analytical performance in terms of precision, reproducibility, linearity, carryover, and time stability was found to be entirely satisfactory. In addition, the results of 500 complete blood counts and differentials correlated perfectly with those obtained by the Coulter STKS (Beckman Coulter, Villapointe, France). The comparison of 500 leukocyte differential count results analyzed in parallel with optical microscopy and the XE-2100 were surprising, and favorable to the XE-2100. This analyzer provides the user with an undeniable feeling of security concerning its reliability in detecting and identifying anomalies in the automated leukocyte differential count. With a sensitivity of 96%, a negative predictive value (NPV) of 98%, and a false-negative (FN) rate of 4%, the XE-2100 has perhaps reached the technological limits for a machine performing morphological recognition of normal and pathological blood cells., (Copyright 2003 Wiley-Liss, Inc.)

Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization. In the present study, we have investigated the relationship between shape change and PAK activation. We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-Fc(gamma)RIIA IV.3 induces an activation of Cdc42 and Rac1. The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1. Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK. Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading. The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation. Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin. These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.

Circulating plasma cells in 10 cases of reactive plasmacytosis had a shared phenotype with early plasma cell (CD19(+) CD38(+) CD138(+) CD40(+) CD45(+) CD11a+ CD49e- CD56(-)). In most cases, a minor subpopulation of CD28(+) plasma cells was also detected. Reactive plasma cells were highly proliferative, suggesting the presence of circulating progenitors (plasmablasts). After CD138(+) plasma cell removal, highly proliferative CD138(-) plasmablasts differentiated into CD138(+) plasma cells within a few days. This differentiation, which was associated with increased CD38 and decreased HLA-DR expression, was further confirmed by a large increase in intracellular Ig content (associated with Ig secretion) and was concomitant with extensive secretion of interleukin-6 (IL-6). The addition of neutralizing anti-IL-6 and anti-CD126 (IL-6 receptor) monoclonal antibodies totally prevented Ig secretion and cell differentiation by inducing apoptosis of plasmablasts, which indicates that IL-6 is an essential survival factor for plasmablasts. This report provides the first characterization of normal plasmablasts and shows that their phenotype is not exactly that of multiple myeloma cells.

Exosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.

B cell chronic lymphocytic leukemias (B-CLL) like other blood cell malignancies are characterized by chromosomal anomalies directly involved in tumor pathogenesis. We report here the molecular characterization of a t(7;14)(q21;q32) chromosomal translocation observed during the course of a B-CLL. We show that this translocation led to the juxtaposition of the immunoglobulin heavy chain locus on chromosome 14 to an endogenous retroviral sequence belonging to the THE family (transposable-like human element) on chromosome 7q21. RT-PCR analysis demonstrated that this sequence is transcribed in most of the tumoral and normal tissue analyzed and in the B-CLL described here. These data raise the question of the role of transposable elements in the pathogeny of some leukemias or at least, in the occurrence of chromosomal rearrangements. Structural rearrangements of the 7q21-22 region are frequently encountered in myeloid disorders, and the work presented here could help in their characterization.

In patients with advanced multiple myeloma (MM) there is an excess of production of interleukin-6 (IL-6) in vivo, and elevated serum levels are associated with plasmablastic proliferative activity and short survival. These data prompted us to perform a clinical trial with a murine anti-IL-6 monoclonal antibody (MoAb) to neutralize the excess of this putatively deleterious factor in these patients. Ten MM patients with extramedullary involvement frequently were treated with anti-IL-6 MoAb. The MoAb was administered intravenously to 9 patients; 1 patient with malignant pleural effusion received intrapleural therapy. Of the 3 patients who succumbed to progressive MM after less than 1 week of treatment (including the only 1 treated locally), 2 with evaluable data exhibited marked inhibition of plasmablastic proliferation. Among the 7 patients remaining more homogeneous receiving the anti-IL-6 MoAb for more than 1 week, 3 had objective antiproliferative effect marked by a significant reduction of the myeloma cell labelling index within the bone marrow. One of these 3 patients achieved a 30% regression of tumor mass. However, none of the patients studied achieved remission or improved outcome as judged by standard clinical criteria. Of major interest, objective antiproliferative effects were associated with complete inhibition of C-reactive protein (CRP) synthesis and low daily IL-6 production in vivo. On the other hand, the lack of effect in 4 patients was associated with a higher IL-6 production and inability of the MoAb to neutralize it. Anti-IL-6 was also associated with resolution of low-grade fever in all the patients and with worsening thrombocytopenia and mild neutropenia. The generation of human antibodies to Fc fragment of the murine anti-IL-6 MoAb observed in 1 patient was associated with dramatic progression. These data show that anti-IL-6 MoAb can suppress the proliferation of myeloma cells and underscore the biologic role of IL-6 for myeloma growth in vivo. Furthermore, suppression of CRP and worsening of neutropenia/thrombocytopenia both indicate that IL-6 is critically involved in acute-phase responses and granulopoiesis/thrombopoiesis.

Background: The active isomer of leucovorin (LV), LV-6S was compared with the racemic form, LV-6R,S and the inactive form, LV-6R., Materials and Methods: Pharmacokinetic studies of LV-6S and 6R,S were performed on normal volunteers and patients. Growth of Pediococcus Cerevisiae (PC), a LV-dependent strain, was measured with the 3 forms of LV. CCRF-CEM, a leukemic human cell line, was used to compare the effect of LV-6S, 6R,S and 6R on the rescue of methotrexate (MTX) and on the cytotoxicity of 5-fluorouracil (5-FU)., Results: LV-6S exhibits pharmacokinetic patterns similar to those obtained with LV-6R,S whatever the route used, oral or intravenous. The growth of PC was similar with the active isomer and the racemic form while the unnatural isomer, LV-6R did not promote any growth. Cells exposed to MTX (10(-7) to 10(-5) M) were rescued from MTX cytotoxicity with LV-6S and LV-6R,S at concentrations 100 and 200 fold higher, whereas LV-6R did not reverse toxic effects of MTX. An enhancement of the cytotoxicity induced by 5-FU (10(-4) M) was obtained after preexposure of cells to LV-6S or LV-6R,S while LV-6R did not exhibit any synergistic effect., Conclusion: LV-6S has similar effects to LV-6R,S in vitro and in vivo but at half doses; its clinical use prevents the possible interference of the inactive isomer, especially in patients receiving high doses of LV.

A new case of terminal deletion 10q26-qter is described. The phenotypic features are compatible with those of the previously reported cases. Deafness is reported for the first time.

Background: Plasmin has been reported both to activate platelets and to inhibit platelet functions. The latter effect was thought to be caused by proteolysis of the main membrane glycoproteins., Methods and Results: We found that incubation of citrated human platelet-rich plasma with streptokinase (SK) (300 IU/ml) does not produce any detectable activation but leads to a time-dependent inhibition of ADP-induced aggregation accompanied by substantial fibrinogenolysis. These effects were abrogated by previous addition of a plasmin inhibitor, aprotinin. Crossover experiments (SK-treated or control platelets mixed with SK-treated or control plasma) demonstrated that the platelets remained functional and that the aggregation defect was caused by fibrinogenolysis. Further experiments (addition of purified fibrinogen to fibrinogen-depleted plasma with either SK or thrombin) suggested that in addition to the low residual level of fibrinogen, fibrinogen degradation products had an inhibitory effect. Under the same conditions, tissue-type plasminogen activator (t-PA) (3,000 ng/ml) had no effect on platelet aggregation, and plasma fibrinogen was not significantly lowered. The effects on glycoproteins IIb-IIIa of incubation with SK, t-PA, or plasmin were assessed with immunoblots with murine monoclonal antibodies directed against either part of the complex, which is the receptor for fibrinogen. Proteolysis was detected only in the presence of EDTA, a potent chelator of divalent cations., Conclusions: The incubation of human platelets in citrated plasma with SK concentrations obtained during therapy leads to an aggregation defect that is related to the decrease in fibrinogen, the adhesive protein involved in this function, and to the impeding effect of fibrinogen degradation products on its binding onto platelets but not to an alteration of the corresponding platelet receptor, the heterodimer glycoproteins IIb-IIIa.

Primary lymphoma of the spleen is characterized by predominant splenomegaly. Lymphoplasmacytic malignant lymphoma of the spleen, of low malignancy in the Kiel classification, low and intermediate grade in the National Cancer Institute Working Formulation (NCIWF), is rare. It is often associated with a monoclonal immunoglobulin M (IgM). Four patients presenting with primary splenic lymphoma of plasmacytic type associated with a high level of monoclonal IgM and a lupus anticoagulant (LA) are described. This association has not previously been reported. In contrast with the usual heterogeneity of LA, this LA is relatively homogeneous with an important prolongation of the prothrombin time (greater than 18 sec for a control of 12), more prolonged partial thromboplastin time (PTT) of the mixture patient + control plasma than PTT of the patient plasma. Despite the important coagulation abnormalities, none of these four patients has presented any hemorrhagic or thrombotic complications, even during major surgery such as splenectomy. The lupus-like anticoagulant effect ran parallel with the monoclonal IgM. Survival, after splenectomy and chemotherapy, appears to be favourable: three patients are alive with survivals of greater than or equal to 7 years. The follow-up is as yet too short for the last patient.

Coexisting deficiencies of both intrinsic factor (IF) and R binder were identified in an Algerian boy who presented with severe megaloblastic anemia, growth retardation, and neurologic dysfunction with typical features of subacute combined degeneration of the spinal cord. The anemia responded completely to cyanocobalamin and folic acid. IF was absent from gastric juice, but acid secretion and gastric mucosa were normal. R binders were absent from gastric juices as well as from serum, saliva, and polymorphonuclear leukocytes. The patient's father exhibited absence of R binder in his serum with a low serum vitamin B12 level and was asymptomatic. This unique case of simultaneous IF and R binder deficiencies suggests a genetic association between these two functionally and immunologically dissimilar, but structurally close vitamin B12-binding proteins.

This is a report of a 62-year-old male patient who had a bleeding disorder due to the presence of a factor VII (proconvertin) inhibitor. After treatment with a high-dose intravenous (IV) immunoglobulin failed and a life-threatening intracranial hemorrhage occurred, plasma exchanges were performed and immunosuppressive therapy was given. The factor VII inhibitor promptly disappeared, and the patient's parameters of hemostasis became normal. Even though a relapse occurred some months later, the patient responded to immunosuppressive therapy. No underlying disorder was evidenced after 17 months of follow-up.