Stem cell transplantation for metastatic breast cancer: analysis of tumor contamination

The purpose of this study was to determine the efficacy, engraftment kinetics, effect of bone marrow tumor contamination, and safety of high-dose therapy and granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cell (PBPC) support for patients with responding metastatic breast cancer. Forty two patients underwent G-CSF (10 μg/kg) stimulated PBPC harvest. PBPC and bone marrow aspirates were analyzed by histologic and immunocytochemical methods for tumor contamination. Thirty-seven patients received high-dose therapy consisting of cyclophosphamide 6 g/m², thiotepa 500 mg/m², and carboplatin 800 mg/m² (CTCb) given as an infusion over 4 d followed by PBPC reinfusion and G-CSF (5 μg/kg) support. No transplant related deaths or grade 4 toxicity was recorded. CD34+ cells/kg infused was predictive of neutrophil and platelet recovery. With a median follow-up of 38 months, three year survival was 44% with relapse-free survival of 19%. Histological bone marrow involvement, found in 10 patients, was a negative prognostic factor and was associated with a median relapse-free survival of 3.5 months. Tumor contamination of PBPC by immunohistochemical staining was present in 22.5% of patients and found not to be correlated with decreased survival. G-CSF stimulated PBPC collection followed by a single course of high dose chemotherapy and stem cell infusion with G-CSF stimulated marrow recovery leads to rapid, reliable engraftment with low toxicity and promising outcome in women with responding metastatic breast cancer.     

单倍体相合造血干细胞移植联合脐带间充质干细胞输注治疗儿童重型再生障碍性贫血的临床观察及安全性分析

目的观察单倍体相合造血干细胞移植（hi-HSCT）联合脐带间充质干细胞（hUC-MSC）输注治疗儿童重型再生障碍性贫血（SAA）的临床效果及安全性。 方法整理海军总医院儿科2010年2月至2014年1月收治的11例接受hi-HSCT联合hUC-MSC输注治疗的SAA患儿的临床资料,进行回顾性分析。对其治疗情况、并发症发生情况及生存情况进行观察,总结该治疗方案的临床效果与安全性,以及治疗体会。 结果患儿全部获得造血重建,移植后1个月复查嵌合体均为70% ~ 100%供者嵌合。白细胞植入时间8 ~ 21 d,中位时间为12 d;血小板植入时间10 ~ 24 d,中位时间为15 d。11例患儿中,2例发生Ⅰ度急性移植物抗宿主病（GVHD）,1例发生Ⅲ度急性GVHD,均经相关治疗后好转;1例发生局限性慢性GVHD,经相关治疗后好转;2例发生广泛性慢性GVHD,发生率为18.18%。患儿移植期间均发生不同程度的恶心、呕吐、纳差和发热等症状,给予对症支持治疗后好转。8例（72.73%）发生口腔黏膜炎,2例（18.18%）发生肺部感染,9例（81.82%）发生病毒感染,2例（18.18%）发生腹泻,均经综合治疗后好转。11例患儿随访时间12 ~ 29个月,中位随访时间16个月,截止末次随访时1例因广泛性慢性GVHD接受持续治疗,2例接受免疫抑制剂减量治疗,其余4例均停用免疫抑制剂;1例患儿因家属自行停用环孢素A发生排异死亡。 结论hi-HSCT联合hUC-MSC输注治疗儿童SAA具有良好临床疗效,值得进一步关注。     

Feasibility and cost analysis of day 4 granulocyte colony-stimulating factor mobilized peripheral blood progenitor cell collection from HLA-matched sibling donors

BackgroundGuidelines recommend treatment with 4–5 days of granulocyte colony-stimulating factor (G-CSF) for optimal donor peripheral blood progenitor cell (PBPC) mobilization followed by day 5 collection. Given that some autologous transplant recipients achieve adequate collection by day 4 and the possibility that some allogeneic donors may maximally mobilize PBPC before day 5, a feasibility study was performed evaluating day 4 allogeneic PBPC collection.MethodsHLA-matched sibling donors underwent collection on day 4 of G-CSF for peripheral blood (PB) CD34⁺ counts ≥0.04 × 10⁶/mL, otherwise they underwent collection on day 5. Those with inadequate collected CD34⁺ cells/kg recipient weight underwent repeat collection over 2 days. Transplant and PBPC characteristics and cost analysis were compared with a historical cohort collected on day 5 per our prior institutional algorithm.ResultsOf the 101 patient/donor pairs, 50 (49.5%) had adequate PBPC collection on day 4, with a median PB CD34⁺ cell count of 0.06 × 10⁶/mL. Day 4 donors were more likely to develop bone pain and require analgesics. Median collected CD34⁺ count was significantly greater, whereas total nucleated, mononuclear and CD3⁺ cell counts were significantly lower, at time of transplant infusion for day 4 versus other collection cohorts. There were no significant differences in engraftment or graft-versus-host disease. Cost analysis revealed 6.7% direct cost savings for day 4 versus historical day 5 collection.DiscussionDay 4 PB CD34⁺ threshold of ≥0.04 × 10⁶/mL identified donors with high likelihood of adequate PBPC collection. Day 4 may be the optimal day of collection for healthy donors, without adverse effect on recipient transplant outcomes and with expected cost savings.     

Efficient removal of platelets from peripheral blood progenitor cell products using a novel micro-chip based acoustophoretic platform

Background

Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties.

Study Design and Methods

PBPC samples were obtained from patients (n = 15) and healthy donors (n = 6) and sorted on an acoustophoresis-chip. The acoustic force was set to separate leukocytes from platelets into a target fraction and a waste fraction, respectively. The PBPC samples, the target and the waste fractions were analysed for cell recovery, purity and functionality.

Results

The median separation efficiency of leukocytes to the target fraction was 98% whereas platelets were effectively depleted by 89%. PBPC samples and corresponding target fractions were similar in the percentage of CD34+ hematopoetic progenitor/stem cells as well as leukocyte/lymphocyte subset distributions. Median viability was 98%, 98% and 97% in the PBPC samples, the target and the waste fractions, respectively. Results from hematopoietic progenitor cell assays indicated a preserved colony-forming ability post-sorting. Evaluation of platelet activation by P-selectin (CD62P) expression revealed a significant increase of CD62P+ platelets in the target (19%) and waste fractions (20%), respectively, compared to the PBPC input samples (9%). However, activation was lower when compared to stored blood bank platelet concentrates (48%).

Conclusion

Acoustophoresis can be utilized to efficiently deplete PBPC samples of platelets, whilst preserving the target stem/progenitor cell and leukocyte cell populations, cell viability and progenitor cell colony-forming ability. Acoustophoresis is, thus, an interesting technology to improve current cell processing methods.     

Autologous transplantation: the viable transplanted CD34+ cell dose measured post-thaw does not predict engraftment kinetics better than the total CD34+ cell dose measured pre-freeze in patients that receive more than 2x10(6) CD34+ cells/kg

BACKGROUND: The aim of the study was to investigate whether the number of viable CD34+ cells in cryopreserved PBPC autografts is a better predictor of engraftment than the total CD34+ cell number determined before freezing. METHODS: A total of 119 patients was treated with autotransplantation for various malignant disorders during the period 1996-2002. All patients were reinfused with at least 2x10(6)/kg total CD34 cells analyzed before programmed freezing in 10% DMSO. The total CD34 cell number determined before freezing was compared with the number of viable cells determined after cryopreservation for 51 of these patients. The number of viable cells was determined by a flow cytometric analysis including triple staining with anti-CD34, anti-CD45 and the viability marker 7-actinomycin D (7-AAD). RESULTS: Simple linear regression analyses showed that both the total transplanted CD34 cell dose measured before freezing and the viable CD34 cell dose determined after cryopreservation were significantly correlated with neutrophil and platelet engraftment. In a multiple regression model the prediction of engraftment was not improved when the transplanted viable CD34 cell dose was included as a variable in addition to the total CD34 cell dose measured immediately after collection. DISCUSSION: Routine estimation of viable CD34 cells after cryopreservation of PBPC autografts is not necessary as long as the total CD34 cell dose is determined before freezing and the patients are reinfused with at least 2x10(6) cells/kg body weight.     

Hematopoietic stem cell transplantation with umbilical cord multipotent stromal cell infusion for the treatment of aplastic anemia—a single-center experience

Background aimsThe purpose of this study was to observe the outcome of co-transfusion of umbilical cord multipotent stromal cells (UC-MSC) and allogeneic hematopoietic stem cells in the treatment of heavily-transfused patients with severe aplastic anemia.MethodsOf the 22 patients, eight cases received haploidentical hematopoietic stem cells from granulocyte colony-stimulating factor–primed bone marrow and peripheral blood grafts; the other patients received granulocyte colony-stimulating factor–mobilized peripheral blood grafts from human leukocyte antigen–matched related (six cases) and unrelated donors (eight cases). MSCs were intravenously infused at a mean dose of 1.2 × 10⁶/ kg (range, 0.27–2.5 × 10⁶/kg). Fludarabine-based conditioning was conducted, and graft-versus-host disease prophylaxis containing cyclosporine A, methotrexate and mycophenolate mofetil with or without addition of anti-CD25 monoclonal antibody was performed. Hematopoietic engraftment, the occurrence of graft-versus-host disease (GVHD) and infections and overall survival were documented.ResultsAll patients had rapid engraftment; mean time for neutrophil and platelet recovery was 13.95 d and 20.27 d, respectively. No acute toxicity associated with UC-MSC transfusion was observed. Acute GVHD developed in seven cases (grade I–II), and none had development of chronic GVHD. Cytomegalovirus reactivation was observed in 11 cases. One patient died of pulmonary complication 6 months after transplantation. Twenty-one patients are currently alive, at a median follow-up of 15 months; they are transfusion-independent and reached full donor chimerism at the time of reporting.ConclusionsUC-MSC infusion might be an alternative option to promote hematopoietic engraftment and reduce the occurrence of GHVD in hematopoietic stem cell transplantation in the treatment of heavily transfused patients with severe aplastic anemia.     

Intravenous immune globulin use in patients with human immunodeficiency virus-related thrombocytopenia who require dental extraction.

Five patients with human immunodeficiency virus (HIV)-related immune thrombocytopenia who were undergoing dental extraction were treated with intravenous immune globulin (IVIG). All patients received IVIG, 1 gram per kg, the day before the dental extraction and again the day of the dental extraction. Four patients had a previous history of minor clinical bleeding. The median baseline platelet count before extraction was 20 X 10(9) per liter (range 13 to 44). The median peak platelet count was 100 X 10(9) per liter (range 56 to 528) following infusion. This peak response was achieved by day 2 in 3 patients and by days 5 and 7 in 1 patient each. No patients had complications or toxicity from the infusions or perioperative bleeding. No patients required blood product transfusions for the surgical procedure. In conclusion, IVIG infusion should be considered in patients with HIV-related immune thrombocytopenia requiring surgical procedures when a prompt rise in platelet count is desired.     

脐带间充质干细胞治疗儿童重型免疫性血小板减少症的探索性研究

目的探讨脐带间充质干细胞（UC-MSC）输注治疗儿童重型免疫性血小板减少症（s ITP）的疗效及安全性。方法采用UC-MSC治疗儿童s ITP 3例。发病年龄为3个月至4岁,初治时血小板计数为（1-7）×10^9/L,3例均为s ITP,均出现严重出血,激素及免疫抑制剂无效或依赖。后给予2-3次（1次/周）静脉输注非血缘UC-MSC,输注细胞量为（1-2）×10^6/kg。输注后密切监测血象及肝肾功能等各项指标,观察疗效及不良反应。结果随访时间15-45个月,3例在输注细胞后渐显效：第1例在输注细胞后20 d血小板达到65×10^9/L,随访4个月,血小板均维持在1×10^11/L以上;第2例在输注细胞后41 d血小板达105×10^9/L,之后血小板一直维持正常;第3例在输注第2次细胞后血小板渐上升至2×10^11/L以上。输注过程中1例出现面色发红,1例出现血压升高,随访至今无明显不良反应。结论 UC-MSC对儿童重型ITP有一定的疗效,能提高儿童的生活质量;有必要扩大病例数,进一步研究UC-MSC治疗儿童ITP的疗效及机制。     

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.     

Cryopreserving human peripheral blood progenitor cells

High-dose chemotherapy followed by autologous peripheral blood progenitor cell (PBPC) transplantation is used in the treatment of chemosensitive malignancies. Cryopreservation of PBPC in 10% dimethyl sulfoxide (DMSO) has been the standard procedure in most institutions. Infusion of PBPC cryopreserved with DMSO can be associated with toxic reactions such as vomiting, cardiac dysfunction, anaphylaxia and acute renal failure. The grade of toxicity experienced by patients is related to the amount of DMSO present in the PBPC. Cryopreservation with lower DMSO concentrations would be expected to reduce the toxicity. In recent studies done with PBPC cells cryopreserved with 5%, 4% and 2% DMSO, using 10% DMSO as a reference control, CD34+ cells were investigated for preservation of viability, apoptosis, and necrosis. Also preservation of mature colony-forming (CFU) cells, specifically mature myeloid, erythroid progenitors, CFU-megakaryocytes and long-term culture-initiating cells (LTC-ICs) were investigated, using 5% and 10% DMSO as cryoprotectant. All samples were frozen in a rate-controlled programmed freezer and stored in the vapor phase of liquid nitrogen until used. Conclusion: 5% DMSO is the optimal concentration for cryopreserving human PBPC in vitro. Consequently, some hospitals have started using 5% DMSO as cryoprotectant for the autologous PBPC as a standard procedure.     

脐血干细胞移植对儿童重组活化基因突变重症联合免疫缺陷病疗效分析

目的探讨脐血干细胞移植(UCBT)治疗儿童重组活化基因(RAG)突变重症联合免疫缺陷病(SCID)的疗效。方法回顾性分析2015年1月至2019年6月于复旦大学附属儿科医院明确诊断为RAG突变并接受UCBT的8例SCID患儿,其中男6例,女2例。所有患儿均接受白消安(BU)/环磷酰胺(CY)为主的减低毒性预处理方案,采用他克莫司单药预防移植物抗宿主病(GVHD),收集脐血干细胞移植前后的临床及实验室资料,评估患儿治疗效果。不符合正态分布的连续性变量资料用中位数(最小值~最大值)表示,分类资料用例数(相对比)表示。结果8例接受UCBT的RAG突变患儿分析结果显示,脐带血输注中位总有核细胞数为14.73(5.06~27.27)×10^7个/kg,中位CD34+细胞数为4.14(1.75~13.30)×10^5个/kg。8例患儿中7例成功植入,中性粒细胞植入时间25(15~45)d,血小板植入时间34(33~43)d。中位随访时间9.2(0.3~31.3)个月。3例患儿移植早期死亡,5例患儿无病生存并获得免疫重建,脱离静脉丙种球蛋白输注的时间为84(63~100)d。结论UCBT治疗RAG突变所致SCID取得一定疗效。     

The use of plerixafor in hematopoietic progenitor cell collection in pediatric patients: a single center experience

Background aimsPlerixafor was recently approved for use in combination with granulocyte–colony-stimulating factor (G-CSF) for hematopoietic progenitor cell (HPC) collection by apheresis in adults with multiple myeloma (MM) or non-Hodgkin lymphoma (NHL). However, its efficacy in pediatric patients is not well-studied; thus, we report on our institutional experience with this population. Methods. A retrospective observational analysis was performed using both stem cell-processing laboratory information as well as apheresis charts and medical records on all pediatric patients who received plerixafor as part of the mobilization regimen between December 2006 and December 2010. The primary outcome was collection yield. Secondary outcomes included the ability to undergo autologous hematopoietic stem cell transplantation (auto-HSCT) and engraftment status. Results. Eighteen HPC collections by apheresis representing seven mobilization courses were performed on five pediatric patients with poor mobilization status (three males, two females; median age 14 years). Median pre-harvest peripheral blood CD34⁺ cell (PB CD34⁺) count was 6.88/μL. A strong correlation between pre-harvest PB CD34⁺ count and collection yield was observed. Median total collection yield was 2.26 × 10⁶ CD34⁺ cells/kg. Four patients achieved a minimum collection of 2 × 10⁶ CD34⁺ cells/kg. Three patients underwent auto-HSCT with a median neutrophil and platelet engraftment of 12 and 34 days, respectively. No major adverse events with plerixafor administration or apheresis collections were reported. Conclusions. Plerixafor in combination with G-CSF is a safe and potentially helpful mobilization agent in poor mobilizers. Further studies should be done to evaluate the true efficacy of plerixafor in the pediatric population.     

Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers

This phase I clinical trial (NCT01935843) is to evaluate the safety, feasibility, and activity of chimeric antigen receptor-engineered T cell (CART) immunotherapy targeting human epidermal growth factor receptor 2 (HER2) in patients with advanced biliary tract cancers (BTCs) and pancreatic cancers (PCs). Eligible patients with HER2-positive (>50%) BTCs and PCs were enrolled in the trial. Well cultured CART-HER2 cells were infused following the conditioning treatment composed of nabpaclitaxel (100–200 mg/m²) and cyclophosphamide (15–35 mg/kg). CAR transgene copy number in the peripheral blood was serially measured to monitor the expansion and persistence of CART-HER2 cells in vivo. Eleven enrolled patients received 1 to 2-cycle CARTHER2 cell infusion (median CAR⁺ T cell 2.1 × 10⁶/kg). The conditioning treatment resulted in mild-to-moderate fatigue, nausea/vomiting, myalgia/arthralgia, and lymphopenia. Except one grade-3 acute febrile syndrome and one abnormal elevation of transaminase (>9 ULN), adverse events related to the infusion of CART-HER2 cells were mild-to-moderate. Post-infusion toxicities included one case of reversible severe upper gastrointestinal hemorrhage which occurred in a patient with gastric antrum invaded by metastasis 11 days after the CART-HER2 cell infusion, and 2 cases of grade 1–2 delayed fever, accompanied by the release of C-reactive protein and interleukin-6. All patients were evaluable for assessment of clinical response, among which 1 obtained a 4.5-months partial response and 5 achieved stable disease. The median progression free survival was 4.8 months (range, 1.5–8.3 months). Finally, data from this study demonstrated the safety and feasibility of CART-HER2 immunotherapy, and showed encouraging signals of clinical activity.     

CD34(+)-selected stem cell boost for delayed or insufficient engraftment after allogeneic stem cell transplantation

BACKGROUND: Poor graft function without rejection may occur after stem cell transplantation (SCT). CD34(+) stem cell boost (SCB) can restore marrow function but may induce or exacerbate GvHD. We therefore investigated the feasibility and efficacy of CD34(+)-selected SCB in some patients with poor graft function. We present the results for eight patients (median age 46 years) transplanted initially for myelofibrosis, acute leukemia, myeloma and NHL. Six patients had received HLA-matched and two mismatched grafts (PB, BM; n=5, 3). After a median of 128 days post-transplant, the median leukocyte and platelet counts were, respectively, 2.05/nL and 18/nL. None had achieved platelet counts >50/nL even though donor chimerism was >95% in seven recipients. METHODS: Positive selection of CD34(+) stem cells was performed on a CliniMACS device, observing GMP and achieving a median of 98.5% purity. The patients received a median of 1.7 x 10(6)/kg CD34(+) cells and 2.5 x 10(3)/kg CD3(+) T lymphocytes. RESULTS: Hemograms at days +30, +60 and +90, respectively, showed steadily increasing median leukocyte (2.55, 3.15 and 4.20/nL) and platelet (29, 39 and 95/nL) counts. After a median follow-up of 144 days, five patients remained alive. No patient had developed acute or chronic GvHD. One patient died of leukemic relapse and two others of systemic mycosis. DISCUSSION: These preliminary results point to the possibility of safely improving graft function using CD34(+) positively selected stem cells without necessarily increasing the incidence of GvHD in patients with poor graft function post-SCT. Experience with more patients and longer follow-up should clarify the optimal role for this procedure.     

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.     

Effects of imidapril, an angiotensin-converting enzyme inhibitor, on insulin sensitivity and responsiveness in streptozotocin-induced diabetic rats.

We have studied the effect of imidapril, an angiotensin-converting enzyme inhibitor, on streptozotocin-induced diabetic rats. A sequential euglycemic hyperinsulinemic clamp procedure was used (insulin infusion rates: 3 and 30 mU/kg BW/min) in 30 diabetic rats. The rats were divided in 6 groups: a control group, a control group with N-monomethyl-L-arginine (L-NMMA, 1 mg/kg/min, a nitric oxide synthase inhibitor) infusion, a streptozotocin-induced diabetic group, a diabetic group with L-NMMA infusion, a diabetic group involving imidapril infusion (5 microg/kg/min), and a diabetic group involving simultaneous imidapril and L-NMMA infusion. Glucose concentrations were maintained around 140 mg/dl during the clamp studies. Plasma insulin levels during the 3 and 30 mU/kg BW/min insulin infusions were 30 and 400 microU/ml, respectively. Glucose infusion rates (GIR) in STZ-induced diabetic rats showed a significant decrease compared to controls. At both insulin infusion rates, imidapril-infused diabetic rats showed an increased GIR, compared with the saline infused ones. There was no significant difference in GIR between L-NMMA and saline infusion in diabetic rats. Simultaneous infusion of imidapril and L-NMMA did not significantly decrease GIR with low-dose insulin infusion, but the increase in GIR induced by imidapril with high-dose insulin infusion was impaired by 100 % by L-NMMA infusion in diabetic rats. These results suggest that imidapril may improve insulin action, in part, via nitric oxide.     

Technique for repeated collection of cerebrospinal fluid from cisterna magna of anesthetized strain 13 guinea pigs

To study biochemical changes in cerebrospinal fluid (CSF), we developed a reliable technique for repeated collection of CSF in anesthetized strain 13 guinea pigs. The animal's head was mounted in a stereotaxic instrument with ventral tilt at 30 degrees, and cisternal puncture was made with an L-shaped, 23-gauge needle through the shaved skin. Clear CSF was collected in a 1-ml syringe surrounded by crushed ice. Each collection procedure lasted for 3 min, and three consecutive collections produced about 0.2 ml of CSF. Sampling was repeated at 3-hr intervals. With intravenous saline infusion (10 ml/kg.hr), a total volume of 0.6-1.0 ml of CSF was collected over 6 to 12 hr. Animals maintained a mean blood pressure, heart rate, and minute volume, with few changes during CSF sampling for the entire collection.     

Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactor

BACKGROUND: One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. METHODS: In comparison with a conventional bag transduction protocol, a 'closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34(+) progenitor cells (PBPC(MPS)) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. RESULTS: A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPC(MPS) over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8-14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPC(MPS)-LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). CONCLUSIONS: MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer.     

儿童获得性再生障碍性贫血造血干细胞移植后Epstein-Barr病毒血症危险因素及预后分析

目的分析儿童获得性再生障碍性贫血(AA)异基因造血干细胞移植(allo-HSCT)后Epstein-Barr病毒(EBV)血症的危险因素、利妥昔单抗的干预效果及EBV相关疾病的临床转归。方法回顾性分析2010年4月至2018年3月于上海儿童医学中心完成allo-HSCT的257例AA患儿,根据是否发生EBV血症分为:EBV血症组(141例,单纯EBV血症组125例和EBV相关疾病组16例)和非EBV血症组(116例)。采用Cox回归分析危险因素,采用Kaplan-Meier法分析累积生存率,定性资料的组间比较采用卡方检验。结果(1)257例患儿行allo-HSCT后发生EBV血症为141例,发生率为54.86﹪(141/257),其中原发感染为5.67﹪(8/257),再激活为94.33﹪(133/141)。EBV血症的中位发生时间为移植后44 d(13~568 d),单纯EBV血症的移植相关死亡率为6.40﹪(8/125);EBV相关疾病的病死率为56.25﹪(9/16)。(2)利妥昔单抗抢先治疗EBV血症的有效率为88.73﹪(63/71)。(3)与单纯EBV血症组比较,EBV相关疾病组患儿生存率降低,差异有统计学意义(Log-rank P<0.001)。(4)多因素分析结果显示,使用全身照射治疗(TBI)预处理的患儿,发生EBV感染的风险是不使用TBI预处理的患儿1.717倍(95﹪的CI:1.160~2.542);患儿移植物中CD34阳性细胞≥3×10^6个/kg是<3×10^6个/kg患儿的1.775倍(95﹪的CI:1.089~2.894)。结论移植后EBV血症的发生和TBI的应用、输注CD34阳性细胞数大于3×10^6个/kg密切相关,EBV血症进展为EBV疾病后移植相关死亡率升高,对高危患儿应积极予以利妥昔单抗抢先治疗改善预后。     

Cryopreservation of marrow, purging and autologous bone marrow transplantation in childhood

Since 1984 bone marrow from 42 children with acute lymphoblastic leukaemia, non Hodgkin's lymphoma and neuroblastoma was cryopreserved. In 5 cases (c-ALL, NHL and B type) the marrow was purged by using a cocktail of three monoclonal antibodies (VIL A1, VIB C5, VIB-E3). Up to now 13 children (ALL/10, neuroblastoma/3) were autografted (one of them after purging) after supralethal chemoradiotherapy. Except one child with early death all patients had engraftment: a level of 1.0.10(9)/l leukocytes was reached at days 10-33 (median, 19); platelet level over 60.10(9)/l at days 32-60 (median, 41). 2 children died on treatment related complications, one on infection after full haematological restitution, 2 patients alive with relapse, 8/13 alive in CCR and well.