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Erythrocytapheresis procedures, increasingly used in the management of patients with severe complications of sickle cell disease, are limited by adequate venous access. We have successfully used short-term femoral catheter insertion, during a 6.5-year period for a total of 443 procedures, to perform long-term erythrocytapheresis in 18 consecutive children with sickle cell disease., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Background: The ACVBP regimen is an efficient induction regimen for poor-risk patients with diffuse large B-cell lymphoma (DLBCL) before consolidative autologous stem cell transplantation. Adjunction of the monoclonal anti-CD20 antibody rituximab (R-ACVBP) was recently found to be superior to ACVBP alone. This study assessed the impact of rituximab on stem cell mobilization in two similar consecutive groups of patients treated with ACVBP in two prospective, controlled trials., Study Design and Methods: The first trial (LNH-98B-3) involved 137 patients treated with ACVBP alone. In the second trial (LNH-03-3B), 91 patients received an R-ACVBP regimen. Stem cell mobilization was performed after a course of (R)-ACVBP., Results: The median peak numbers of blood CD34+ cell counts recorded before the first apheresis procedure in the ACVBP and R-ACVBP groups were 69×10(6) and 63×10(6) /L, respectively (p=0.55). The median numbers of CD34+ cells collected were 7.1×10(6) and 6.0×10(6) CD34+ cells/kg for the ACVBP and R-ACVBP groups, respectively (p=0.13). The median number of apheresis procedures required for gathering the minimum amount of CD34+ cells (2×10(6) /kg) was the same in the two groups., Conclusion: When compared with ACVBP alone, adjunction of rituximab does not impair stem cell mobilization., (© 2012 American Association of Blood Banks.)

The methods used to collect hematopoietic stem cells in their natural environment (bone marrow or cord blood) or in the peripheral blood after stimulation are well-defined and ruled both to ensure the donor security and perform a quality hematopoietic transplantation. Safety of the familial or non-familial donor must be ensured not only during the collection but also on a medium- or a long-term basis. The stem cells amount in a graft and its characterisation depend on the collection site of hematopoietic stem cells and on the technique used. The knowledge of conditions influencing these amounts allows optimising the hematopoietic stem cells collection while preventing conditions in which the donor safety could be decreased. The collection site also influences the collection of significant amounts of other blood cells. This knowledge conditions the preparation procedures of the graft in cell therapy units or the management of per- or post-transplantations complications in haematology units. Thus, hematopoietic transplantations concern not only hematological units but also the teams involved in various stages of donor selection, hematopoietic stem cells collection and graft preparation. In order to allow an appropriate care of both donor and recipient, a concomitant knowledge of all the stages involved in hematopoietic collection conditions, characterisation of collected cells, hematological diseases and conditioning must be brought to hematological, collection and cell therapy teams., (Copyright © 2011. Published by Elsevier SAS.)

Background: The short-term effects of granulocyte-colony-stimulating factor (G-CSF) have been extensively studied, but recent reports of G-CSF-induced genetic perturbations raised concerns regarding its long-term safety. In this respect, duration of G-CSF-induced perturbations has been less studied than short-term effects and needs to be evaluated., Study Design and Methods: G-CSF mobilization-induced immunologic alterations were prospectively analyzed in a cohort of 24 healthy donors. Blood samples were taken before G-CSF administration; at the time of administration; and at 1, 3, 6, and 12 months and analyzed for blood cell counts and in vitro cytokines (interleukin [IL]-2, -8, and -10) and immunoglobulin production, quantified in the culture supernatant of peripheral blood mononuclear cells (PBMNCs) after, respectively, phytohemagglutinin and pokeweed mitogen stimulation., Results: Platelet, granulocyte, monocyte, B, and dendritic blood cell counts as well as the IL-2, -8, and -10 secretion by PBMNCs, perturbed at the time of G-CSF mobilization, returned to baseline values at 1 month, with T-cell and natural killer cell counts recovering at 3 months. In vitro immunoglobulin production was increased up to 6 months after mobilization., Conclusion: Although assessment of the potential long-term risk of G-CSF administration will require prolonged observation of larger cohorts, our data show that the duration of immunologic perturbations may be more persistent than previously anticipated, especially for B-cell functional alterations. Most perturbations remain, however, transient with a return to baseline values within 1 year., (© 2010 American Association of Blood Banks.)

Background: Hypoxia is a major cause of painful vaso-occlusive crisis in sickle cell disease (SCD). Simple transfusion and red blood cell (RBC) exchange are commonly used as preventive therapies whose aim is to dilute hemoglobin (Hb)S-containing RBCs (SS-RBCs) with normal RBCs (AA-RBCs) to prevent sickling. We hypothesized that the effectiveness of transfusion could be improved by the encapsulation of inositol hexaphosphate (IHP), an allosteric Hb effector, in transfused AA-RBCs. Indeed, apart from their diluting effect on SS-RBCs, IHP-loaded RBCs (IHP-RBCs) with increased oxygen release capacity could palliate in vivo oxygen deprivation and reduce sickling., Study Design and Methods: The study was designed to investigate the therapeutic effect of IHP-RBCs transfusion on in vitro sickling of SS-RBCs collected from 20 SCD patients. Patients' RBCs were diluted with various proportions of IHP-RBCs or AA-RBCs (processed or stored RBCs as controls). Resulting suspensions were subjected to deoxygenation followed by partial reoxygenation at 5% oxygen. Sickling was evaluated by microscopy., Results: Stored RBCs (50% dose) used to mimic simple transfusion exhibited a poor antisickling effect (5.6%) and a low response rate (65%). In contrast, IHP-RBCs treatment was seven times more effective resulting in 35% of sickling reduction and a 94% response rate. Sickling was inhibited in a dose-dependent manner: 9.9, 25.1, and 35.0% for IHP-RBCs in percentages of 10, 30, and 50%, respectively., Conclusion: Our results indicate that IHP-RBCs prevent in vitro sickling and suggest that it could improve conventional transfusion therapy in terms of transfused volume, frequency, and efficacy., (© 2010 American Association of Blood Banks.)

Background: Extracorporeal photopheresis (ECP) is a powerful therapy currently used to treat various hematological disorders as in graft versus host disease. Clinical data clearly demonstrate its efficacy and immunomodulation toward the pathogenic T cells. However, ECP mechanism of action is still poorly understood. Monocytes represent up to 30% of the total amount of treated cells and are known to play an important role in adaptive immunity. However, data from previous reports analyzing the effect of psoralen and UV-A irradiation (PUVA) on their functions are heterogeneous. In this study, we focused on the effect of PUVA on human monocytes functions in adaptive immunity., Design and Methods: Purified human monocytes were treated in vitro by PUVA. We measured their kinetic of apoptosis after the treatment. We also determine whether their phenotype and functionalities were modified. Finally, we assessed the functionalities of PUVA-treated monocytes-derived dendritic cells (DC)., Results: PUVA treatment sentenced purified monocytes to die in 6 days and immediately altered their migratory capacities without impairing their ability of endocytosis. It also up-regulated co-stimulatory molecules and production of inflammatory cytokines on activation and consequently stimulated allogeneic or autologous T cells as efficiently as untreated monocytes. Moreover, PUVA-treated monocytes retained their ability to differentiate into fully functional DC that maturated and stimulated T cells as well as normal DC., Conclusions: Our data demonstrate that monocytes undergo apoptosis and loose a part of their migratory capacity after ECP and the surviving cell functionalities are not impaired, suggesting that monocytes have a minor effect on ECP-mediated immunomodulation.

Twenty-seven patients (14 girls, 13 boys) affected by familial hypercholesterolemia who had begun low-density lipoprotein (LDL) apheresis treatment before the age of 15 were studied. The median age at diagnosis was 4 years and the blood LDL cholesterol level was 704 +/- 163 mg/dL. Screening was performed for homozygous or double heterozygous mutations of the LDL cholesterol receptor gene and mutations were found in 24 of the patients. The mean age at the beginning of treatment was 8.5 years and the mean length of follow up was 12.6 years. The two main procedures used were direct adsorption of lipoproteins and dextran sulfate cellulose adsorption. Nine patients experienced anaphylactic reactions due to bradykinin and six had to have their treatment changed. The LDL cholesterol level before the session was lowered by 45 +/- 11% of the value at diagnosis. The LDL cholesterol reduction in a session was 72 +/- 10%. Tendinous xanthomas disappeared or diminished dramatically in 62% of the children. In 22 patients no cardiovascular event occurred during LDL apheresis treatment. Three had angina pectoris; two others had surgical management of aortic stenosis, but no clinical manifestations. Seven children had normal cardiovascular pictures while on treatment. Eleven had abnormalities of the aortic root or coronary arteries, which in six cases had appeared before treatment; the other five children did not undergo prior cardiac evaluation. In five children the abnormalities appeared during treatment. Based on these data, LDL-apheresis can be recommended for the treatment of homozygous familial hypercholesterolemia, even in young children, with good efficiency on biological parameters, cutaneous lesions and cardiovascular events.

The mechanisms underlying the immunomodulatory functions of mesenchymal stem cells (MSC) on dendritic cells (DC) have been shown to involve soluble factors, such as IL-6 or TGF-beta, or cell-cell contact, or both depending on the report referenced. In this study, we intend to clarify these mechanisms by examining the immunosuppressive effect of human adult MSC on adult DC differentiated from CD34(+) hemopoietic progenitor cells (HPC). MSC have been shown to inhibit interstitial DC differentiation from monocytes and umbilical CD34(+) HPC. In this study, we confirm that MSC not only halt interstitial DC but also Langerhans cell differentiation from adult CD34(+) HPC, as assessed by the decreased expression of CD1a, CD14, CD86, CD80, and CD83 Ags on their cell surface. Accordingly, the functional capacity of CD34(+) HPC-derived DC (CD34-DC) to stimulate alloreactive T cells was impaired. Furthermore, we showed that 1) MSC inhibited commitment of CD34(+) HPC into immature DC, but not maturation of CD34-DC, 2) this inhibitory effect was reversible, and 3) DC generated in coculture with MSC (MSC-DC) induced the generation of alloantigen-specific regulatory T cells following secondary allostimulation. Conditioned medium from MSC cultures showed some inhibitory effect independent of IL-6, M-CSF, and TGF-beta. In comparison, direct coculture of MSC with CD34(+) HPC resulted in much stronger immunosuppressive effect and led to an activation of the Notch pathway as assessed by the overexpression of Hes1 in MSC-DC. Finally, DAPT, a gamma-secretase inhibitor that inhibits Notch signaling, was able to overcome MSC-DC defects. In conclusion, our data suggest that MSC license adult CD34(+) HPC to differentiate into regulatory DC through activation of the Notch pathway.

Background: We performed the first human partial face allograft on November 27, 2005. Here we report outcomes up to 18 months after transplantation., Methods: The postsurgical induction immunosuppression protocol included thymoglobulins combined with tacrolimus, mycophenolate mofetil, and prednisone. Donor hematopoietic stem cells were infused on postoperative days 4 and 11. Sequential biopsy specimens were taken from a sentinel skin graft, the facial skin, and the oral mucosa. Functional progress was assessed by tests of sensory and motor function performed monthly. Psychological support was provided before and after transplantation., Results: Sensitivity to light touch, as assessed with the use of static monofilaments, and sensitivity to heat and cold had returned to normal at 6 months after transplantation. Motor recovery was slower, and labial contact allowing complete mouth closure was achieved at 10 months. Psychological acceptance of the graft progressed as function improved. Rejection episodes occurred on days 18 and 214 after transplantation and were reversed. A decrease in inulin clearance led to a change in immunosuppressive regimen from tacrolimus to sirolimus at 14 months. Extracorporeal photochemotherapy was introduced at 10 months to prevent recurrence of rejection. There have been no subsequent rejection episodes. At 18 months, the patient is satisfied with the aesthetic result., Conclusions: In this patient who underwent the first partial face transplantation, the functional and aesthetic results 18 months after transplantation are satisfactory., (Copyright 2007 Massachusetts Medical Society.)

Objectives: To analyze the consequences of lymphocyte donations on lymphopoiesis in donors having previously undergone hematopoietic stem cell collection for allogeneic stem cell transplantation., Methods: Repeated analysis of leukocyte subsets in the peripheral blood of 76 hematopoietic stem cell donors undergoing lymphocyte donation(s) for DLI., Results: Grade I/II lymphopenia was present in 22 donors (29%) just before first apheresis for lymphocyte collection, demonstrating that former stem cell donation induced prolonged lymphopenia in a subset of donors. The monocytic lineage was not affected. Older age and history of PBSC harvest constituted 2 independent factors of lymphopenia, but had no influence on monocytopenia. The first apheresis induced lymphopenia in 36 donors (47%) and monocytopenia in 23 donors (39%). Lymphopenia before first apheresis and prior history of PBSC harvest were independent factors of apheresis-induced lymphopenia while those factors had no influence on monocytopenia. A time-dependent decrease in lymphocyte counts was observed in donors undergoing repeated aphereses, resulting in persistent and prolonged lymphopenia in 50% of donors. No persistent monocytopenia over time and aphereses was observed. Kaplan-Meier estimate of the risk to develop persistent lymphopenia after multiple aphereses was 21% +/- 6% at 2 months, 38% +/- 8% at 4 months, and 64% +/- 10% at 12 months. After Cox regression analysis, previous PBSC harvest remained the unique factor associated with the risk for persistent lymphopenia., Conclusions: Monitoring the potential long-term effects of repeated aphereses in hematopoietic stem cell donors appears important. Selecting young bone marrow donors for subsequent DLI significantly reduces the risk for acute and prolonged lymphopenias.

CD4 regulatory cells have been postulated to prevent autologous graft-vs.-host disease (GVHD). In order to test this hypothesis, we used BALB/c mice, a strain known to be resistant to autologous GVHD, which had received autologous stem cell transplantation (ASCT) and cyclosporine A (CsA). As expected, ASCT/CsA-treated BALB/c mice did not develop any sign of acute or chronic GVHD. However, depletion of CD4 T cells induced a skin disease with clinical and histological features of alopecia areata (AA), a CD8 T-cell-mediated human autoimmune skin disease. The hair loss in mice developing AA was associated with the infiltration of the skin by activated CD8 T cells. Analysis of the T-cell recovery in ASCT- and ASCT/CsA-treated mice showed that CsA induced an increase in the number of CD4+ 25+ T cells, suggesting that the lack of GVHD in ASCT/CsA treated-mice could be related to the expansion of this CD4 T-cell subset. Collectively these data show that CD4 T cells comprise regulatory cells controlling the onset of autologous GVHD and suggest that the naturally occurring CD4+ 25+ subset may be responsible for this effect.

The purpose of this study was to evaluate retrospectively the effect of a second treatment with rituximab for patients who progressed after a response to a first treatment. We analysed the charts of 41 patients treated at CHU Lyon-Sud between 1997 and May 2003. Patients were treated with rituximab alone or with a combination of rituximab and chemotherapy. The overall response rate (complete and partial response) was 73% for the second treatment. The median time to progression was longer but not significant for the second treatment in comparison with the first one (15.2 versus 11.3 months, P = 0.09). The second treatment was well tolerated. Thus, a second treatment with rituximab should be considered, alone or in combination with chemotherapy, for patients who progress after a first response to rituximab.

Objective: Immunoparalysis has recently emerged as a possible cause explaining the failure of clinical trials in septic shock. Because human peripheral blood CD4+CD25+ T cells have been characterized as suppressor T cells, we hypothesized they might be increased in sepsis-induced immunoparalysis., Design: Prospective, observational, clinical study., Setting: Adult intensive care units in a university hospital., Subjects: Patients with septic shock (n = 16) and healthy individuals (n = 36)., Interventions: None., Measurements and Main Results: In patients with septic shock (mortality rate at 28 days, 56%; mean admission Simplified Acute Physiology Score II, 47), we first illustrated immunoparalysis by showing a severe diminished monocytic human leukocyte antigen (HLA)-DR expression. Afterward, compared with control values, we found in these patients a marked elevation of circulating CD4+CD25+ T cells that were also CD45RO+ and CD69- and overexpressed CTLA-4. Importantly, nonsurvivors (n = 9) presented prolonged lower monocytic HLA-DR expression and higher percentage of CD4+CD25+ T-suppressor T cells., Conclusions: These data support the concept that the persistence of a pronounced immunoparalysis after septic shock is associated with a poor outcome. Whether CD4+CD25+ T cells directly participate in sepsis-induced immunoparalysis remains to be investigated.

The aim of this study was to retrospectively analyze survival and tumor response data in patients with localized gastric MALT lymphoma treated by different treatment modalities other than anti-Helicobacter pylori treatment (diagnosis made before 1993, or after failure of antibiotics + anti-acid), including surgery, chemotherapy or combined treatment. Here we studied a series of 48 patients with stage IE or IIE disease treated during the past 11 years. These patients received different treatments: chemotherapy was proposed to 19 (40%) patients; gastric surgery to 21 (43%) patients, consisting of partial gastrectomy of 7 patients and total gastrectomy in 14 patients; combined treatment to 8 (17%) patients, consisting of surgery + chemotherapy in 7 patients and surgery + chemotherapy + radiotherapy in 1 patient. At diagnosis, 85% of the patients had good PS and no B symptoms. Complete response after treatment was reached in 45 (94%) patients (chemotherapy: 84% of the patients; surgery alone: 95%; combined treatment: 100%). Progression was observed in 16 (33%) patients. No statistical difference in the survival was found among the different therapeutic modalities: 5-year overall survival year FFP survival was 81% for chemotherapy, 86% for surgery alone and 95% for combined treatment. Prognostic factors for survival were age, performance status and hemoglobin level at diagnosis. Considering the natural bias of a retrospective analysis, surgery or chemotherapy was associated with a similar outcome in patients with MALT lymphoma after antibiotics failure.

Splenic marginal zone B-cell lymphoma (MZL), with or without villous lymphocytes, is an indolent lymphoma, presenting with massive splenomegaly, generally associated with bone marrow dissemination. In patients requiring therapy, splenectomy has been reported as the treatment of choice. We reviewed the cases of 81 patients with splenic MZL. Patients presented with stage IV disease at diagnosis in 95% of the cases. Autoimmune events (hemolytic anemia, immune thrombocytopenia, acquired coagulation disorders, positive Coomb's test) were observed in 16 patients, and a monoclonal (M) serum component was detected in 46% of the patients. Twenty patients did not receive any initial treatment at diagnosis. Splenectomy was proposed in 79% of the treated patients, with adjuvant chemotherapy in 47% of patients. Median survival was 10.5 years and was significantly shorter in the presence of an M component, an elevated b2-microglobulin level, leukocyte count > 20000/microL, and lymphocytes > 9000/microL. Disease progression was significantly more frequent in patients presenting an immunological event or an M component. Seventy percent of the patients had persistent involvement of bone marrow and/or peripheral blood after splenectomy. Disease progression was significantly more frequent in partial responders than in complete responders (P < 0.005), but overall survival, risk of histologic transformation, and risk of death from lymphoma were not different in the 2 groups. Moreover, patients with cytopenia at diagnosis treated by splenectomy alone rapidly recovered normal hematological parameters. We conclude that splenectomy is an efficient treatment for splenic MZL, but that it may be delayed until the occurrence of symptoms or cytopenia.

Purpose: Non-Hodgkin's lymphoma occurs frequently in patients with human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). We determined the association between the clinical and histologic features of HIV-related lymphoma., Subjects and Methods: We reviewed the medical records of 291 patients with noncerebral HIV-related lymphoma who had been treated in multicenter trials coordinated by the Groupe d'Etude des Lymphomes de l'Adulte between 1988 and 1997. This study was performed mainly before the availability of combination antiretroviral therapy., Results: The main histologic subtypes were centroblastic lymphoma in 131 patients (45%), immunoblastic lymphoma in 39 patients (13%), and Burkitt's lymphoma (including the classical form and the variant with plasmacytic differentiation) in 115 patients (40%). Burkitt's lymphoma was the most aggressive form, whereas immunoblastic lymphoma occurred in severely immunodeficient patients. Two-year survival after enrollment was 15% in immunoblastic lymphoma, 32% in Burkitt's lymphoma, and 31% in centroblastic lymphoma (P = 0.006), but multivariate analysis did not confirm the independent prognostic value of histologic subtype. Instead, five independent pretreatment factors increased the risk of mortality: age 40 years or older [relative risk (RR) = 1.5; 95% confidence interval (CI), 1.1 to 2.1; P = 0.005], elevated serum lactate dehydrogenase level (RR = 1.5; 95% CI, 1.1 to 2.1; P = 0.02), having a diagnosis of AIDS before lymphoma (RR = 1.8; 95% CI, 1.2 to 2.6; P = 0.006), CD4(+) cell count less than 100 x 10(6)/L (RR = 1.8; 95% CI, 1.3 to 2.6; P = 0.0004), and impaired performance status (RR = 2.4; 95% CI, 1.7 to 3.4; P <0.0001)., Conclusion: Several pretreatment characteristics of HIV-related lymphoma were linked to the histologic form, but HIV disease parameters other than those of lymphoma were the main determinants of outcome, so the histologic features of the lymphoma were not associated with prognosis.

Patients receiving high-dose cyclophosphamide as a conditioning regimen for peripheral stem cell collection are subjected over a short period of time to significant exposure to reactive oxygen species (ROS). All these patients undergo profound leucopenia. Various other short-term toxicities are observed in a fraction of the patients, including febrile aplasia requiring hospitalization, thrombocytopenia and mucositis. Although stem cell collection is feasible in the majority of patients stimulated with haematopoietic growth factors, in some instances, graft collection cannot be performed because of insufficient concentrations of stem cells in peripheral blood. There is currently no predictive assay to determine which patients treated with high-dose cyclophosphamide have a high risk of febrile aplasia or will successfully undergo cytaphereses for stem cell collection. In order to identify such predictive factors, we analysed the level of expression before treatment of various ROS detoxification mechanisms in the peripheral blood of 37 patients receiving high-dose cyclophosphamide for lymphoproliferative diseases. Various parameters involved in the metabolism of ROS were measured in plasma and/or erythrocytes, including superoxide dismutase, glutathione, glutathione peroxidase, glutathione reductase and malondialdehyde. High levels of erythrocyte superoxide dismutase before cyclophosphamide therapy were correlated with an increased risk of hospitalization for febrile aplasia (65% vs. 29%, P = 0.013). High superoxide dismutase and low erythrocyte glutathione reductase were associated with lower CD34 yields. These data suggest that components of the ROS detoxification system modulate the degree of short-term toxicity of cyclophosphamide and could be used as predictive markers in individual patients.