A mathematical model of haemopoiesis as exemplified by CD34 cell mobilization into the peripheral blood

A mathematical model for the kinetics of haemopoietic cells, including CD34+cells, is proposed. This minimal model reflects the known kinetics of haemopoietic progenitor cells, including peripheral blood CD34+ cells, white blood cells and platelets, in the presence of granulocyte colony-stimulating factor. Reproducing known perturbations within this system, subjected to granulocyte colony-stimulating factor treatment and apheresis of peripheral blood progenitor cells (CD34+ cells) in healthy individuals allows validation of the model. Predictions are made with this model for reducing the length of time with neutropenia after high-dose chemotherapy. Results based on this model indicate that myelosuppressive treatment together with infusion of CD34+ peripheral blood progenitor cells favours a faster recovery of the haemopoietic system than with granulocyte colony-stimulating factor alone. Additionally, it predicts that infusion of white blood cells and platelets can relieve the symptoms of neutropenia and thrombocytopenia, respectively, without drastically hindering the haemopoietic recovery period after high dose chemotherapy.     

Sepsis, low platelet nadir at mobilization and previous IFN use predict stem cell mobilization failure in patients with multiple myeloma

BACKGROUND: Successful stem cell mobilization is a prerequisite for autologous blood cell transplantation. We analyzed factors that may predict the success of stem cell mobilization in patients with multiple myeloma (MM). METHODS: We analyzed 124 consecutive patients and compared those who failed to mobilize a sufficient amount of CD34(+) cells (peak blood CD34(+) cell count <20x10(6)/L) (n=20) with those with successful mobilization (n=104). The peak blood CD34(+) cell count after mobilization was used as the marker of mobilization success against which the various predictive factors were tested. RESULTS: In univariate analysis the best predictive factors for mobilization failure were the number of different chemotherapy regimens (P<0.001), number of chemotherapy cycles (P<0.001), time from diagnosis to mobilization (P<0.001) and previous use of IFN (P<0.001). The distributions of treatment responses at mobilization were similar in the groups with successful and unsuccessful mobilization, and were CR or VGPR in 10% of all patients, PR in 54% and stable or progressive disease in 36%. Regarding the mobilization-related factors, lower leukocyte nadir (P<0.001), longer duration of leukocyte counts <1x10(9)/L (P<0.001), lower platelet nadir (P=0.001), longer duration of platelet counts <20x10(9)/L (P<0.001) and the occurrence of sepsis after the mobilization therapy (P=0.001) were significantly associated with mobilization failure. In multivariate analysis, the amount of earlier chemotherapy cycles (P=0.002), low platelet nadir (P=0.020), occurrence of sepsis at mobilization (P=0.040) and previous use of IFN (P=0.052) remained as significant predictive factors for mobilization failure. DISCUSSION: Predicting the success of stem cell mobilization beforehand may have important practical consequences. By identifying those patients who will fail to mobilize stem cells, unnecessary mobilization and collection attempts can be avoided.     

自体CIK细胞联合化疗治疗老年急性白血病的临床疗效评估

目的:探讨自体细胞因子诱导杀伤(CIK)细胞联合化疗方案治疗老年急性白血病的临床疗效。方法:采集6例在我院进行化疗治疗的老年急性白血病患者的静脉血制备自体CIK细胞,成熟后回输入体内,持续注射10天为一个疗程。观察培养前和培养13天后白细胞各亚型细胞所占百分比,并比较CIK细胞回输前后患者白细胞亚群、感染次数及输血量的变化。结果:培养后CD3~+、CD8~+、CD3~+CD8~+、CD3~+CD56~+亚型细胞百分比均显著高于培养前(P0.05),CIK细胞回输后患者体内CD3~+、CD3~+CD8~+、CD3~+CD56~+亚群细胞百分比显著高于回输前(P0.05);CIK细胞回输后,患者感染次数和持续时间均显著少于回输前(P0.05),疾病稳定期输血量显著低于回输前(P0.05),疾病进展期输血量与回输前差异无显著性意义(P0.05)。结论:自体CIK细胞联合化疗治疗老年急性白血病患者有效且安全,可维持病情的稳定、提高机体细胞免疫功能和抗感染能力,值得临床推广。     

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+cells in the bone marrow. The clonogenicity as, well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38—) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+and CD34+CD38—populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+ CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.     

Increased DNA single-strand break joining activity in UV-irradiated CD34+ versus CD34- bone marrow cells

The kinetics of UV-irradiation-induced (254 nm) DNA single-strand breaks (SSBs) were studied in single human hematopoietic cells using alkaline comet assay. Three cell populations were investigated: (i) Bone marrow mononuclear cells (BMMNCs) isolated by density gradient centrifugation, (ii) CD34- cells, and (iii) CD34+ cells. The two latter populations were purified from BMMNCs by negative and positive selection, respectively, using anti-CD34 immunobeads. SSBs were induced faster by 10 and 50 J/m2 than by 2 J/m2 and those caused by 2 J/m2 were joined faster that those caused by 10 or 50 J/m2. During the first 1.5 h after irradiation with a dose of 10 J/m2, CD34+ cells joined SSBs faster than did BMMNCs. The superior joining capacity of CD34+ cells was further substantiated with a higher UV dose. The comet lengths, indicating the extent of DNA repair, among 8/8 study subjects were shorter in CD34+ than in CD34- cells when assessed 24 h after a dose of 50 J/m2. Overall, the comet lengths at 24 h after irradiation were: CD34+ cells; 39+/-12 *m, and CD34- cells; 65+/-18 *m (8 subjects, 50 cells measured from each donor, mean+/-S.D.; p=0.0087, Mann-Whitney U-test). These results strongly suggest that nucleotide excision repair, the major mechanism responsible for the repair of UV-irradiation-induced DNA lesions in mammalian cells, is increased in CD34+ cells compared with CD34- cells and with BMMNCs. These results may have implications in stem cell purging, clinical chemotherapy and carcinogenesis.     

Lymphocyte subpopulations in pulmonary tuberculosis patients

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4(+) and CD8(+) T-cell subsets. The aim of this study was to evaluate CD4(+) and CD8(+) T-cell profiles and CD19(+) and CD3(+)CD(16 + 56)(+) populations in patients with pulmonary tuberculosis. CD4(+) and CD8(+) T cells, B-lymphocytes, and natural killer (NK) cells were evaluated in 75 active (APTB) and 25 inactive (IPTB) pulmonary tuberculosis cases and 20 healthy subjects (HCs). The results were compared at different stages of antituberculosis treatment in the APTB patients and also according to X-ray findings in the newly diagnosed APTB patients. The percentages of CD4(+) T cells were significantly lower (P < .01) and those of CD3(+)CD(16+56)(+) cells were significantly higher (P < .01) in APTB patients than in HCs. CD8(+) T cells were significantly decreased (P < .05), and CD3(-)CD(16+56)(+) cells were significantly increased (P < .01), in IPTB patients compared to HCs. The percentages of CD4(+), CD8(+), CD3(-)CD19(+), and CD3(-)CD(16+56)(+) cells showed no differences at different times of the antituberculosis regimen, and different stages of newly diagnosed APTB patients. APTB patients have a reduced percentage of circulating CD4(+) T cells and an increased percentage of NK cells compared with healthy individuals. These cells could play important roles in the immune response to M tuberculosis infection.     

Lack of dendritic cell mobilization into the peripheral blood of cancer patients following standard- or high-dose chemotherapy plus granulocyte-colony stimulating factor

BACKGROUND: Dendritic cells (DC), the most specialized antigen-presenting cells, can be detected in the peripheral blood (PB) and divided into two subsets of populations, DC1 and DC2, endowed with different functions. The aim of this study was to evaluate the effect on DC release and on their subsets of three regimens utilized to mobilize CD34+ cells into the PB in cancer patients and in normal CD34+ cell donors. PATIENTS AND METHODS: The mobilizing sequences were: standard-dose epirubicin+taxol+granulocyte-colony-stimulating factor (G-CSF; 15 patients with advanced breast cancer), high-dose cyclophosphamide (CTX)+G-CSF (10 patients with breast cancer patients and 7 with non-Hodgkin's lymphoma, NHL), and G-CSF alone (5 normal donors of CD34+ cells for allogeneic transplantation). Comparative data were obtained from the steady-state PB of 20 healthy volunteers. For flow cytometric analysis, DC were gated as negative for specific lineage markers (CD3, CD11b, CD14, CD16, CD56, CD19, CD20, CD34) and positive for HLA-DR. The DC1 and DC2 subsets were defined as CD11c and CDw123 positive, respectively. RESULTS: The percentages of DC at baseline and the time of CD34+ cell peak were: 0.48 and 0.51 for standard-dose chemotherapy (CT); 0.55 and 0.63 for breast cancer after high-dose CTX+G-CSF; 0.53 and 0.71 for NHL after high-dose CTX+G-CSF; and 0.51 and 0.54 for normal donors of CD34+ cells after G-CSF alone (all p=n.s.).Mean DC1/DC2 ratios in each study group at the time of CD34+ cell peak were 0.10, 0.12, and 0.18, respectively. Finally, in the group of healthy volunteers, the percentage of circulating DC was 0.95 and the mean DC1/DC2 ratio was 1.28. CONCLUSION: To our knowledge, this is the first report that demonstrates that both standard-dose or high-dose CT, when utilized together with G-CSF, do not induce DC mobilization into the PB, whereas a reversed DC1/DC2 ratio is observed. Furthermore, a lack of significant DC mobilization after G-CSF alone was also seen, in contrast to what was previously observed by others. These data should be taken in account when evaluating clinical correlations between DC number and CPC engraftment in both the transplantation setting, when monitoring the effects on the immune system of combinations of new drugs and/or cytokines, and when high numbers of DC are required for both experimental and clinical applications.     

香菇多糖注射液对直肠癌根治术后患者纤维蛋白水平及免疫功能的影响

目的:研究香菇多糖注射液对直肠癌根治术后患者纤维蛋白水平及免疫功能的影响。方法:将2010年5月-2014年10月期间在我院接受直肠癌根治术的患者随机分为两组,观察组术后接受香菇多糖联合FOLFOX-4化疗,对照组术后接受FOLFOX-4化疗,观察两组患者化疗过程中的不良反应,测定化疗前后纤维蛋白水平及免疫功能指标。结果:观察组患者在化疗过程中消化道反应、肝功能损伤、骨髓抑制、周围神经毒性、口腔黏膜损伤、脱发的发生率均显著低于对照组(P0.05);化疗后,对照组患者外周血中CD3~+CD4~+CD8~-细胞、CD3~+CD4~-CD8~+细胞、CD19~+细胞、CD3~-CD56~+细胞的含量以及血清Ig M、Ig A、Ig G、FIB的含量显著低于化疗前(P0.05),观察组患者外周血中CD3~+CD4~+CD8~-细胞、CD3~+CD4~-CD8~+细胞、CD19~+细胞、CD3~-CD56~+细胞的含量以及血清Ig M、Ig A、Ig G的含量与化疗前无显著性差异(P0.05),血清FIB含量显著低于化疗前(P0.05);观察组患者化疗后外周血中CD3~+CD4~+CD8~-细胞、CD3~+CD4~-CD8~+细胞、CD19~+细胞、CD3~-CD56~+细胞的含量以及血清Ig M、Ig A、Ig G、FIB的含量显著高于对照组(P0.05),血清FIB含量显著低于对照组(P0.05)。结论:香菇多糖注射液辅助治疗能够减轻直肠癌根治术后FOLFOX-4化疗的毒副作用、改善免疫功能。     

Ex vivo expansion of neutrophil precursor cells from mobilized peripheral blood cells: similar results in cancer patients and normal donors

BACKGROUND: Infusion of ex vivo differentiated myeloid progenitors may reduce or abrogate severe neutropenia following mobilized peripheral blood transplantation. We compared the ex vivo expansion of myeloid progenitor cells starting from cancer patients (CP) and from normal donors (ND) and evaluated the influence of the CD34(+) cell mobilization on the capacities of cells to be expanded. METHODS: The ex vivo-expanded cells were evaluated for their phenotype, the presence of primary and secondary granules and their functional capacities (oxidative burst activity and phagocytosis). RESULTS: We did not observe significant differences between ND and CP for the total leukocyte and CD34(+) cell expansions nor for the myeloid progenitor production. In CP as well as in ND, the expanded cells were functionally competent. DISCUSSION: This suggests that the capacities of CD34(+) cells to proliferate and differentiate ex vivo are not impaired by prior chemotherapy and/or disease status. On the other hand, we did not observe any significant correlation between the number of mobilized CD34(+) cells before apheresis and the cell expansion. In conclusion, the ex vivo expansion of CP and ND cells is comparable and achievable even with a low CD34(+) cell number in mobilized peripheral blood.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

Distribution of peripheral blood leukocytes in acute heatstroke.

We examined 11 heatstroke patients (mean rectal temperature 41.4 +/- 0.3 degrees C) and 40 healthy subjects to determine the effects of hyperthermia on peripheral blood leukocyte distribution. Precooling samples were taken on admission. Whole blood was incubated with conjugated monoclonal antibodies, and erythrocytes were eliminated by FACS lysing solution. Lymphocyte subsets were detected by specific mouse monoclonal antibodies: Leu-4/CD3+ (T-cells), Leu-3a/CD4+ (T-helper cells), Leu-2a/CD8+ (T-suppressor-cytotoxic cells), Leu-11/19/CD16+/CD56+ (natural killer cells), and Leu-12/CD19+ (B-cells). Immunofluorescence was measured with a flow cytometer. The number of circulating leukocytes and lymphocytes was significantly increased in heatstroke patients. This lymphocytosis was mainly due to an increase in T-suppressor-cytotoxic cells and natural killer cells. The absolute number of lymphocytes and T-suppressor-cytotoxic cells significantly correlated with the degree of hyperthermia (r = 0.62, P = 0.04; r = 0.751, P = 0.007, respectively). There was a significant decrease in the percentages of T-, B-, and T-helper cells and increase in T-suppressor-cytotoxic and natural killer cells, giving a marked decrease in the ratio of T-helper to T-suppressor-cytotoxic cells. We conclude that heatstroke is associated with leukocytosis and significant alteration in absolute number and percentage of circulating lymphocyte subpopulations.     

CD34+ cells homing: quantitative expression of adhesion molecules and adhesion of CD34+ cells to endothelial cells exposed to shear stress

To investigate the function of the main adhesion receptors (CD62L, CD49d, CD49e, CD11b and CD18) on CD34+ cells during homing, their expression was quantified by flow cytometry using calibration beads. CD34+ cells were isolated from bone-marrow (BM), cord blood (CB) or peripheral blood (PB) from patients with myeloma. As this process might mimic the mature leukocyte migration, we also observed the effect of exposing endothelial cells to shear stress (7 dyn/cm(2)) on the adhesion of CB CD34+ cells.The proportion of CD34+/CD62L+ cells was greater in PB than in BM (p<0.05). Likewise, we found a significantly greater expression of CD62L receptor on PB cells compared to BM cells (p<0.05) and on BM cells compared to CB cells (p<0.05). The proportions of CD34+/CD49d+ cells and CD34+/CD49e+ cells were significantly higher in the BM and CB than in PB. However, no significant difference in CD49d or CD49e antigen densities was observed. The beta_2 integrins (CD11b and CD18) receptors are also implicated in CD34+ cells homing to BM. No significant variation in CD34+/CD11b+ and CD34+/CD18+ cells frequency was noted. However quantitative analysis revealed that CD18 was more strongly expressed on BM cells than on PB and CB cells.The adhesion assay showed that fluid flow may favour a firm adhesion of CB CD34+ cells to endothelial cells whereas static conditions just allowed CD34+ cells sedimentation.In conclusion, quantitative expression of the main receptors on CD34+ cells indicates that the three main sources of CD34+ cells currently used for transplantation have neither the same phenotype nor the same number of antigenic sites for a receptor. So, we hypothesize that migrational capacity of these cells might be different. Moreover, it seems that shear stress could favor adhesion of CD34+ cells to endothelial cells.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

Flow cytometric detection of circulating dendritic cells in healthy subjects

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.     

Maturation-dependent adhesion of human B cell precursors to the bone marrow microenvironment

Murine B cell precursors can be induced to proliferate in culture if allowed to bind to bone marrow derived adherent cells prepared under specific conditions. We studied the binding of human B cell precursor subpopulations to various in vitro microenvironments to determine which conditions may potentially be suitable models for human B precursor differentiation. Using the markers CD10, CD34, and CD20, B lineage populations of increasing maturation were quantitated: CD10+/CD34+, CD10+/CD20-, CD10+/CD20+, and CD10-/CD20+ cells in marrow, and CD10-/CD20+ mature B cells in peripheral blood. The adhesion of subpopulations of blood and marrow-derived light density cells to adherent cell layers or matrix was studied following a 2-h incubation in 24-well plates. The absolute number of bound B lineage cells was determined by cell counts and flow cytometry analysis. The adherence of B lineage cells to passaged human marrow fibroblasts (BM-FB) was highest in the most immature CD10+/CD34+ cells (34.3 +/- 4.2%), decreasing steadily with each stage of maturation to the peripheral blood B cells (11.2 +/- 2.4%). Increased adhesion of CD10+ B cell precursors relative to CD10-/CD20+ marrow B cells was confirmed by adhesion studies using sorted cells. The two most immature B lineage cells (CD10+CD34+ and CD10+/CD20-) showed more adherence to BM-FB than any other cell type tested, except for monocytes. Only B lineage precursor cells, erythroid precursors and CD10-/CD34+ cells showed significantly greater binding to BM-FB than to plastic. B lineage precursors bound equally well to primary and passaged human marrow fibroblasts, but bound significantly less well to passaged human foreskin fibroblasts, primary human marrow stroma, extracellular matrix of marrow fibroblasts, or fibronectin. These results suggest that specific binding to marrow fibroblasts is part of the differentiation program of early B lineage precursors. This binding activity gradually and predictably decreases during B lineage differentiation, in contrast to expression of other binding receptors, such as LFA-1 and CD44, which increase during B lineage maturation.     

Immunophenotyping of hematopoietic progenitor cells: Comparison between cord blood and adult mobilized blood grafts

AIM:To study the immunophenotype of hematopoietic progenitor cells from cord blood (CB) grafts (n = 39) in comparison with adult apheresis grafts (AG, n = 229) and pre-apheresis peripheral blood (PAPB) samples (n = 908) using flow cytometry analysis.METHODS: First, we performed a qualitative analysis of CD34+ cell sub-populations in both CB and PAPB grafts using the standardized ISHAGE protocol and a wide panel of 20 monoclonal antibodies. Next, we stud-ied some parameters, such as the age of mothers and the weight of newborns, which can influence the qual-ity and the quantity of CD34+ cells from CB. RESULTS: We found that the percentage of apoptotic cells was high in CB in comparison to PAPB (PAPB: 4.6% ± 2.6% vs CB: 53.4% ± 5.2%, P < 0.001). In CB, the weight of newborn and the age of the mother have the influence on CD34+ cells. The follow-up of Ag CD133in the ISHAGE double platform protocol in association with CD45, CD34 and the 7’AAD shows an equal rate between the two cell populations CD133+CD45+CD34+ high and CD34+CD45+ high with a higher percentage. So, is the inclusion of Ac CD133 necessary in the pres-ent panel included in the ISHAGE methodflLast part, we showed a signif icant presence of interferon γ in CB in comparison to PAPB, the annexin showing the high number of apoptotic cells in CB. CONCLUSION: This study demonstrates that many different obstetric factors must be taken into account when processing and cryo-banking umbilical CB units for transplantation.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

Immune recovery after allogeneic stem cell transplantation: study of 19 patients

The aim of this study was to access average delays for novogeneration of myeloid and lymphoid cells after allogeneic bone marrow transplantation (BMT) outcome and factors affecting this organization. A prospective analysis over 2 years (01/01/07 to 31/12/08) enrolling 19 children treated with allogeneic intrafamilial bone marrow transplantation. Indications for bone marrow transplantation were: aplastic anemia (3 cases), bemoglobinopathies (9 cases), myelodysplastic syndrome (1 case) and primary immunodeficiency (6 cases). Different conditioning regiments were used according to the indication. The study of immune reconstitution was based on the quantitative determination of immunoglobulin and lymphocyte subpopulation. These tests were routinely requested to 1 month, 2 months, 3 months, 6 months, 9 months and 12 months. The average time of engraftment was 18 days (12-24). A rate of CD4+T lymphocytes>200/mm3 was provided within an average of 2,5 months (1-7). The average time to obtain CD8+T lymphocytes>200/mm3 was 2 months (1-5). The humoral immune reconstitution was made within an average of 2 months (1-4). A report of CD4+/CD8+T lymphocytes>I was obtained within 10 months and a half (1-24). Univaried analysis showed a correlation between the bone marrow sex matched and the faster reorganization of CD8+T cells (p=0.042). A quantity of CD34+>6 10(6)/kg was significantly associated with the recapture of a formula lymphocyte CD4+/CD8+T>1 (p=0.03) Immune recovery post bone marrow transplantation in children begins with myeloid lineage then lymphoid B then lymphoid T The inversion of the report CD4+/CD8+T lymphocytes, seems to be influenced by the high contain of CD34+cells in the graft as well as the type of conditioning.     

Macrophage-inflammatory protein-1alpha receptor expression on normal and chronic myeloid leukemia CD34+ cells

We have assessed expression of MIP-1alpha binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 +/- 6.2% in NBM and 9 +/- 4% in CML), which has specific macrophage-inflammatory protein-1alpha (MIP-1alpha) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1alpha, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1alpha-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1alpha receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy- progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1alpha are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.     

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O*2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34+/CD38-, CD34+/CD38+ and CD34-/CD38+ cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O*2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34+/CD38- cell population, where the level of O*2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34+/CD38- cell population, and this intracellular pH decreased as early as 4 h post-irradiation, virtually simultaneous with the significant elevation of O*2- generation. These results suggest that the CD34+/CD38- stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O*2-, compare to more differentiated CD34+/CD38+ and CD34-/CD38+ cells and that its intracellular pH declines at an early phase in the apoptosis process.