TRANSPLANTATION AND CELLULAR ENGINEERING: Compensated variability in the expression of globin‐related genes in erythroblasts generated ex vivo from different donors

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced. STUDY DESIGN AND METHODS: Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four β⁰‐thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of α‐, β‐, and γ‐globin genes and of adult hemoglobin‐stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann‐Whitney rank sum tests. RESULTS: The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2‐log range. With maturation, the levels of γ‐globin and BCL11A mRNA did not decrease while those of α‐globin, γ + β‐globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two‐ to fourfold) observed between α‐ and γ + β‐globin mRNA was fully compensated by AHSP expression. Thus, the levels of α‐globin mRNA were correlated with those of γ + β‐globin (R² = 0.93, p < 0.0001) and AHSP (R² = 0.86, p < 0.0001). CONCLUSIONS: Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of α‐globin and γ + β‐globin fully compensated by AHSP expression, likely ensuring normal function and survival.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Increasing the economic efficacy of peripheral blood progenitor cell collections by monitoring peripheral blood CD34+ concentrations

BACKGROUND: After mobilization, the collection of peripheral blood progenitor cells (PBPCs) can either be started a fixed number of days after having passed the white blood cell nadir (fixed‐day scheme) or be based on monitoring of CD34+ cells. This study was conducted to compare both approaches and to assess possible financial consequences. STUDY DESIGN AND METHODS: For 29 patients daily enumeration of CD34+ cells was used to guide leukapheresis timing. In a retrospective analysis for the same group of patients, application of a fixed‐day scheme was assumed. For scenarios of beginning apheresis 2, 3, 4, or 5 days after WBC nadir, the number of apheresis days and granulocyte–colony‐stimulating factor (G‐CSF) application days that could be saved was calculated. RESULTS: A total of 44 apheresis procedures were performed resulting in a mean CD34+ cell content per apheresis product of 10.4 × 10⁶ (range, 0.1 × 10⁶‐49.5 × 10⁶)/kg of body weight. The smallest number of deviation days compared to a fixed‐day scheme was found for beginning an apheresis on Day 3. In comparison to this, CD34+ monitoring reduced the number of G‐CSF days by 9 and the number of apheresis procedures by 11 overall, resulting in savings of €19,965 (US$28,788) in comparison to expenses of €826 (US$1191) for CD34+ monitoring. CONCLUSIONS: Measurement of CD34+ cells has reached a precision enabling a prediction of the harvest success. In comparison to a fixed‐day scheme, daily CD34+ monitoring reduces the donor's exposition to G‐CSF, enables collection of a sufficient number of PBPCs in the least possible number of apheresis sessions, and improves the economic efficacy of the institution.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Prolonged isolated red blood cell transfusion requirement after allogeneic blood stem cell transplantation: identification of patients at risk

BACKGROUND: Delayed donor red blood cell chimerism (DRCC), pure red blood cell aplasia (PRCA), and autoimmune hemolytic anemia (AIHA) are poorly documented complications after hematopoietic cell transplantation (HCT). The clinical variable “prolonged isolated red blood cell transfusion requirement” (PRTR) was evaluated as a trigger for an extended diagnostic workup. STUDY DESIGN AND METHODS: PRTR was defined as the need for red blood cell (RBC) transfusions beyond Day 60 after HCT. We analyzed 487 patients transplanted between 2000 and 2006. Median age was 37 years (range, 0‐70 years). Peripheral blood stem cells (n = 344), marrow (n = 138), and cord blood (n = 5) from 278 unrelated and 209 family donors were used. RESULTS: Univariate analysis identified age (incidence of 18.3% among elderly patients, 10.5% in adults, and 2.0% among children [p = 0.002]), ABO incompatibility (16.4% after major incompatible, 2.9% after minor incompatible, and 9.4% after ABO‐compatible transplantations [p = 0.003]), conditioning (15.2% after reduced‐intensity regimens vs. 7.3% after myeloablative conditioning; p = 0.006), donor type (13.2% after HLA‐matched unrelated, 13.6% after mismatched unrelated, 5.7% after matched related, and 0.0% after mismatched related grafts; p = 0.026), and acute graft‐versus‐host disease (aGVHD; 7.1% with aGVHD vs. 12.5% without aGVHD; p = 0.046) as predisposing factors. In multivariate analysis minor ABO incompatibility (odds ratio [OR] = 0.2, p = 0.01), younger age (OR = 0.1, p = 0.02), and matched related HCT (OR = 0.4, p = 0.02) remained independent protective factors. CONCLUSIONS: PRTR could serve as a trigger for a standardized screening for DRCC, PRCA, and AIHA after HCT.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Adipose tissue mesenchymal stem cell expansion in animal serum‐free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL (PL 22.9 ± 1.5 hr vs. FBS 106.7 ± 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical‐scale applications mitigating the potential untoward side effects associated with the use of animal‐derived reagents.     

TRANSPLANTATION AND CELLULAR ENGINEERING: Long‐term storage does not alter functionality of in vitro generated human erythroblasts: implications for ex vivo generated erythroid transfusion products

BACKGROUND: Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage. STUDY DESIGN AND METHODS: Using mononuclear cells from four adult blood donors, erythroblasts were generated ex vivo in expansion cultures supplemented with stem cell factor, interleukin‐3, erythropoietin (EPO), dexamethasone, and estradiol. The viability and in vitro function of freshly expanded or short (1‐2 months)‐ and long (8 years)‐term‐stored erythroblasts cryopreserved in dimethyl sulfoxide were compared. Erythroblast function was defined as ability to proliferate in expansion media and mature in response to EPO. Cell number was determined manually and expressed as fold increase. Viability was assessed by trypan blue and propidium iodide exclusion. Maturation was evaluated by morphologic analyses and CD36/CD235a expression profiling. Cytogenetic evaluation included karyotype and multicolor fluorescence in situ hybridization analyses. RESULTS: Equivalent numbers (>80%) of erythroblasts were viable after short‐ and long‐term storage. Freshly expanded and short‐ and long‐term‐stored erythroblasts equally doubled in number (fold increase, 2.4) retaining an immature phenotype (23% of the cells were CD36^highCD235a^neg) when cultured for 4 days under expansion conditions. The numbers of freshly expanded and short‐term‐stored erythroblasts that matured when exposed for 4 days to EPO were also similar (approx. 22% of the cells became CD36^negCD235a^high). In spite of the massive amplification, ex vivo generated erythroblasts demonstrated a normal (46,XY) karyotype with no obvious genomic rearrangements. CONCLUSION: Ex vivo expanded erythroblasts remain functional and genetically normal after long‐term storage.     

剖宫产术后再次妊娠妇女分娩方式意愿及相关因素分析王悦,王滟,张铮,王霞

目的 调查剖宫产术后再次妊娠妇女的分娩方式意愿,分析影响其分娩决策的因素。方法 采用自制问卷对2018年8月~11月在上海市某三级甲等妇产专科医院进行产检的121例剖宫产后再次妊娠的妇女进行调查,采用单因素分析法分析影响分娩意愿决策的因素。结果 最终收集有效问卷110例,持阴道分娩意愿的有54例(49.1%);持剖宫产意愿的46例(41.8%);尚不确定本次分娩意愿的10例(9.1%)。单因素分析显示年龄、户口类型、文化程度、家庭经济水平、上一胎剖宫产原因、孕前有无慢性疾病,配偶及周围朋友对分娩方式的建议、本人分娩知识掌握情况对分娩方式选择具有影响,差异有统计学意义(P﹤0.05)。结论 剖宫产后再次妊娠妇女持剖宫产意愿的比率较高,分娩意愿除了受社会人口学资料的影响,还受孕妇分娩认知水平及社会支持因素的影响。强化孕期健康教育,积极调动社会支持系统,大力推广无痛分娩技术,提供专业分娩咨询和评估决策支持,有望降低剖宫产后再次妊娠妇女非医学指征剖宫产率。     

Evolution of the adaptogenic concept from traditional use to medical systems: Pharmacology of stress‐ and aging‐related diseases

Adaptogens comprise a category of herbal medicinal and nutritional products promoting adaptability, resilience, and survival of living organisms in stress. The aim of this review was to summarize the growing knowledge about common adaptogenic plants used in various traditional medical systems (TMS) and conventional medicine and to provide a modern rationale for their use in the treatment of stress‐induced and aging‐related disorders. Adaptogens have pharmacologically pleiotropic effects on the neuroendocrine‐immune system, which explain their traditional use for the treatment of a wide range of conditions. They exhibit a biphasic dose‐effect response: at low doses they function as mild stress‐mimetics, which activate the adaptive stress‐response signaling pathways to cope with severe stress. That is in line with their traditional use for preventing premature aging and to maintain good health and vitality. However, the potential of adaptogens remains poorly explored. Treatment of stress and aging‐related diseases require novel approaches. Some combinations of adaptogenic plants provide unique effects due to their synergistic interactions in organisms not obtainable by any ingredient independently. Further progress in this field needs to focus on discovering new combinations of adaptogens based on traditional medical concepts. Robust and rigorous approaches including network pharmacology and systems pharmacology could help in analyzing potential synergistic effects and, more broadly, future uses of adaptogens. In conclusion, the evolution of the adaptogenic concept has led back to basics of TMS and a new level of understanding of holistic approach. It provides a rationale for their use in stress‐induced and aging‐related diseases.     

Association of coagulation‐related and inflammation‐related genes and factor VIIc levels with stroke: the Cardiovascular Health Study

Summary. Background: Thrombosis and inflammation are critical in stroke etiology, but associations of coagulation and inflammation gene variants with stroke, and particularly factor VII levels, are inconclusive. Objectives: To test the associations between 736 single‐nucleotide polymorphisms (SNPs) between tagging haplotype patterns of 130 coagulation and inflammation genes, and stroke events, in the 5888 participants aged ≥ 65 years of the observational Cardiovascular Health Study cohort. Patients/Methods: With 16 years of follow‐up, age‐adjusted and sex‐adjusted Cox models were used to estimate associations of SNPs and FVIIc levels with future stroke. Results: Eight hundred and fifteen strokes occurred in 5255 genotyped participants without baseline stroke (748 ischemic strokes; 586 among whites). Among whites, six SNPs were associated with stroke, with a nominal P‐value of < 0.01: rs6046 and rs3093261 (F7); rs4918851 and rs3781387 (HABP2); and rs3138055 (NFKB1A) and rs4648004 (NFKB1). Two of these SNPs were associated with FVIIc levels (units of percentage activity): rs6046 (β = ? 18.5, P = 2.38 × 10^?83) and rs3093261 (β = 2.99, P = 3.93 × 10^?6). After adjustment for age, sex, race, and cardiovascular risk factors, the association of FVIIc quintiles (Q) with stroke were as follows (hazard ratio; 95% confidence interval): Q1, reference; Q2, 1.4, 1.1–1.9); Q3, 1.1, 0.8–1.5); Q4, 1.5, 1.1–2.0); and Q5, 1.6, 1.2–2.2). Associations between SNPs and stroke were independent of FVIIc levels. Conclusions:   Variations in FVII‐related genes and FVIIc levels were associated with risk of incident ischemic stroke in this elderly cohort, suggesting a potential causal role for FVII in stroke etiology.     

Enhancing effects of co-transduction of both human erythropoietin receptor and c-kit cDNAs into hematopoietic stem/progenitor cells from cord blood on proliferation and differentiation of erythroid progenitors

Steel factor (SLF) and erythropoietin (Epo) play critical roles in erythropoiesis. To evaluate interactive effects of Epo and SLF receptors (R) in erythropoiesis, CD34+ and CD34 cord blood cells were transduced with human EpoR and c-kit cDNAs by retroviral mediated gene transfer. Erythroid (BFU-E) colonies derived from CD34+ or CD34 cells transduced with either the EpoR or c-kit gene were significantly increased in the presence of interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), Epo, and different concentrations of SLF compared with that from mock transduced cells. This number was further enhanced by co-transduction of both genes. Enhancement was more apparent in the absence of SLF. Cell numbers in individual erythroid colonies were also significantly increased in cells transduced with both genes compared with cells transduced with a single gene. Short-term liquid culture showed that ex vivo expansion for five days and numbers of CD34+CD71+ cells in expanded cells from single CD34 cells co-transduced with both EpoR and c-kit genes were increased compared with those of EpoR or c-kit-transduced cells. These results demonstrate that co-transduction of both c-kit and EpoR enhances the proliferative capacity of erythroid progenitors under cytokine stimulation above that of single-gene transduced cells.     

Reduced hemoglobin on day of peripheral blood progenitor cell collection is associated with low graft content of vascular progenitors and increased toxicity after autologous hematopoietic stem cell transplantation

BACKGROUND: Tissue damage after hematopoietic stem cell transplantation (HSCT) occurs as a result of high‐dose chemotherapy and radiation. The aim was to determine the importance of pretransplant anemia on toxicity and red blood cell (RBC) transfusion requirements after autologous HSCT. STUDY DESIGN AND METHODS: A total of 350 patients undergoing autologous HSCT were included in the analysis. Patient factors and pretransplant laboratory values of possible relevance were assessed in multivariate regression analysis. RESULTS: Reduced hemoglobin (Hb) on the first day of peripheral blood progenitor cell (PBPC) collection was significantly associated with increased organ toxicity after HSCT, as measured by the Seattle criteria. Lower Hb levels at baseline before transplantation, but not at PBPC collection, were significantly associated with increased RBC transfusion requirements. In a second cohort of 28 patients, higher Hb levels on the day of PBPC collection were significantly associated with increased levels of endothelial‐like vascular progenitor cells in PBPC grafts. CONCLUSION: Our observations suggest that higher Hb levels on the day of PBPC collection may be a marker of reduced toxicity associated with HSCT and increased vascular progenitors in PBPC collections. Further, baseline anemia before transplant may reflect an unfavorable hematopoietic microenvironment that leads to increased RBC transfusion requirements.     

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum-free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells, 相似文献