Effects of bestatin on myelopoietic stem cells in normal and cyclophosphamide-treated mice

Summary The effect of bestatin on hematopoietic parameters and bone marrow progenitor activity (colony-forming unit — granulocyte/macrophage: CFU-GM) was examined in normal and myelosuppressed C₅₇BL/6 mice. CFU-GM frequency and absolute number were determined with a limiting dilution analysis of bone marrow cells in soft agar using recombinant murine colony-stimulating factor — granulocyte/macrophage. We report that bestatin increased splenic, bone marrow, and peripheral blood cellularity and the number of CFU-GM over a dose range from 2.5 mg/kg through 100 mg/kg following i.p., i.v., or oral administration. The greatest myeloid stimulation was observed following multiple injections of bestatin. Bestatin also increased the recovery from cyclophosphamide-induced myelodepression as measured by these parameters. The hematopoietic properties of bestatin following oral administration is of potential importance for clinical application.By acceptance of this article, the publisher or recipient acknowledges the right of the U. S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. This research was supported by the DHHS, under contract no. N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

A mathematical model for reconstitution of granulopoiesis after high dose chemotherapy with autologous stem cell transplantation

High dose chemotherapy supported with hematopoietic progenitor cells gives a characteristic neutropenic period (blood neutrophils <0.510⁹ c/l) ranging from 10 to 16 days. The question of a correlation between the CFU-GM content of the transplanted CD34+ cells and time to neutrophil recovery by patients having been given high-dose chemotherapy (HD-CT) with stem cell support was addressed by means of a mathematical model of granulopoiesis. The model utilizes a convection-reaction partial differential equation (PDE) with feedback from a cytokine compartment on proliferation, maturation, and mobilization of granulocytes from bone marrow to blood. The observed number of CFU-GM cells in the transplanted CD34+ cell autograft was used as input to the model. Using this approach, the observed gross relationship between CFU-GM content in the reinfused blood product and engraftment time could be reproduced. At the same time, the effects of assumed physiological mechanisms, especially some of the effects of G-CSF on proliferation rate, maturation rate, mobilization, and cell death, could be investigated and discussed relative to observed engraftment. The model makes it possible to explain how cytokines interfere with progenitor cell mobilization from bone marrow to blood, and it points out the implications of a regulating mechanism for the granulocyte maturation rate.     

Repopulating potential of hematopoietic precursor cells

Experiments were performed in 1800 cGy whole-body x-irradiated dogs. Mononuclear cells were collected from bone marrow, peripheral blood, and fetal liver. They were cryopreserved in -196 degrees C liquid nitrogen until used for transplantation. The thawed transfusates were adjusted to contain 1.5-1.6 x 10(5) CFU-GM per kg body weight. The blood granulocyte recovery was rapid after transfusion of blood-derived stem cells as compared to the use of bone-marrow-derived stem cells. In both instances, however, normal values were not reached for several weeks. In contrast, the use of fetal-liver-derived stem cells resulted in a very rapid initial granulocyte increase with a return of values to normal (or even overshoot) within 3 weeks after transplantation. A biomathematical granulocyte renewal simulation system is described that permits calculation of the absolute number of pluripotent stem cells in the transfusate. The data after fetal liver stem cell transplantation can be fitted only if an initial stem cell replication rate of 0.95 is assumed (in contrast to 0.65 using bone marrow or blood-derived stem cells).     

Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth.

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.     

Early bone marrow hematopoietic defect in simian/human immunodeficiency virus C2/1-infected macaques and relevance to advance of disease

To clarify hematological abnormalities following infection with human immunodeficiency virus (HIV), we examined the hematopoietic capability of bone marrow by using cynomolgus monkeys infected with pathogenic simian/human immunodeficiency virus (SHIV) strain C2/1, an animal model of HIV infection. The relationship between the progress of the infection and the CD4/CD8 ratio of T lymphocytes or the amount of SHIV C2/1 viral load in the peripheral blood was also investigated. A colony assay was performed to assess the hematopoietic capability of bone marrow stem cells during the early and advanced phases of the infection. Colonies of granulocytes-macrophages (GM) were examined by PCR for the presence of the SIVmac239 gag region to reveal direct viral infection. There was a remarkable decrease in the CFU-GM growth on days 1 and 3 postinoculation, followed by recovery on day 56. During the more advanced stage, the CFU-GM growth decreased again. There was minimal evidence of direct viral infection of pooled cultured CFU-GM despite the continuously low CD4/CD8 ratios. These results indicate that the decrease in colony formation by bone marrow stem cells is reversible and fluctuates with the advance of the disease. This decrease was not due to direct viral infection of CFU-GM. Our data may support the concept that, in the early phase, production of inhibitory factors or deficiency of a stimulatory cytokine is responsible for some of the bone marrow defects described in the SHIV C2/1 model.     

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

Summary The effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.     

Hematopoiesis in the rat: quantitation of hematopoietic progenitors and the response to iron deficiency anemia

To determine the quantitative effects of iron deficiency on erythropoiesis and to assess the response of erythroid progenitors to sustained anemia, we developed quantitative assays for various hematopoietic progenitors in the adult, Sprague-Dawley rat including erythroid colony- and burst-forming cells (CFU-E and BFU-E), granulocyte/macrophage colony-forming cells (CFU-GM), and megakaryocytic colony-forming cells (CFU-Meg). CFU-E were cultured in methylcellulose and grew best in the presence of fetal calf serum. CFU-GM, BFU-E, and CFU-Meg grew better in normal rat plasma and required the presence of pokeweed mitogen-stimulated rat spleen cell conditioned medium. The numbers of progenitors and nucleated erythroblasts in total marrow were estimated by the ratios of radioactivity in the humerus to the total skeleton as determined by radioiron dilution. The numbers of progenitors and erythroblasts in the spleen were measured by simple dilution. Sustained anemia was brought about through chronic iron deficiency. The response to iron deficiency anemia (IDA) was monitored by the numbers of the various progenitors and their cell cycle characteristics as measured by the tritiated thymidine suicide technique. With IDA, the number of CFU-F in the body (marrow plus spleen) was increased to 3.5 times control, whereas the numbers of BFU-E and CFU-GM were unchanged. There was no difference in the percentage of CFU-E, BFU-E, and CFU-GM in DNA synthesis (68%, 19.4%, and 18.8%, respectively). With iron therapy of IDA, CFU-E numbers in marrow began to decrease by day 1 and fell in a manner reciprocal to changes in the hematocrit. Marrow and spleen erythroblasts, 1.7 times control in IDA, increased further to 3.9 times control by the fourth day after iron administration. There was no change in BFU-E or CFU-GM numbers in response to iron repletion, although the fraction of progenitors increased in the spleen. Thus, IDA does not limit the increase in CFU-E seen with anemia, but does restrict erythroid maturation. Furthermore, the increase in CFU-E and the state of chronic anemia occur without detectable changes in the number of cell cycle state of the more primitive BFU-E.     

Inhibitor of granulopoiesis in human cyclic neutropenia.

Peripheral blood mononuclear cells of a patient with cyclic neutropenia release a high molecular weight substance (over 300,000) inhibiting normal CFU-GM cells to enter the S-phase of cell cycle. The inhibitor was released predominantly in the neutropenic phase of the disease, while in the period of normal granulocyte count the release was lower or undetectable. Also sensitivity of patient's bone marrow CFU-GM cells to similar high molecular weight inhibitor produced by ML-2 cell liner or to human placental ferritin varied within the disease cycle. CFU-GM in the normal granulocyte count period were sensitive to the inhibitors, but CFU-GM in the neutropenic phase were resistant.     

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34⁺ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34⁺ cells as well as peripheral blood CFU-GM was observed (P<0.01). Thus, the measurement of CD34⁺ cells is useful for predicting the number of circulating CFU-GM.     

Smad3基因剔除对小鼠造血功能的影响

研究Smad3基因剔除对小鼠造血功能的影响。实验小鼠分为 5组 ,每组有Smad3基因剔除小鼠(Smad3 - - )和其同窝孪生的野生型小鼠 (Smad3 + + )各 1只。小鼠的造血功能用 14天形成的脾结节 (CFU S1 4 )、多系祖细胞 (CFU GEMM)、粒 单系祖细胞 (CFU GM)、红系祖细胞 (BFU E)测定及外周血象、骨髓象等实验血液学指标来确定。每组小鼠取尾血作白细胞、红细胞和血小板计数 ,涂片作白细胞分类计数。将一侧股骨的骨髓冲出 ,制成单细胞悬液 ,计数其中有核细胞数 ,测定CFU GM、BFU E、CFU GEMM值。将每只小鼠的 4× 10 4个骨髓有核细胞 ,经尾静脉注入 3只 8～ 10周经致死量射线照射的同系雌性小鼠体内 ,测定 14天的CFU S。取一部分胸骨、肝脏、脾脏固定做病理切片 ,其余胸骨冲出骨髓 ,涂片作分类计数。结果Smad3 - - 小鼠外周血白细胞和血小板计数明显高于Smad3 + + 小鼠 ,红细胞数无显著差异。外周血白细胞分类结果也表明粒细胞显著增高。骨髓有核细胞数无显著差异 ,CFU GM显著增高 ,BFU E无显著差异 ,CFU GEMM明显减少 ,CFU S显著减少。病理形态学观察发现骨髓增生极度活跃 ,以粒系为主 ,肝脾无显著差别。骨髓涂片分类表明粒系增多 ,粒系 :红系比例增高。因此得出结论Smad3基因剔除使小鼠造血干祖细胞数目     

Pulmonary complications in patients receiving granulocyte transfusions and amphotericin B.

To evaluate the possibility that in febrile granulocytopenic patients amphotericin B given along with granulocyte transfusions could increase the incidence of pulmonary complications, we studied 43 severely granulocytopenic patients during 46 episodes of fever. Granulocytes were administered as part of the clinical protocol to all 19 patients who had clinically or microbiologically documented infection; the other 24 patients were randomly allocated to treatment with granulocytes (13 patients) or without granulocytes (11 patients). In all, 32 patients received granulocyte transfusions during 35 episodes of fever. Pulmonary complications developed in six patients in each of the two randomized groups. The incidence of pulmonary complications was not influenced by the number of granulocyte transfusions or by the number of granulocytes per transfusion. Pulmonary complications were significantly more likely to occur in patients with fungal infections. Amphotericin B was given according to clinical indications; 21 patients in all received it. Survival was significantly poorer in patients with pulmonary complications, but the administration of amphotericin B was not related either to survival or to the incidence of pulmonary complications. We conclude that pulmonary complications and poor prognosis are related to underlying pulmonary fungal infection and not to any interaction between amphotericin B and granulocyte transfusions.     

Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human "mobilized" peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma

Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel "grooved" perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocyte-macrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34% and 64% of input in stroma-containing and stroma-free perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications. (c) 1996 John Wiley & Sons, Inc.     

Maitake beta-glucan promotes recovery of leukocytes and myeloid cell function in peripheral blood from paclitaxel hematotoxicity

Bone marrow myelotoxicity is a major limitation of chemotherapy. While granulocyte colony stimulating factor (G-CSF) treatment is effective, alternative approaches to support hematopoietic recovery are sought. We previously found that a beta-glucan extract from maitake mushroom Grifola frondosa (MBG) enhanced colony forming unit-granulocyte monocyte (CFU-GM) activity of mouse bone marrow and human hematopoietic progenitor cells (HPC), stimulated G-CSF production and spared HPC from doxorubicin toxicity in vitro. This investigation assessed the effects of MBG on leukocyte recovery and granulocyte/monocyte function in vivo after dose intensive paclitaxel (Ptx) in a normal mouse. After a cumulative dose of Ptx (90–120 mg/kg) given to B6D2F1mice, daily oral MBG (4 or 6 mg/kg), intravenous G-CSF (80 µg/kg) or Ptx alone were compared for effects on the dynamics of leukocyte recovery in blood, CFU-GM activity in bone marrow and spleen, and granulocyte/monocyte production of reactive oxygen species (ROS). Leukocyte counts declined less in Ptx + MBG mice compared to Ptx-alone (p = 0.024) or Ptx + G-CSF treatment (p = 0.031). Lymphocyte levels were higher after Ptx + MBG but not Ptx + G-CSF treatment compared to Ptx alone (p < 0.01). MBG increased CFU-GM activity in bone marrow and spleen (p < 0.001, p = 0.002) 2 days after Ptx. After two additional days (Ptx post-day 4), MBG restored granulocyte/monocyte ROS response to normal levels compared to Ptx-alone and increased ROS response compared to Ptx-alone or Ptx + G-CSF (p < 0.01, both). The studies indicate that oral MBG promoted maturation of HPC to become functionally active myeloid cells and enhanced peripheral blood leukocyte recovery after chemotoxic bone marrow injury.     

阿德福韦酯与拉米夫定治疗慢性乙肝的疗效对比

目的:比较阿德福韦酯与拉米夫定治疗慢性乙肝的远期疗效。方法:回顾性分析2012年5月至2014年5月我院收治的80例慢性乙肝患者的临床资料,随机分为研究组和对照组,每组各40例。研究组患者给予阿德福韦酯治疗,对照组患者给予拉米夫定治疗。观察两组患者不同时段肝功能指标复常率、病毒量转阴率、乙肝E抗原(HBe Ag)转阴率、粒细胞降低率及乙肝病毒脱氧核糖核酸(HBV-DNA)抑制率,并比较两组患者不良反应发生率。结果:研究组患者肝功能指标复常率、血病毒量转阴率、Hbe Ag转阴率均显著高于对照组同期对应值,差异具有统计学意义(P0.05),但两组患者血粒细胞降低率无显著差异(P0.05);研究组患者HBV-DNA水平总抑制率高于对照组,差异有统计学意义(P0.05);两组患者均无严重不良反应发生(P0.05)。结论:阿德福韦酯治疗慢性乙肝的疗效更显著,能有效抑制HBV-DNA复制且安全性较高。     

GROWTH OF STEM CELL CONCENTRATES IN DIFFUSION CHAMBERS (DC)

The growth in diffusion chamber (DC) of normal murine marrow and marrow separated by discontinuous albumin centrifugation was studied. The colony-forming cells (CFC) assayed in soft agar, total cell counts and differentials were measured in the DC over a 19 day period after intraperitoneal implantation into CF₁ mice. Growth of implants of normal marrow or fraction 3 (F3) in which CFC had been concentrated 1.7–3.9-fold were compared at an initial cell concentration of 1 × 10⁵. There was a good correlation between the number of CFC implanted with granulocyte production but not with macrophage production. When higher cell concentrations of normal unfractionated marrow were implanted growth was reduced as was recovery of CFC. In two experiments in which both CFU and CFC concentrations were measured there was a general correlation between the two.     

The effect of vitamin D3 metabolites on normal and leukemic bone marrow cells in vitro

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.     

Effect of continuous, whole-body gamma irradiation upon canine lymphohematopoietic (CFU-GM, CFU-L) progenitors and a possible hematopoietic regulatory population

Clonogenic assays for granulocytes-macrophages (CFU-GM) in bone marrow and for T lymphocytes (CFU-L) in peripheral blood were performed on dogs continuously exposed to 60Co irradiation (0.02, 0.04, or 0.11 Gy/day). When decreased numbers of CFU-GM were observed they correlated well with the clinical status of the dogs but were not generally associated with increasing cumulative doses of absorbed irradiation. In clinically normal, irradiated animals, decreased CFU-GM values and myeloid-erythroid ratios were observed, suggesting that chronic irradiation may affect the granulocytic series well before decreased peripheral blood values are seen. In hypocellular dogs the number of CFU-GM were significantly decreased compared to values obtained from control or clinically normal irradiated dogs, while virtually no CFU-GM were observed in the leukemic dogs. Only the CFU-GM values of the hypocellular group showed an association, e.g., a suggestion of an abortive regenerative effort, with increasing absorbed dose. Proliferative capacity of T lymphocytes (CFU-L) was not affected by either increasing absorbed irradiation or the presence of leukemia. D0 values were determined on marrow fibroblastic cells to ascertain whether a radioresistant subpopulation of stromal elements would result from continuous in vivo irradiation. No correlation was found between absorbed dose and increased D0 values. However, seven of eight dogs which developed acute nonlymphocytic leukemia displayed marrow fibroblastic cells with elevated D0 values. These radioresistant marrow fibroblastic cells were assayed for their ability to support normal granulopoiesis and found to be not significantly different from control fibroblasts.     

Serum concentrations of complement anaphylatoxins and proinflammatory mediators in patients with 2009 H1N1 influenza

Anaphylatoxins (C5a, C4a, and C3a) are fragments of activated complement and are leading mediators of the inflammatory response for controlling viral infection. However, an excessive response may increase the severity of infectious diseases. Serum concentrations of proinflammatory mediators, including cytokines, high-mobility group box 1 and anaphylatoxins, were measured in pediatric 2009 H1N1 influenza patients in order to investigate the pathology of this new influenza. The concentrations of all three anaphylatoxins were significantly enhanced by 2009 H1N1 influenza infection. However, there were no significant differences in anaphylatoxin concentrations between 2009 H1N1 influenza patients with and without severe complications during the early stages of the disease. C3a concentrations dropped significantly during the recovery phase, whereas there were no significant differences between the acute and recovery phases in C5a and C4a concentrations. There was a correlation between C5a and IL-2. C4a was associated with IL-1ra, eotaxin, MCP-1, PDGFbb, and VEGF. C3a was correlated with IL-2 and IFN-γ. Taken together, these findings indicate that complement activation occurs in patients infected with 2009 H1N1 influenza virus and demonstrate that anaphylatoxins are involved in increased production of proinflammatory mediators in this new influenza.     

锂对小鼠高增殖潜能集落形成细胞和粒巨噬系祖细胞体外增殖…

本文观察了锂对ＢＡＬＢ／Ｃ小鼠骨髓高增殖潜能集落形成细胞和粒巨噬系祖细胞ＣＦＵ－ＧＭ体外增殖的影响。ＨＰＰ－ＣＦＣ集落由ＩＬ－１,ＩＬ－６,ＷＥＨＩ３条件培养液及Ｌ９２９条件培养液所支持,而ＣＦＵ－ＧＭ由ＷＥＨＩ３－ＣＭ所支持。结果显示,ＬｉＣｌ浓度在０．４－２ｍｍｏｌ／Ｌ时呈现剂量依赖性抑制ＨＰＰ－ＣＦＣ增殖;而在０．４－１ｍｍｏｌ／Ｌ的浓度范围内,则对ＣＦＵ－ＧＭ的增殖起剂量依赖性促进作用。     

A French single-center experience on allogeneic stem cell transplant cryopreservation during severe acute respiratory syndrome coronavirus 2 pandemic

Background aimsAllogeneic hematopoietic stem cell transplantation (allo-SCT) is a curative treatment for chemo-resistant hematological malignancies. Because of transport restriction imposed by the coronavirus disease 2019 pandemic, regulatory bodies and societies recommended graft cryopreservation before recipient conditioning. However, the freezing and thawing processes, including washing steps, might impair CD34+ cell recovery and viability, thereby impacting the recipient engraftment. Over 1 year (between March 2020 and May 2021), we aimed to analyze the results of frozen/thawed peripheral blood stem cell allografts in terms of stem cell quality and clinical outcomes.MethodsTransplant quality was evaluated by comparing total nucleated cells (TNCs), CD34+ cells and colony-forming unit–granulocyte/macrophage (CFU-GM)/kg numbers as well as TNC and CD34+ cell viabilities before and after thawing. Intrinsic biological parameters such as granulocyte, platelet and CD34+ cell concentrations were analyzed, as they might be responsible for a quality loss. The impact of the CD34+ cell richness of the graft on TNC and CD34 yields was evaluated by designing three groups of transplants based on their CD34 /kg value at collection: >8 × 10 ⁶/kg, between 6 and 8 × 10⁶/kg and <6 × 10⁶/kg. The consequences of cryopreservation were compared in the fresh and thawed group by evaluating the main transplant outcomes.ResultsOver 1 year, 76 recipients were included in the study; 57 patients received a thawed and 19 patients a fresh allo-SCT. None received allo-SCT from a severe acute respiratory syndrome coronavirus 2–positive donor. The freezing of 57 transplants led to the storage of 309 bags, for a mean storage time (between freezing and thawing) of 14 days. For the fresh transplant group, only 41 bags were stored for potential future donor lymphocyte infusions. Regarding the graft characteristics at collection, median number of cryopreserved TNC and CD34+ cells/kg were greater than those for fresh infusions. After thawing, median yields were 74.0%, 69.0% and 48.0% for TNC, CD34+ cells and CFU-GM, respectively. The median TNC dose/kg obtained after thawing was 5.8 × 10⁸, with a median viability of 76%. The median CD34+ cells/kg was 5 × 10⁶, with a median viability of 87%. In the fresh transplant group, the median TNC/kg was 5.9 × 10⁸/kg, and the median CD34+ cells/kg and CFU-GM/kg were 6 × 10⁶/kg and 276.5 × 10⁴/kg, respectively. Sixty-one percent of the thawed transplants were out of specifications regarding the CD34+ cells/ kg requested cell dose (6 × 10⁶/kg) and 85% of them would have had this dose if their hematopoietic stem cell transplant had been infused fresh. Regarding fresh grafts, 15.8% contained less than 6 × 10⁶ CD34+ cells /kg and came from peripheral blood stem cells that did not reach 6 × 10⁶ CD34+ cells /kg at collection. Regarding the factor that impaired CD34 and TNC yield after thawing, no significant impact of the granulocyte count, the platelet count or the CD34+ cells concentration/µL was observed. However, grafts containing more than 8 × 10 ⁶/kg at collection showed a significantly lower TNC and CD34 yield.ConclusionsTransplant outcomes (engraftment, graft-versus-host disease, infections, relapse or death) were not significantly different between the two groups.