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  • Efficacy of cascade-primed cell infusion as an adjuvant immunotherapy with concurrent chemotherapy for patients with non–small-cell lung cancer: A retrospective observational study with a 5-year follow-up
    Cytotherapy (IF 4.297) Pub Date : 2020-01-03
    Hong Li; Zhen Zhang; Xiaoran Duan; Nomathamsanqa Resegofetse Maimela; Shuangning Yang; Xuan Zhao; Jianmin Huang; Yi Zhang

    Background Clinical studies have shown the efficacy of combination therapy for various malignancies. In this study, the characteristics, safety and feasibility of use of cascade-primed (CAPRI) cells for the combination treatment of non–small-cell lung cancer (NSCLC) were evaluated both in vitro and in vivo. Methods Sixty-five patients with stage II–IV NSCLC were recruited. Of these patients, 31 patients received CAPRI cell therapy combined with chemotherapy (CAPRI group), and the other 34 patients constituted the control group and received chemotherapy alone. This study primarily aimed to evaluate the overall survival (OS), progression-free survival (PFS), short-term responses and treatment efficacy. Results CD83, CD1a, CD80 and CD86 marker levels were significantly upregulated in CAPRI cells. Interferon-γ expression levels were highest in CD3+CD8+ cells (33.77% ± 4.40%). Furthermore, interleukin-2 levels were highest in CD3+CD56+ cells (26.73% ± 6.63%), whereas perforin expression levels were similar in CD3+CD8+ and CD3+CD56+ cells. Furthermore, CAPRI cells had a better anti-tumor potential in CD3+CD56+ cells and displayed the highest expression levels of CD107a to H460 and A549 cell lines. The 5-year OS was significantly greater in the CAPRI group than in the control group (P = 0.008), and the PFS of two groups exhibited a significant difference (P = 0.007). Median OS (48 versus 31.6 months; P = 0.004) and PFS (48 versus 36.4 months; P = 0.016) differed between these two groups. Moreover, treatment-associated toxicities were mild and well-tolerated by patients with NSCLC. Conclusion CAPRI cell therapy potentially prolongs the survival of patients with NSCLC when combined with chemotherapy.

    更新日期:2020-01-04
  • Reliable isolation of human mesenchymal stromal cells from bone marrow biopsy specimens in patients after allogeneic hematopoietic cell transplantation
    Cytotherapy (IF 4.297) Pub Date : 2019-12-27
    Thomas Krüger; Jan Moritz Middeke; Friedrich Stölzel; Anke Mütherig; Catrin List; Kalina Brandt; Katharina Heidrich; Raphael Teipel; Rainer Ordemann; Ulrich Schuler; Uta Oelschlägel; Martin Wermke; Martin Kräter; Maik Herbig; Rebekka Wehner; Marc Schmitz; Martin Bornhäuser; Malte von Bonin

    Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit–fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 106 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 106; range, 0–2.3 × 107 P0 MSCs versus 5.4 × 104; range, 0–8.9 × 106; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions.

    更新日期:2019-12-27
  • High incidence of Pneumocystis jirovecii pneumonia in allogeneic hematopoietic cell transplant recipients in the modern era
    Cytotherapy (IF 4.297) Pub Date : 2019-12-27
    Christopher Evernden; Michelle Dowhan; Rosy Dabas; Ahsan Chaudhry; Amit Kalra; Poonam Dharmani-Khan; Daniel Gregson; Andrew Johnson; Jennifer Jupp; Victor Jimenez-Zepeda; Kareem Jamani; Peter Duggan; Jason Tay; Faisal Khan; Andrew Daly; Jan Storek

    Background International guidelines for Pneumocystis jirovecii pneumonia (PJP) prevention recommend prophylaxis for ≥6 months following allogeneic hematopoietic cell transplantation, and longer in patients with graft-versus-host disease (GVHD) or on immunosuppressive therapy (IST). These recommendations are based on cohorts of patients who did not routinely receive anti-thymocyte globulin (ATG) for GVHD prophylaxis. Methods We performed a retrospective chart review of 649 patients, all of whom received ATG as part of GVHD prophylaxis. Results The cumulative incidence of definite PJP was 3.52% at both 3 and 5 years (median follow up, 1648 days for survivors). PJP occurred in 13 non-GVHD patients between days 207 and 508, due in part to low CD4 T-cell counts (<200 CD4 T cells/µL). PJP occurred in eight GVHD patients between days 389 and 792, due in part to non-adherence to PJP prophylaxis guidelines (discontinuation of PJP prophylaxis at <3 months after discontinuation of IST). Breakthrough PJP infection was not observed in patients receiving prophylaxis with cotrimoxazole, dapsone or atovaquone, whereas three cases were observed with inhaled pentamidine. Discussion In conclusion, for non-GVHD patients receiving ATG-containing GVHD prophylaxis, 6 months of PJP prophylaxis is inadequate, particularly if the CD4 T-cell count is <200 cells/µL or if there is a high incidence of PJP in the community. For patients with GVHD receiving ATG-containing GVHD prophylaxis, continuing PJP prophylaxis until ≥3 months post-discontinuation of IST is important. Cotrimoxazole, dapsone and atovaquone are preferred over inhaled pentamidine.

    更新日期:2019-12-27
  • Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units
    Cytotherapy (IF 4.297) Pub Date : 2019-12-27
    Diane Fournier; Antoine Lewin; Carl Simard; Patrick Trépanier; Sonia Néron; Lara Ballerini; Margarita Codinach; Heidi Elmoazzen; Mike Halpenny; Gesine Kogler; Stefanie Liedtke; Isabelle Louis; Carmen Azqueta Molluna; Nicolas Pineault; Arun Prasath; Sergio Querol; Riccardo Saccardi; D. Robert Sutherland; Serena Urbani

    Background aims In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. Methods Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. Results Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. Conclusions The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.

    更新日期:2019-12-27
  • Low baseline platelet count predicts poor response to plerixafor in patients with multiple myeloma undergoing autologous stem cell mobilization
    Cytotherapy (IF 4.297) Pub Date : 2019-12-24
    Mohammed Bakeer; Abba C. Zubair; Vivek Roy

    Background aims Baseline platelet count has been shown to be a sensitive predictor of autologous peripheral blood progenitor cell collection yield in patients with multiple myeloma mobilized with granulocyte colony-stimulating factor (G-CSF). Patients who mobilize poorly with G-CSF are often treated with plerixafor to enhance mobilization. There are no surrogate markers available to predict response to plerixafor. Methods We retrospectively analyzed data from 73 patients with multiple myeloma who did not have adequate mobilization with G-CSF alone and were treated with plerixafor as a rescue agent. Results We found that baseline platelet count directly correlated with peripheral blood CD34+ (PB-CD34+) count after plerixafor treatment (r = 0.36, P < 0.0001) and the number of PB-CD34+ cells collected on the first day of apheresis and inversely correlated with the number of apheresis sessions needed to collect the target number of PB-CD34+ cells (P = 0.0015). Baseline platelet count of 153 000/µL or less was associated with 90% specificity of predicting poor response to plerixafor with a sensitivity of 33%. Conclusions Baseline platelet count is a good predictor of mobilization response to plerixafor in patients with multiple myeloma.

    更新日期:2019-12-25
  • Spanish Cell Therapy Network (TerCel): 15 years of successful collaborative translational research
    Cytotherapy (IF 4.297) Pub Date : 2019-12-19
    Fermín Sánchez-Guijo; Damián García-Olmo; Felipe Prósper; Salvador Martínez; Agustín Zapata; Francisco Fernández-Avilés; Juan José Toledo-Aral; Miguel Torres; Isabel Fariñas; Lina Badimón; José Luis Labandeira-García; Javier García-Sancho; José M. Moraleda

    In the current article we summarize the 15-year experience of the Spanish Cell Therapy Network (TerCel), a successful collaborative public initiative funded by the Spanish government for the support of nationwide translational research in this important area. Thirty-two research groups organized in three programs devoted to cardiovascular, neurodegenerative and immune-inflammatory diseases, respectively, currently form the network. Each program has three working packages focused on basic science, pre-clinical studies and clinical application. TerCel has contributed during this period to boost the translational research in cell therapy in Spain, setting up a network of Good Manufacturing Practice–certified cell manufacturing facilities– and increasing the number of translational research projects, publications, patents and clinical trials of the participating groups, especially those in collaboration. TerCel pays particular attention to the public-private collaboration, which, for instance, has led to the development of the first allogeneic cell therapy product approved by the European Medicines Agency, Darvadstrocel. The current collaborative work is focused on the development of multicenter phase 2 and 3 trials that could translate these therapies to clinical practice for the benefit of patients.

    更新日期:2019-12-19
  • Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture
    Cytotherapy (IF 4.297) Pub Date : 2019-12-11
    Seshu Tyagarajan, David Schmitt, Christopher Acker, Erik Rutjens

    Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)–T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.

    更新日期:2019-12-11
  • Towards a common framework for defining ancillary material quality across the development spectrum
    Cytotherapy (IF 4.297) Pub Date : 2019-12-11
    Oliver Ball, Claudia Zylberberg

    Ancillary materials (AMs) play a critical role in the manufacture of cell and gene therapies, and best practices for their quality management are the subject of ongoing discussion. Given that the final product cannot be sterilized, AM quality becomes increasingly critical to the clinical advancement of cell and gene therapies. Despite a lack of direct legislative direction regarding AM quality, internationally harmonized guidance is available from several industry-standard bodies that describe the principles and application of a risk-based approach to AM qualification and related supply-chain risk management. According to a best-practice risk-based approach, AMs must be adequately qualified to a degree that reflects the level of risk the material presents to patient safety and the drug product's specification. This general approach can be implemented in different ways, and balancing quality with cost of goods is critical to the cost-effective manufacture of advanced therapy medicinal products. In some cases, it may be preferable or necessary to use AMs that are produced in compliance with current Good Manufacturing Practice. However, developers may be able to suppress manufacturing costs without undermining safety or regulatory compliance in the case that a material presents a lower risk profile. Despite a great deal of attention and interest in the quality of AMs in the cell and gene therapy space, there is still a need for greater harmonization to create a shared understanding of what constitutes a risk-based approach to AM production and sourcing. In this article, we propose a staged approach to AM quality that achieves a balance between the competing demands of risk mitigation and cost of goods containment at the various stages of AM quality development. Our novel, heuristic framework for communication among AM suppliers, users and regulators aims to bring down development and manufacturing costs and lessen the workload around regulatory compliance.

    更新日期:2019-12-11
  • Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors
    Cytotherapy (IF 4.297) Pub Date : 2019-12-11
    Christine Gagliardi, Mariam Khalil, Aaron E. Foster

    Background Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. Methods Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. Results The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. Discussion This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.

    更新日期:2019-12-11
  • Cell and gene therapy manufacturing capabilities in Australia and New Zealand
    Cytotherapy (IF 4.297) Pub Date : 2019-12-03
    GABRIELLE M. O'SULLIVAN, ZLATIBOR M. VELICKOVIC, MICHELLE W. KEIR, JANET L. MACPHERSON, JOHN E.J. RASKO

    Cell and gene therapy products are rapidly being integrated into mainstream medicine. Developing global capability will facilitate broad access to these novel therapeutics. An initial step toward achieving this goal is to understand cell and gene therapy manufacturing capability in each region. We conducted an academic survey in 2018 to assess cell and gene therapy manufacturing capacity in Australia and New Zealand. We examined the following: the number and types of cell therapy manufacturing facilities; the number of projects, parallel processes and clinical trials; the types of products; and the manufacturing and quality staffing levels. It was found that Australia and New Zealand provide diverse facilities for cell therapy manufacturing, infrastructure and capability. Further investment and development will enable both countries to make important decisions to meet the growing need for cell and gene therapy and regenerative medicine in the region.

    更新日期:2019-12-03
  • Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol
    Cytotherapy (IF 4.297) Pub Date : 2019-12-03
    Katherine N. MacDonald, Sabine Ivison, Keli L. Hippen, Romy E. Hoeppli, Michael Hall, Grace Zheng, I. Esme Dijke, Mohammed Al Aklabi, Darren H. Freed, Ivan Rebeyka, Sanjiv Gandhi, Lori J. West, James M. Piret, Bruce R. Blazar, Megan K. Levings
    更新日期:2019-12-03
  • Mesenchymal stem cells in the treatment of articular cartilage degeneration: new biological insights for an old-timer cell
    Cytotherapy (IF 4.297) Pub Date : 2019-11-26
    Alessandra Colombini, Carlotta Perucca Orfei, Dimitrios Kouroupis, Enrico Ragni, Paola De Luca, Marco ViganÒ, Diego Correa, Laura de Girolamo

    Osteoarthritis (OA) is a debilitating, degenerative joint disease characterized by progressive destruction of articular cartilage. Given the poor repair capacity of articular cartilage and the associated local destructive immune/inflammatory responses involving all joint structures, OA frequently ends up as a “whole joint failure” requiring prosthetic replacement. Current pharmacological efforts, belatedly started, mainly aim at symptomatic pain relief, underscoring the need for novel therapeutic schemes designed to modify the course of the disease. Mesenchymal stem cell (MSC)–based therapy has gained significant interest, sparking the design of multiple trials proving safety while providing promising preliminary efficacy results. MSCs possess ‘medicinal signaling cell’ properties related to their immunomodulatory and anti-inflammatory effects, which induce the establishment of a pro-regenerative microenvironment at the injured tissue. Those trophic effects are paralleled by the long-established chondroprogenitor capacity that can be harnessed to ex vivo fabricate engineered constructs to repair damaged articular cartilage. The present review focuses on these two aspects of the use of MSCs for articular cartilage damage, namely, cell therapy and tissue engineering, providing information on their use criteria, advancements, challenges and strategies to overcome them.

    更新日期:2019-11-27
  • Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease
    Cytotherapy (IF 4.297) Pub Date : 2019-11-26
    Lydia N. Raines, Matthew M. Hsieh, Tina Nassehi, Claire M. Drysdale, John F. Tisdale, Naoya Uchida

    Background aims Allogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. Methods In this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. Results In co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. Conclusions In summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

    更新日期:2019-11-27
  • Tumorigenicity assessment of cell therapy products: the need for global consensus and points to consider
    Cytotherapy (IF 4.297) Pub Date : 2019-11-09
    Y. Sato, H. Bando, M. Di Piazza, G. Gowing, C. Herberts, S. Jackman, G. Leoni, S. Libertini, T. MacLachlan, J.W. McBlane, L. Pereira Mouriès, M. Sharpe, W. Shingleton, B. Surmacz-Cordle, K. Yamamoto, J.W. van der Laan

    Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

    更新日期:2019-11-11
  • Small extracellular vesicle loading systems in cancer therapy: Current status and the way forward
    Cytotherapy (IF 4.297) Pub Date : 2019-11-05
    Yue-Feng Zhang, Jin-Bo Shi, Chao Li

    Systemic chemotherapy is a conventional and important strategy for inhibition of cancer progression, but it is usually accompanied by various adverse effects. Targeting drug delivery systems, effective tools to avoid the adverse effects of chemotherapy, have been intensively studied and developed. Recently, the emerging application of exosomes and exosome-mimics (small extracellular vesicles [sEVs]) in targeted drug delivery and therapeutics has been widely appreciated. The sEVs-based delivery system comprises three basic components: vesicles, cargoes and surface decorations. In this article, we review the current status, existing challenges and future directions in this field from the following aspects: selection and production of vesicles; cargoes and methods to load them into vesicles; modifications to the surfaces of vesicles; as well as ways to prolong the half-life of sEVs in the circulation. Existing and emerging data indicate that sEVs are promising nanocarriers for clinical use, but additional efforts are needed to translate research findings into therapeutic products.

    更新日期:2019-11-06
  • Minimal residual cancer.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    K Pantel,T J Moss

    更新日期:2019-11-01
  • Assays to determine hematopoietic stem cell content in blood or marrow grafts.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    C M Verfaillie,R Ploemacher,J Di Persio,R Sutherland,S Serke,H Johnsen,S Noga,R Negrin,

    更新日期:2019-11-01
  • Selection of CD34+ cells from cryopreserved PBPC can be significantly improved by the addition of recombinant human DNase (Pulmozyme).
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    M J Alcorn,L J Richmond,E Farrell,J Barr,C Pearson,R Schupp,I M Franklin

    BACKGROUND It has been reported previously that PBPC can be recovered from cryopreservation and can be efficiently CD34-selected, to provide a product of high purity (> 80% CD34) with good yield (> 50% recovery). METHODS In this study, we have investigated the effects of thawing and CD34-selecting cryopreserved PBPC in the presence of recombinant human deoxyribonuclease (rhDNase; Pulmozyme) and magnesium chloride (MgCl2 injection). RESULTS The addition of Pulmozyme and MgCl2 significantly improves the yield of CD34+ cells, compared with the standard procedure (65.2% and 39.7%, respectively). Following CD34 selection, significantly greater recovery of CFC in the selected fraction can be obtained from Pulmozyme-treated cells, compared with standard cells. The use of recombinant human Pulmozyme and i.v. grade MgCl2 should facilitate the application of this procedure to the clinical setting. CD34+ cells selected from cryopreserved PBPC, can in turn be cryopreserved for a second time. When thawed, these cells still retained good viability (> 80%). DISCUSSION Cells originally processed in the presence of Pulmozyme gave significantly superior yields of CD34+ cells and CFC compared with standard cells. The functional ability of these CD34+ cells was demonstrated further in an ex vivo expansion culture system with extensive proliferation of cells and CFC. In addition, the presence of significant numbers of primitive hemopoietic cells could be readily demonstrated in a cobblestone-area forming assay.

    更新日期:2019-11-01
  • Influence of T cell depletion method on circulating gammadelta T cell reconstitution and potential role in the graft-versus-leukemia effect.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    L S Lamb,A P Gee,L J Hazlett,P Musk,R S Parrish,T P O'Hanlon,S S Geier,R S Folk,W G Harris,K McPherson,C Lee,P J Henslee-Downey

    BACKGROUND Our laboratory previously reported that leukemia patients who developed > or = 10% gammadelta+ T cells during the first six months after receiving an anti-TCRalphabeta T-cell-depleted (TCD) graft from a partially mismatched related donor (PMRD) had a disease-free survival (DFS) advantage. These gammadelta+ T cells were V81+CD3+CD4-CD8-CD69+HLADR+ and are cytotoxic to K562 cells. METHODS In order to determine whether the anti-alphabeta TCD regimen was associated with these findings, we compared the reconstitution of gammadelta+ T cells from patients who received TCD PMRD grafts using the anti-TCRc4 MAb TIOB9-1A31 (previously reported) with similar patients who received grafts using the anti-CD3 MAb OKT3. RESULTS Increased cytotoxic Vdelta1+ T cells were seen in 10 of 43 T10B9 TCD patients compared to 7 of 100 in the OKT3 TCD group (23% versus 7%, p = 0.010). T10B9 patients with increased gammadelta+ T cells also exhibited a higher range of increased gammadelta+ T cells and the length of time the gammadelta+ T cells remained high was longer when compared to OKT3 patients. Patients with increased gammadelta+ T cells whose grafts were T-cell depleted with T10B9 showed a significant decrease in relapse (p = 0.038). Similar rates and reduction in relapse were seen in OKT3 TCD patients, although significance was not reached due to the small number of patients with increased gammadelta+ T cells. Estimated 3 year disease-free survival was significantly improved in T10B9 patients with increased gammadelta+ T cells (0.79 versus 0.31, p = 0.009), a trend also seen in OKT3 patients (p = 0.091). DISCUSSION These observations indicate that Vdelta1+CD4-CD8-cytotoxic T cells are associated with lower relapse rates and improved survival, and thus may have a role in a graft-versus-leukemia effect.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • FDA wrokshop summary: FDA-NCI workshop on tumor vaccines.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    D Przepiorka

    更新日期:2019-11-01
  • Quantitation of the proliferative potential of highly enriched human primitive hematopoietic progenitor cells using a stroma-free limiting dilution assay with automated scoring.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    J C Young,S Wu,M Travis,K M Luens,L Osborne,R Scollay,B Hill

    BACKGROUND Increasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation. METHODS We have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34+Thy-1+ were purified by fluorescence-activated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34+Thy-1+ cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serum-deprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing > or = 500 cells). RESULTS Progenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r = 0.80, r2 = 0.65, and r = 0.76, r2 = 0.58, respectively; these correlations were highly significant (p < 10(-7)). DISCUSSION This limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.

    更新日期:2019-11-01
  • The CD34+ cell concentration in peripheral blood predicts CD34+ cell yield in the leukapheresis product.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    K L Hollingsworth,T M Zimmerman,T Karrison,A Oliver,S F Williams

    BACKGROUND Measurement of the stem cells collected by leukapheresis has undergone marked improvement through the recent advent of CD34 analysis with flow cytometry. METHODS The relationship between CD34+ cell count in the peripheral blood (PB) and the leukapheresis product CD34+ cell yield was examined. One hundred patients with hematologic and non-hematologic malignancies underwent mobilization, with either growth factors combined with chemotherapy, or growth factors alone. Prior to each leukapheresis, PB was obtained for measuring the WBC, differential and percentage of CD34+ cells. The same tests were then performed for the corresponding leukapheresis products and the following correlations quantified: PB to product CD34+%, PB to product CD34+ cell concentration and PB CD34+ cell concentration, WBC and mononuclear cell (MNC) concentration to product CD34+ cell yield/kg. RESULTS The best predictor of product yield of CD34+ cells/kg x 10(6) was the PB CD34+ cell concentration with r = 0.93. The resulting regression formula (on log-log scale), log10 yield/kg = 1.52 + (0.99 x log10 PB CD34+ cell concentration x 10(6)/mL), predicts, with 50% probability, a minimally acceptable yield of 0.2 x 10(6) CD34+ cells/kg with a CD34+ cell concentration equal to 0.006 CD34+ cells x 10(6)/mL. A cell concentration of > or = 0.023 CD34+ cells/mL will ensure that a very high fraction (> 97%) of the patient population exceeds the minimally-acceptable yield. DISCUSSION The CD34+ cell concentration measured in the PB prior to leukapheresis is an excellent predictor of the yield of CD34+ cells generated in the PB stem cell product and should be used to signal the initiation of leukapheresis for post-mobilized patients.

    更新日期:2019-11-01
  • Improvement of breast cancer cell detection by immunomagnetic enrichment.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    W Krüger,F Tögel,S Rössing,N Kröger,A R Zander

    BACKGROUND Contaminating breast cancer cells in leukapheresis harvested for reinfusion to rebuild hemopoiesis after high-dose therapy have been described by several investigators. Methods for tumor cell detection are conventional immunocytochemistry, culture techniques and reverse transcriptase PCR. The percentage of tumor cell positive leukaphereses shows a wide variation. An approach to clarify if these cells can induce systemic relapse is to characterize them molecular-genetically and immunologically, but these techniques require a sufficient cell count. METHODS We compared conventional immunocytochemistry with immunocytochemistry after immunomagnetic enrichment of cancer cells by HEA-125 magnetic microbeads for the detection of micrometastatic tumor cells. A total of 25 samples, consisting of 16 samples from G-CSF-mobilized peripheral stem cell harvests, eight BM aspirations and one peripheral blood sample were investigated without [median 2, range 1-3 x 10(6) MNCs] and after [median 5, range 1-10 x 10(7) MNCs] HEA-microbead selection. Additionally 10 buffy coat samples from healthy subjects were investigated. RESULTS Using conventional immunocytochemistry, tumor cells could be detected in nine stem cell samples. Two BM samples and the blood sample (48%) were positive, with a median tumor cell load of 0 (0-12) cells per sample (mean: 2.4). By HEA-bead selection the rate of positivity could be increased to 88% (13 stem cell samples, eight marrow samples and one blood sample) with a median load of 6 (0-47) (mean 10.6) suspected cells (p < 0.007). However, calculation of recovery revealed tumor cell losses by immunobead selection. False positive results were not seen. DISCUSSION We conclude first that immunomagnetic selection is an excellent and highly sensitive tool to enrich contaminating cancer cells from marrow and stem cell samples; second that the existence of real tumor cell negative stem cell harvests is doubtful; and third that immunobead selection delivers sufficient tumor cell counts for their further characterization by molecular and immunological methods.

    更新日期:2019-11-01
  • Development of an infusible-grade solution for non-cryopreserved hematopoietic cell storage.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    S R Burger,A Hubel,J McCullough

    BACKGROUND Various tissue culture medium formulations currently are used to transport, store and process stem and progenitor cells for transplantation, although these solutions are not manufactured for clinical use. METHODS We have developed an infusible-grade solution, STM-Sav, for short-term liquid cell storage and tested its ability to maintain viable functional hematopoietic cells, compared with commonly-used tissue culture solutions. G-CSF-stimulated normal-donor PBPC were stored in alpha-MEM, IMDM, RPMI-1640, AIM 5, X-VIVO 10, PlasmaLyte A and STM-Sav, in gas-permeable Cryocyte bags, at ambient temperature and 4 degrees C. The percentage of viable cells, percent recovery of viable mononuclear cells (MNC), percentage of CD34+ cells, CFU-GM frequency and solution pH were determined for each solution, at time points ranging from 0 to 72 h. RESULTS Cells were slightly better maintained at 4 degrees C than at ambient temperature. No solution was superior to any other overall, although PlasmaLyte A was significantly inferior to all other solutions at preserving viable MNC. STM-Sav consistently maintained viable, functional cells as well as, or better than in-vitro-use-only culture media, or PlasmaLyte A. DISCUSSION Cells could be stored in STM-Sav for at least 24 h at 4 degrees C or ambient temperature, with recovery of approximately 90% of initial viable MNC, unchanged percentage CD34+ cells and stable solution pH.

    更新日期:2019-11-01
  • Bi-phasic CD34+ cell mobilization of a syngeneic donor during prolonged G-CSF delivery.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    S M Arnold,G Van Zant,G Phillips

    BACKGROUND G-CSF administration over 10 days and neutrophil cytapheresis have been reported in the literature, but the kinetics of CD34+ cells in this situation is unclear. CASE A 42-year-old female underwent syngeneic transplantation for metastatic breast cancer. The recipient was in critical condition peri-transplant, therefore the donor received G-CSF for 13 days, during which eight cytaphereses for both PBPC and neutrophils were performed. Two peaks in CD34+ cells were noted; the first on Day 5 and the second on Days 10-13 of G-CSF administration; a total of 11.6 x 10(6)/kg CD34+ cells and 38.11 x 10(8)/kg neutrophils were infused. The recipient's ANC exceeded 0.1 x 10(9)/L on Day +3. DISCUSSION To our knowledge, this is the longest reported cytapheresis of CD34+ cells from a normal donor The bi-phasic pattern in the cytapheresis product is also of interest. It is an unusual pattern that suggests a profound and complicated alteration in the marrow progenitor cell pool. If substantiated, this finding may offer an alternative cytapheresis schedule for donors.

    更新日期:2019-11-01
  • Debulking blood stem cell collections by density gradient centrifugation in a closed-vessel system.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    D Przepiorka,J G Lu,P Anderlini,M Körbling,M Donato,R Champlin,A P Gee,P van Vlasselaer

    BACKGROUND Blood stem cells collected by apheresis are largely mononuclear cells in nature, so manipulation of blood stem-cell components frequently requires more time and reagents than for a marrow harvest. Reducing the nucleated cell number in mobilized blood stem-cell collections, while preserving hematopoietic progenitor content, would make such manipulations simpler and less costly, but only if debulking procedures were not complex. METHODS We evaluated separation of light-density cells and enrichment of CD34+ cells from mobilized peripheral blood stem-cell collections by density gradient centrifugation over buoyant density solution 60 (BDS 60) in a single, closed vessel. RESULTS Fifteen apheresis products from five normal volunteers and eight cancer patients contained 3.44 (range, 1.19-5.51) x 10(10) nucleated cells. Following processing and washing, there was a median 29% recovery of nucleated cells, 79% recovery of CD34+ cells, 2.49-fold enrichment of CD34+ cells, 0.96-log depletion of CD3+ cells, 0.48-log depletion of CD56+ cells, and 0.72-log depletion of CD19+ cells. Results of processing were affected by the variability in composition of the apheresis products. The enrichment of CD34+ cells varied by donor type, and there was a logarithmic relationship between the preprocessing percentage of CD19+ cells and the log reduction in CD19+ cells. Recovery of cells after thawing and washing was acceptable for processed cells cryopreserved at concentrations over the range of 0.01-1.5 x 10(8)/mL. DISCUSSION These results demonstrate a simple method by which an apheresis product of 1-5 x 10(10) cells can be debulked effectively in a single, closed vessel.

    更新日期:2019-11-01
  • Nuclei-size distributions as predictive tools of hematopoietic cell proliferation.
    Cytotherapy (IF 4.297) Pub Date : 1999-01-01
    P C Collins,S D Patel,E T Papoutsakis,W M Miller

    BACKGROUND Current protocols for transplantation of hematopoietic stem and progenitor cells may be limited by donor-cell availability and the long time needed to restore neutrophil and platelet counts to normal levels. Ex vivo expansion of hematopoietic cells has the potential to decrease the required harvest size, and to enhance the transplant outcome, by providing greater numbers of progenitor and post-progenitor cells. However, widespread application of ex vivo expansion in the clinical setting is complicated by sample-to-sample variability in the extent and kinetics of cell expansion. For example, the lag time before active cell expansion may vary by several days and some samples may never expand under the culture conditions employed. An early determination regarding the fate of a culture would save time and resources, and would allow corrective action to be taken if desired. Furthermore, anticipation of the onset of cell cycling should prove useful in the development of culture-feeding strategies, as well as for maximizing transduction efficiency in gene-therapy protocols that employ retroviral vectors. METHODS We demonstrate that the nuclei-size distribution, which is obtained at the same time as the total nucleated cell concentration, can be used to predict the onset of cell proliferation. The formation of a second peak (with diameter > 4 microm) in the nuclei-size distribution, in addition to the smaller diameter peak (< 4 microm) present for quiescent cells, precedes total cell expansion. RESULTS In particular 94% of all MNC and CD34+ cell cultures that we have observed to exhibit a second peak in the nuclei-size distribution have realized total cell expansion. Furthermore, only one of 67 observed cultures that did not exhibit the formation of a second peak realized total cell expansion. The formation of a second peak in the nuclei-size distribution is evident, either before or on the same day as the presence of a significant fraction of cells in the S-phase of the cell cycle.

    更新日期:2019-11-01
  • Screening of genes responsible for differentiation of mouse mesenchymal stromal cells by DNA micro-array analysis of C3H10T1/2 and C3H10T1/2-derived cell lines.
    Cytotherapy (IF 4.297) Pub Date : 2007-03-17
    I Oh,M Ozaki,A Miyazato,K Sato,A Meguro,K Muroi,T Nagai,H Mano,K Ozawa

    BACKGROUND The molecular mechanisms underlying the biologic effects or differentiation of mesenchymal stromal cells (MSC) have not been clarified. Screening for genes differentially expressed at different stages is an important step in determining these molecular mechanisms. METHODS In this study, we analyzed the gene expression profiles of C3H10T1/2 (10T1/2) cells and two sublines, A54 (pre-adipocyte) and M1601 (myoblast), as a model of MSC and downstream committed progenitors. RESULTS We found up-regulated expression of delta-like-1 (Dlk), Wnt-5a and IL-1 receptor-like-1 (ST2) in 10T1/2 cells; stem cell factor (SCF) and stromal derived factor-1 (SDF-1) in A54 cells; and cardiac muscle-specific gene in M1601 cells. Overexpression of Dlk in A54 cells did not induce any effects on their differentiation into adipocytes. After differentiation into adipocytes, A54 cells reduced the expression of SCF, SDF-1 and Ang-1 as well as the ability to support the formation of a cobblestone appearance. DISCUSSION The results suggest that these three lines hae different gene profiles and are a useful system for analyzing the differentiation and function of MSC and progenitor cells.

    更新日期:2019-11-01
  • Comparison of cellular functionality of human mesenchymal stromal cells and PBMC.
    Cytotherapy (IF 4.297) Pub Date : 2007-03-17
    H Schmal,P Niemeyer,M Roesslein,D Hartl,T Loop,N P Südkamp,G B Stark,A T Mehlhorn

    BACKGROUND Human mesenchymal stromal cells (MSC) and PBMC play significant roles in repair processes following inflammation. Mechanisms of recruitment are still under investigation. METHODS AND RESULTS MIP-1alpha induced the chemotactic migration of MSC but not of PBMC. Correlating with this, 7.7% of MSC expressed the chemokine receptor CCR-1, as shown by FACS analysis. In contrast, PBMC did not express CCR-1 or CCR-2 but did express CXCR-4 (81.9%) and CCR-7 (42.2%). Setum induced the chemotaxis of both cell types, and zymosan activation increased the migration of PBMC but not of MSC. Corresponding with this, C5a induced the migration of PBMC but not of MSC. Dose-dependent and -specific adhesion to fibronectin, fibrinogen, collagen type I and collagen type II could be demonstrated for MSC; in contrast, PBMC did not adhere to any of the investigated proteins. Real-time PCR of receptor expression revealed a 12.2-fold higher expression of alphav in MSC compared with PBMC. Incubation of MSC with tumor necrosis factor-alpha (TNFalpha) induced NFkappaB activation and increased the chemotactic response to serum and adhesion to fibronedtin. DISCUSSION Chemotaxis and adhesion are crucial and differing cell fundtons of MSC and PBMC.

    更新日期:2019-11-01
  • Quality assessment of autografting by probability evaluation: model estimation by clinical end-points in newly diagnosed multiple myeloma patients.
    Cytotherapy (IF 4.297) Pub Date : 2006-04-28
    O Roer,J Hammerstrøm,S Lenhoff,A K Mylin,L M Knudsen,T Rasmussen,H E Johnsen,

    BACKGROUND Pre-transplant clinical evaluation of autografting is an important step in predicting post-transplant support, complications and safety. Today, unfavorable outcomes such as early death or graft failure are rare, making them unsuitable for quality assessment of supportive autografting. However, end-points constructed from frequently occurring clinical events may estimate clinically relevant prognostic models. METHODS The present retrospective analysis was based on two consecutive clinical trials in the Nordic area including up to 640 newly diagnosed multiple myeloma patients. RESULTS In the model, the efficacy (time on antibiotics and use of transfusions) was influenced by pre-transplant variables, including sex, nationality, serum creatinine, hemoglobin, disease stage at diagnosis, response following induction therapy, length of priming and average graft CD34+ cell number per day of harvest. The toxicity end-point (time to blood cell recovery) was influenced by nationality, marrow plasma cell percentage, serum creatinine, M-component isotype, response to induction therapy, length of priming and graft CD34+ cell number. The safety (early disease recurrence or death) was influenced by serum creatinine, hemoglobin, treatment response and CD34+ cell number. DISCUSSION In conclusion, the model illustrates that intervention strategies in quality assessment of autografting may benefit from probability estimates of graded clinical end-points.

    更新日期:2019-11-01
  • Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation.
    Cytotherapy (IF 4.297) Pub Date : 2005-07-29
    P J Amrolia,G Mucioli-Casadei,H Huls,H E Heslop,J Schindler,P Veys,E S Vitetta,M K Brenner

    Poor immune reconstitution after haplo-identical stem cell transplantation results in high mortality from viral infections and relapse. One approach to overcome this problem is to deplete alloreactive cells selectively by deleting T cells activated by recipient stimulators, using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient PBMC, and this can result in GvHD. We have shown that using recipient EBV-transformed LCL as stimulators to activate donor alloreactive T cells results in more consistent depletion of in vitro alloreactivity while preserving T-cell responses to viral and potential myeloid tumor Ag. Based on these data, we have embarked on a phase I clinical dose escalation study of add-back of allo-LCL-depleted donor T cells in the haplo-identical setting, to determine if the allodepletion we achieve to allow infusion of sufficient T cells to restore useful antiviral/anti-leukemic responses without causing GvHD. Fifteen patients have so far been treated. The incidence of significant acute or chronic GvHD has been low (2/15), as has mortality from infection (1/15). Preliminary data show accelerated immune reconstitution in dose level 2 patients. Infused allodepleted donor T cells appear able to expand significantly in the face of viral reactivations, and doses as low as 3 x 10(5)/kg may be sufficient to confer useful antiviral immunity in this setting. At a median follow-up of 19.5 months, nine of 15 patients are alive and disease-free. Five patients have relapsed, all of whom have died.

    更新日期:2019-11-01
  • Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging.
    Cytotherapy (IF 4.297) Pub Date : 2005-03-19
    J A Frank,S A Anderson,H Kalsih,E K Jordan,B K Lewis,G T Yocum,A S Arbab

    Superparamagnetic iron oxide (SPIO) nanoparticles are being used for intracellular magnetic labeling of stem cells and other cells in order to monitor cell trafficking by magnetic resonance imaging (MRI) as part of cellular-based repair, replacement and treatment strategies. This review focuses on the various methods for magnetic labeling of stem cells and other mammalian cells and on how to translate experimental results from bench to bedside.

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  • Clinical-grade manufacturing of DC from CD14+ precursors: experience from phase I clinical trials in CML and malignant melanoma.
    Cytotherapy (IF 4.297) Pub Date : 2005-03-19
    A B Dietz,D J Padley,G W Butler,M L Maas,C W Greiner,D A Gastineau,S Vuk-Pavlović

    BACKGROUND We evaluated a clinical-grade protocol for the manufacture of mature DC from CD14 + precursors derived from normal donors and patients suffering from CML and stage IV malignant melanoma. We manufactured six products for CML patients and five for melanoma patients and administered them as vaccines in phase I clinical trials. METHODS We isolated CD 14+ cells from apheresis products by immunomagnetic separation and incubated them in X-VIVO 15' medium supplemented with human AB serum, GM-CSF and IL-4 for 7 days, and with additional tumor necrosis factor (TNF)-a, IL-lIf, IL-6 and prostaglandin E2 for 3 days. Some cells were electroporated and transfected with mRNA isolated from melanoma tissue. DC were characterized by flow cytometry for the expression of CD83, CD86 andCD14. RESULTS CD14+ cells constituted 14.4+/-6.2% (mean + SD) of nucleated cells in apheresis products and 98.3+/- 3.6% of isolated cells. Normal DC and CML DC were 77.4+/-7.3% CD83+ and 93.5+/- 7.0% CD86+. Corresponding values for electroporated DC from melanoma patients were 66.1 + 7.2% and 94.1 + 7.8%. The yield of CD83+ DC from isolated CD14+ cells was 18.1 + 7.2% for normal and CML patients and 9.8 + 3.7% for melanoma patients. DC viability was 92.7 + 5.8%; after cryopreservation and thawing it was 77+/-13.5%. DISCUSSION Our method yielded viable and mature DC free of bacteria and mycoplasma. This robust and reproducible method provides cells of consistent phenotype and viability. Cryopreservation in single-dose aliquots allows multiple DC vaccine doses to be manufactured from a single apheresis product.

    更新日期:2019-11-01
  • Mesenchymal stem cell-based cartilage tissue engineering: cells, scaffold and biology.
    Cytotherapy (IF 4.297) Pub Date : 2005-03-19
    L Song,D Baksh,R S Tuan

    Cartilage repair and regeneration by stem cell-based tissue engineering could be of enormous therapeutic and economic potential benefit for an aging population. However, to use stem cells effectively, their natural environment must be understood in order to expand them in vitro without compromising their multilineage potential and their specific differentiation program. Collaboration between diverse academic disciplines and between research and regulatory government agencies and industry is crucial before cell-based cartilage tissue engineering can achieve its full therapeutic potential.

    更新日期:2019-11-01
  • Factors affecting engraftment of allogeneic hematopoietic stem cells after reduced-intensity conditioning.
    Cytotherapy (IF 4.297) Pub Date : 2005-03-19
    J Walshe,M R Bishop

    Several factors influence the engraftment of allogeneic hematopoietic stem cells (HSC). Recently, there has been increased utilization of transplant-conditioning regimens that use reduced doses of chemotherapy and radiation that are considered to be non-myeloablative. These non-myeloablative (or reduced-intensity) allogeneic HSC transplants (RIST) decrease early post-transplant complications, but they are associated with higher incidences of mixed chimerism and graft rejection compared with transplantation after myeloablative condition-ing. RIST provides a unique opportunity to study allogeneic HSC engraftment. In particular, host immune status and stem cell graft composition have emerged as important factors affecting engraftment after RIST Based on these observations, it has been hypothesized that conditioning regimens and allograft composition can be tailored to an individual patients immune and disease status prior to transplant.

    更新日期:2019-11-01
  • Isolation and characterization of mesenchymal progenitor cells from chorionic villi of human placenta.
    Cytotherapy (IF 4.297) Pub Date : 2005-03-18
    K Igura,X Zhang,K Takahashi,A Mitsuru,S Yamaguchi,T A Takashi

    BACKGROUND BM-derived mesenchymal stem cells (MSC) are attractive sources for autotransplantation with no risk of rejection, but the use of these cells bas problems, including the necessity of harvesting BM from donors, the donors' age-dependency, limitation to autologous use and difficulty of use for patients with hereditary diseases. We report a method of isolating placenta-derived mesenchymal progenitor cells (PDMPC) that can be used as an alternative source of MSC. METHODS We isolated PDMPC from human fetal chorionic villi using the explant culture method, from placentas collected after neonatal delivery (38-40 weeks of gestation). The PDMPC were characterized by morphologic and immunophenotypic analysis. The differentiation ability of mesenchymal and neural lineages was detected using specific culture conditions and determined by morphology, reverse transcription(RT)-PCR, histochemical staining and immunocytostaining. RESULTS The PDMPC all originated from fetal chorionic villi, as confirmed by fluorescence in situ hybridization analysis. The PDMPC population consisted of spindle-shaped cells and large flat cells. The PDMPCexpressed CD13, CD44, CD73, CD90, CDIO5 and HLA class I as surface epitopes, but not CD31, CD34, CD45 and HLA-DR. These cells differentiated into osteocytes, chondrocytes and adipocytes under specific culture conditions, and were also induced to form neural-like cells. DISCUSSION Our study shows that PDMPC can differentiate into mesenchymal lineages and be induced to form neural-like cells. Thus, PDMPCisolated from chorionic villi of placenta may provide a novel source for the research of stem and progenitor cells in placenta, cell therapy and regenerative medicine, particularly as a source of allogenic mesenchymal stem and progenitor cells with little ethical conflict and various advantages

    更新日期:2019-11-01
  • Collection of autologous PBSC in patients with polycythemia vera.
    Cytotherapy (IF 4.297) Pub Date : 2003-10-28
    L Isola,M Gorsky,V Najfeld,E Scigliano,Y Sinitsyna,S Fruchtman

    BACKGROUND Allogeneic stem-cell transplantation (SCT) can eradicate myelofibrosis (MF), but is limited by donor availability and toxicity. We previously reported normalization of counts and resolution of MF after ablative, syngeneic SCT in spent phase polycythemia vera (PV). Hence, GvL is not required to eradicate MF. Autologous SCT may advance treatment for spent phase PV by restoring effective hematopoiesis. The influence of organomegaly, myelosuppression and MF on PBSC collection has not been studied in the setting of PV. METHODS Sixteen patients with PV underwent PBSC collection. Mobilization was with filgrastim alone, with a target cell content of 2.5 x 10(6) CD34(+)/kg. All myelosuppression was discontinued 2 weeks prior to collection. RESULTS Median ages at diagnosis and collection were 47 and 57 years, respectively. Organomegaly, MF and use of myelosuppressive therapies were present in 10 (63%), 4 (25%) and 7 (44%) patients. Median total nucleated cells (TNC) and CD34(+) counts were 8.3 x 10(8)/kg and 4.98 x 10(8)/kg. MF had an adverse effect on TNC (p=0.05) but not on the CD34(+) content. Time from diagnosis and the use of myelosuppresion had no influence on TNC and CD34(+) contents. Four patients had CD34(+) contents <2.5 x 10(6)/kg. Complete blood count (CBC) parameters were not predictive of CD34(+) content. DISCUSSION Autologous PBSC collection is feasible in PV several years after diagnosis. Organomegaly and MF are not absolute contraindications for collection. Discontinuing myelosuppresion for 2 weeks before mobilization appears sufficient to collect adequate numbers of CD34(+) progenitors.

    更新日期:2019-11-01
  • A comparison of ex vivo expanded DCs derived from cord blood and mobilized adult peripheral blood plastic-adherent mononuclear cells: decreased alloreactivity of cord blood DCs.
    Cytotherapy (IF 4.297) Pub Date : 2003-10-28
    F Bracho,C van de Ven,E Areman,R M Hughes,V Davenport,M B Bradley,J-W Cai,M S Cairo

    BACKGROUND Cord blood (CB) has been used as an alternative source of transplantable allogeneic stem cells for a variety of malignant and non-malignant diseases. However, we have demonstrated delayed recovery of T- and B-cell function, and T-cell subsets post unrelated CB transplantation (UCBT), and deficiencies of CB mononuclear cells (MNC) in producing cytokines, including G-CSF, GM-CSF, M-CSF, IL-12, and IL-15. In this study we have investigated the ex vivo generation of DC from CB versus mobilized adult peripheral blood (APB) for later use as adoptive cellular immunotherapy. METHODS CB and APB-adherent MNC were cultured in serum-free media with GM-CSF IL-4, FLT-3 ligand, tumor growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) for 7 days. Morphology, phenotype, immunohistochemistry, clonogenic activity, and alloreactivity in MLR were evaluated. RESULTS CB and APB monocyte-derived ex vivo expanded DC expressed similar DC markers CD83 (31.27+ 11.7% versus 34.0+ 5.2%, CB versus APB), CD1a (23.4+ 4.2% versus 27.6+ 6.3%), and CD80 (21.97+ 12.01% versus 27.7+ 5.95). Immunohistochemistry showed that cells with DC morphology expressed CDla but not CD14. Neither FLT-3 ligand nor TGF-fl enhanced DC expansion. Addition of 10% autologous plasma to CB cultures promoted greater cell survival and a 150% increase in CDla + /CD80+ cell recovery. CB DC were 62% as effective stimulators of adult allogeneic T-cels as APB DC (p < .05) in allogeneic MLR. DISCUSSION While phenotypically similar, CB and APB DC have differential potency in allogeneic MLR, which may account for the difference in GvHD and infection incidence and severity between UCBT and allogeneic stem cell transplantation, and may require a different approach for adoptive cellular immunotherapy. The mechanism(s) associated with these differences require further elucidation.

    更新日期:2019-11-01
  • Endogenous microbial contamination of cultured autologous preparations in trials of cancer immunotherapy.
    Cytotherapy (IF 4.297) Pub Date : 2003-05-29
    D J Padley,C W Greiner,T L Heddlesten,M K Hopkins,M L Maas,D A Gastineau

    BACKGROUND Standardization of the manufacturing and processing of cellular immunotherapy products is necessary to ensure patient safety, establish efficacy, and demonstrate potency. Recognition of the autologous donor as a likely source of microbial contamination of cellular immunotherapy products may improve patient care and reduce expense to the laboratory. METHODS Data on 243 immunotherapy products manufactured in the authors' institution between December 1997 and June 2001 were retrospectively reviewed. Also reviewed were the case reports of four patients whose autologous immunotherapy products were contaminated. RESULTS Twenty-five (10%) of the 243 immunotherapy products processed were positive on one or more tests for microbial contamination. In six (24%) of the products, the source of microbial contamination was the autologous donor. In 17 of the remaining 19 products, test results were judged to be false-positive. DISCUSSION The unique processing techniques and stringent controls involved in the manufacture of cellular immunotherapy products may result in changes in the sources of microbial contamination routinely encountered. The identification of the autologous donor as a potential source of the microbial contamination of the product may assist the clinician and the laboratory in troubleshooting products with positive results on microbial sterility testing. Also, the number of false-positive results in this study indicates that further research is needed to maximize the specificity of testing while maintaining the present high sensitivity.

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  • New approaches to clinical harvest manipulation.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    J G Sharp

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  • Megadose and minitransplantation: what is the biology?
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    R Handgretinger

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  • Current methods and new developments in graft evaluation.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    S J Noga,R Sutherland

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  • Nonhematopoietic mesenchymal stem cells: what are they?
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    E M Horwitz,A Keating

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  • Blood and marrow transplantation activities among adult patients in Turkey.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05

    BACKGROUND Since 1978, 856 hematopoietic stem-cell transplantations have been performed for adult patients in Turkey. METHODS AND RESULTS Data were collected on the source of hematopoietic progenitor cells, recipient diagnosis, conditioning regimen, DFS and mortality. The data were analyzed by the national registry system of the Blood and Marrow Transplantation Subcommittee of the Turkish Society of Hematology. This analysis allowed the examination of national trends in survival, outcome and transplant-related complications in certain diseases. DISCUSSION In the allogeneic setting, the results of transplants for acute and chronic leukemia, with the exception of severe aplastic anemia, were similar to those found in the European and international registries. Likewise, in the autologous setting, the transplant results from Turkey paralleled those in European and international registries.

    更新日期:2019-11-01
  • Comparison of three methods of CD34+ cell enumeration in peripheral blood: dual-platform ISHAGE protocol versus single-platform, versus microvolume fluorimetry.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    P Chapple,H M Prince,D Wall,R Filshie,D Haylock,M Quinn,M Bretell,D Venter

    BACKGROUND Quantitation of peripheral blood (PB) CD34(+) cells is now an established method for timing PBPC harvesting. Recent refinements to the dual-platform ISHAGE gating strategy for CD34(+) cells has seen the introduction of microbeads to enable absolute counting of cells on a single instrument platform. This eliminates the need for total WBCC performed on an automated hematology analyzer and potentially increases the analytical precision of the methodology. At the same time, alternative methods for CD34(+) cell enumeration have started to emerge, notably microvolume fluorimetry, which forms the basis of the fully-automated STELLer CD34 method using the Imagn 2000. METHODS We performed a three-way evaluation of these methods. Sixty-eight samples of PB from 42 patients undergoing PBPC mobilization were analyzed by all three methods and correlations between all three calculated. The two-platform ISHAGE method was used as the reference method. RESULTS Precision and linearity of the single-platform and STELLer CD34 assays were excellent. Correlation with the dual-platform reference method was also excellent (single-platform method slope = 1.03, intercept = -0.03 and R(2) = 0.9325, STELLer CD34 assay slope = 0.827, intercept = 4.27, R(2) =0.8215). Bias, determined by Bland-Altman analysis, was 1.16 and -1.62 for single platform and STELLer CD34 assay respectively. CONCLUSION The three methods of CD34(+) cell enumeration gave equivalent results. The single-platform methodology negated the need for a separate white cell analyzer, while the STELLer CD34 methodology was technically the simplest.

    更新日期:2019-11-01
  • CD34(+) immunoselected cells for poor graft function following allogeneic BMT.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    M Mohty,C Faucher,C Chabannon,N Vey,A M Stoppa,P Ladaique,G Novakovitch,S Olivero,R Bouabdallah,J A Gastaut,D Maraninchi,D Blaise

    BACKGROUND Poor graft function without signs of graft rejection following allogeneic BMT (allo-BMT) occurs in around 9% of patients. A high incidence of hazardous complications may be encountered, leading to life-threatening situations. METHODS We describe three patients who underwent allo-BMT for acute leukemia in first complete remission and untreated myelodysplastic syndrome. The three patients experienced prolonged and profound granulocytopenia, anemia and thrombocytopenia, despite growth factors and transfusions. This was not corrected by donor leukocytes infusion. They received a boost of CD34(+) positively-selected cells from their HLA-identical sibling donors. RESULTS A rapid improvement of peripheral blood cell counts was observed in both patients who were in full donor chimerism status at time of boost infusion, whereas the patient with mixed chimerism did not show any signs of improvement. Neither patient suffered further exacerbation of GvHD. DISCUSSION Allogeneic positively-immunoselected CD34(+) cells can represent an interesting alternative treatment for poor graft function following allo-BMT, in the absence of graft rejection signs.

    更新日期:2019-11-01
  • Therapeutic uses of MAbs directed against CD20.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    J M Vose

    BACKGROUND There are two main classes of Abs directed against the CD20 Ag that have been developed for therapeutic intent: unconjugated and radio-labeled Abs. METHODS The clinical results available from the large clinical trials utilizing both the unconjugated and radiolabelled Abs are summarized in this article. DISCUSSION Both of these classes of agents have shown promise in clinical trials both alone and in conjunction with conventional chemotherapy or high-dose chemotherapy and transplantation. Ongoing research with these agents will provide further evidence of the place in clinical practice for these agents.

    更新日期:2019-11-01
  • In vitro and in vivo purging of B lymphoma cells from stem-cell products using anti-CD20 Abs.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    H G Derigs

    BACKGROUND Autologous stem-cell transplantation has proved curative therapy for relapsed NHL. However, recurrence of underlying disease remains the major cause of treatment failure in this setting. METHODS Development of effective MAb therapy directed against the B cell surface antigen CD20 has added a valuable tool of clearing contaminating lymphoma cells from stem-cell products by either in vitro or in vivo application. RESULTS Transplantation of successfully in vitro purged bone marrow using Mabs has been correlated with prolonged survival in large Phase-II study. So far, no randomized trial could demonstrate a therapeutic benefit for in vitro purging. The anti-CD20 Mab rituximab has been used for in vivo purging at the time of stem cell collection or peritransplantation. This method has been shown to be safe and feasible. In the majority of patients the combination of rituximab with anti-lymphoma chemotherapy meant the collected stem cell products were free of molecularly-detectable lymphoma cells. DISCUSSION The increasing ability to kill all lymphoma cells in vivo by regimens including myeloablative therapy renders contaminating lymphoma cells of the autologous stem cell product the main source for disease recurrence. Clearing of these cells remains a prerequisite for curative stem-cell transplantation. Establishment of safe and effective therapeutic schedules using Mabs will enhance the chance for collection of lymphoma-free hematopoietic stems cells.

    更新日期:2019-11-01
  • Umbilical cord blood processing with the Optipress II blood extractor.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    M I Godinho,M E de Sousa,A Carvalhais,I L Barbosa

    BACKGROUND We have previously shown that UC blood (UCB) units can be volume-reduced manually, in a closed system, without major losses of nucleated and CD34(+) cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the 'buffy-coat' collection in a standardized volume. METHODS After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34(+) cells, was set at 30 mL. The nucleated and CD34(+) cell content of the UCB collections and the resulting BC were evaluated. RESULTS The UCB units were grouped according to the volume collected: Group I < 80 mL and Group II > or = 80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34(+) cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%. DISCUSSION This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor-cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.

    更新日期:2019-11-01
  • Optimized retroviral transduction protocol for human progenitor cells utilizing fibronectin fragments.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    T Trarbach,S Greifenberg,W Bardenheuer,A Elmaagacli,H Hirche,M Flasshove,S Seeber,T Moritz

    BACKGROUND Retroviral transduction in the presence of fibronectin (FN) fragments has proven an efficient and clinically-applicable procedure for gene transfer into hematopoietic cells. So far, FN-based transduction protocols have been optimized primarily for transduction of stem cells, whereas for several therapeutic applications transduction of clonogenic progenitors (CFU) may be sufficient. METHODS Transduction protocols for CFU were optimized by evaluating the effect of growth factors, timing of retroviral transduction, CD34-selection and heparin, using a neomycin-phosphotransferase (neo(R))-expressing retroviral vector. RESULTS The presence of multiple growth factors during prestimulation and transduction, including the differentiating cytokines G-CSF or GM-CSF, substantially enhanced transduction of CFU. Best results were achieved when 24 h of prestimulation were followed by a 24-48 h transduction period in the presence of the CH-296 FN-fragment and IL-3, IL-11, SCF, erythropoietin (EPO), and GM-CSF. With this proto-col we observed highly efficient transduction of BM-derived CFU (90.7 +/- 8.8 % G 418-resistant colonies), even with retrovirus preparations of moderate infectious titer (5 x 10(4) - 2 x 10(5) CFU/mL). The number of CFU increased on average 2.6-fold (range 1.5-3.8) during the transduction procedure. Selection of CD34(+) cells prior to transduction did not improve transduction efficiency. Heparin, even in concentrations as low as 2.0 microg/mL, significantly inhibited transduction of CFU on FN-fragments. DISCUSSION An optimized protocol for retroviral gene transfer into human clonogenic progenitor cells that allows highly efficient transduction, even with moderate titer retroviral vectors, is presented.

    更新日期:2019-11-01
  • An analysis of the effect of chronic GvHD on relapse and survival following allogeneic PBSC transplantation.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    G Miflin,N H Russell,I Franklin,G Cook,D W Milligan,R M Hutchinson,M N Potter,G J Morgan,A Pagliuca,J Marsh,A Bell

    BACKGROUND PBSC are increasingly being used as the source of stem cells in allogeneic transplantation. An increased incidence of chronic GvHD has been suggested following unmanipulated allogeneic PBSC transplantation (PBSCT), however, how this affects overall survival is not yet clear. Our aim was to study the impact of chronic GvHD on survival and relapse following allogeneic PBSCT. METHODS We have analyzed data from 73 patients undergoing HLA-matched allogeneic PBSCT. GvHD prophylaxis was with CYA and MTX in 97% of patients. We have studied the incidence of chronic GvHD and its affect on relapse and survival in these patients. All patients were at least 100 days post-transplant at the time of analysis. RESULTS Seventy-three patients were evaluable for analysis of chronic GvHD. The overall incidence of chronic GvHD was 55% (limited in 18% and extensive in 37%). Overall median survival was 991 days, with a 4 year survival rate of 48%. Twelve patients relapsed. Patients with chronic GvHD had a significantly lower incidence of disease relapse (p = 0.005) with a relapse probability of 8% at 3 years, compared with 40% in patients with no chronic GvHD. In addition, the extent of chronic GvHD had a marked effect on survival, patients with limited chronic GvHD had a 4 year survival rate of 83%, compared with 45% in patients with extensive chronic GvHD and 38% in patients with no chronic GvHD. This difference was primarily due to the low incidence of relapse and low mortality seen in patients with limited chronic GvHD. DISCUSSION The presence and extent of chronic GvHD is an important predictor of outcome following allogeneic PBSCT, in that patients who developed either limited or extensive chronic GvHD had a low risk of disease relapse.

    更新日期:2019-11-01
  • Cell separation improves the sensitivity of detecting rare human normal and leukemic hematopoietic cells in vivo in NOD/SCID mice.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-05
    T L Holyoake,C Horrocks,T Thomas,C J Eaves,A C Eaves

    BACKGROUND This report describes a novel cell-separation procedure developed to improve detection and analysis of rare human hematopoietic populations, obtained from NOD/SCID mice engrafted with normal and/or leukemic stem cells. METHODS In preliminary experiments, artificial mixtures of murine and human BM cells were labeled with a combination of Abs specific for murine hematopoietic cells, prior to immunomagnetic negative selection using StemSep. In subsequent experiments, BM was harvested from individual NOD/SCID mice transplanted 6-12 weeks earlier with either human cord blood or primary CML cells and a similar immunomagnetic selection procedure was applied to enrich human cells present. RESULTS Application of this selection procedure to mixtures of murine and human hematopoietic cells using anti-mouse CD45 and Ter-119 allowed a > 1000-fold depletion of murine cells with > 50% recovery of human cells, including progenitors. This level of depletion and recovery were found to be reproducible for NOD/SCID mice transplanted and engrafted with human cord blood stem cells, thus facilitating detection of human progenitors, including colony-forming cells (CFC) and LTCIC. For NOD/SCID mice previously transplanted with CML cells, this procedure increased the sensitivity of detecting rare human cell subsets by up to > 100-fold. This, in turn, improved the sensitivity of RT-PCR for BCR-ABL and made possible the identification by FACS of various minor subsets of human cells, including CD34(-)CD19/20(+) B-lineage cells, CD34(+) progenitors, mature CD15(+) myeloid cells and CD3(+) T cells present in the mice. DISCUSSION This simple cell-depletion procedure should facilitate future investigations of normal and CML stem cell populations in vitro and in NOD/SCID mice.

    更新日期:2019-11-01
  • Process control: application to the cell processing laboratory.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-04
    C A Keever-Taylor

    更新日期:2019-11-01
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  • 更新日期:2019-11-01
  • A new United States Pharmacopeia (USP) Chapter 1046: cell and gene therapy products.
    Cytotherapy (IF 4.297) Pub Date : 2002-06-04
    S Seaver

    The United States Pharmacopeia (USP) has published the first draft of a new information chapter on cell and gene therapy products in January, 2000. The chapter discusses the manufacturing and testing cell and gene therapy products. It is intended for people working in the field as well as for pharmacists and clinicians who would like more information on this subject. This article outlines the background of USP and explains how it became involved with cell and gene therapy and what an information chapter is. It details the subjects covered by the chapter and the philosophy behind the chapter. This draft will be revised based on comments received from the public and then published as a revision subject to further comments before it becomes an official chapter in a supplement to USP 24-NF 19.

    更新日期:2019-11-01
Contents have been reproduced by permission of the publishers.
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