Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures

There is a growing consensus that clinical practice in the areas of bone marrow (BM) transplantation and gene therapy will rely on the ex vivo expansion of hematopoietic cells. We report here on the development of continuously perfused culture systems (bioreactor systems) that expand human stem and progenitor cells from BM mononuclear cell (MNC) populations obtained without cell enrichment. In three separate experiments, 10 bioreactors were each inoculated with 3 x 10(7) BM MNC from patients undergoing marrow harvest for autologous transplantation. At various times thereafter (between days 6 and 16), duplicate bioreactors were harvested and cells were analyzed. The bioreactors contained three cell populations that were analyzed separately: nonadherent cells; cells that were loosely adherent to the endogenously formed stromal layer; and an adherent cell layer that required trypsinization for removal. Total cell numbers increased continuously to give an overall 10-fold (range, 8- to 11-fold) expansion by day 14. The adherent stromal layer significantly expanded to more than 2 x 10(7) cells, but remained less than 6% of the total cell population. Colony-forming unit-granulocyte-macrophage (CFU-GM) numbers expanded 21-fold (range, 12- to 34-fold) by day 14 and, because this expansion was greater than that for total cells, CFU-GM were enriched by as much as fourfold by day 14. Burst-forming unit-erythroid (BFU-E) numbers peaked earlier than did CFU-GM numbers, with a 12-fold (range, 6- to 18-fold) expansion obtained on day 8. In contrast to CFU- GM, which were predominantly nonadherent, BFU-E were more evenly distributed between the three cell populations. Stem cell activity was measured by the long-term culture-initiating cell (LTC-IC) limiting dilution assay. The number of LTC-IC per reactor consistently increased with time in all cultures, resulting in a 7.5-fold (range, 3.4- to 9.8- fold) expansion. In summary, more than 3 billion cells, containing 12 million CFU-GM, were reproducibly generated from the equivalent of a 10 to 15 ml BM aspirate. These data indicate that small numbers of BM MNC can be readily expanded ex vivo in continuous perfusion cultures, and that such ex vivo expansion may have direct applications in clinical and experimental BM transplantation.     

高+Gx对猴肝细胞c-fos表达的影响

目的:观察航天应急返回过程中高正加速度( Gx)对肝脏细胞c-fos基因表达的影响．方法:选用♂猕猴(共9只)为对象,随机分为4组,对照组承受 1Gx,300 s的超重作用;实验组根据承受过载峰值的大小分为3个亚组,其承受过载峰值分别为 15Gx,200 s; 18Gx, 165 s; 21Gx,140 s．观察高 Gx对猴肝脏细胞c-fos基因表达的影响．结果:实验组肝脏细胞胞质呈现不同程度的水肿及泡状变性,c-fos基因表达明显增强,呈弥漫性细胞质内棕黄色着色;肝细胞c-fos基因表达程度随超重剂量的增加有增强趋势．对照组肝脏组织病理学改变程度明显较实验组轻微,c-fos基因表达亦明显减弱．结论: Gx可引起猴肝脏组织细胞c-fos基因表达增强,提示有早期肝脏组织损伤．     

Murine thymocytes proliferate in direct response to interleukin-7

The ability of interleukin-7 (IL-7) to stimulate murine thymocyte proliferation was investigated. IL-7, either alone or in concert with lectin, induced proliferation of adult thymocytes as well as day 13 fetal and adult CD4-/CD8-thymocytes. The IL-7-induced proliferative response of unfractionated thymocytes could not be inhibited by antibodies to IL-2, or IL-4, IL-6, or the IL-2 receptor. In addition, IL-2, IL-4, and IL-6 were not produced by thymocytes activated with IL- 7, as judged by the absence of biologically active cytokine in IL-7- stimulated culture supernatants. IL-7 could act in concert with IL-2 and IL-4 or with IL-4 to enhance the proliferative response of thymocyte cultures. Thus, IL-7 may cause proliferation of thymocytes directly, not indirectly, through production of IL-2, IL-4, or IL-6. IL- 7 may then play a significant role in differentiation of T lymphocytes.     

Iron incorporation into ferritin and hemoglobin during differentiation of murine erythroleukemia cells

Hemoglobin and ferritin iron content have been followed during differentiation in tissue cultures of murine erythroleukemia cells (MELC) using the techniques of Mossbauer spectroscopy and electron microscopy. In undifferentiated cells grown without DMSO, only iron stored in ferritin was detected. The amount of iron in a cell grown in the presence of iron citrate is approximately 1.2 X 10(-14) g, whereas in a cell grown in the presence of transferrin the amount is approximately 0.28 X 10(-14) g. These quantities do not depend on the iron concentration in the nutrition medium in a range from 0.3 to 2.0 microgram Fe/ml and are the same for growth times between 8 hr and 7 days. Cells grown with DMSO contain, in addition to ferritin, increasing concentrations of hemoglobin. Chase experiments prove that ferritin iron participates in hemoglobin synthesis. The amount of ferritin iron reaches saturation within less than 8 hr in MELC grown with or without DMSO. In differentiating cells grown with iron citrate there is a decrease with time in ferritin iron content concomitant with the increase in hemoglobin. Cells grown with transferrin incorporate additional amounts of iron, which are approximately equal to the amounts used for hemoglobin synthesis maintaining a constant ferritin iron level. In the electron microscope, iron is seen only as ferritin within lysosomes. The density of the ferritin in lysosomes correlates with the ferritin iron concentrations determined by Mossbauer spectroscopy.     

Bone marrow donor registries: the relation between registry size and probability of finding complete and partial matches [see comments]

In a registry of volunteer bone marrow donors, the relation between registry size and probability of finding an exact or partial match for a random recipient cannot be theoretically derived because it depends on specifics of the human leukocyte antigen (HLA) haplotype frequencies in the donor and recipient populations. The relation must be explicitly calculated using empirically determined HLA haplotype frequency data for all possible pairings between a donor and a recipient population. This report describes a general solution to this problem. The method shows that the relation of the probability of matching to registry size is sigmoidal, with small increases in probability at the extremes of registry size and a middle range of registry size within which the probability of matching increases most sharply. This range determines the approximate size of the most cost-effective registry. In addition, for any pairing of donor and recipient populations, there is a maximum probability of identifying a match of a given quality for a random recipient, which cannot be exceeded even if registry size were infinite. This upper limit is a function of the frequency of blank (or unknown) alleles in the donor and recipient populations; the higher that frequency, the lower the maximum probability of achieving any given quality of match. The determinants of the probability of achieving a given quality of match with a given registry size are (1) the genetic heterogeneity within the recipient and donor populations, which increases the registry size required to achieve a given probability of matching, and (2) the degree of genetic homology between the donor and recipient populations, which increases the maximum probability of matching and also lowers registry size requirements. The method described here can be used to estimate donor pool size requirements using any donor and recipient populations for which HLA frequency data are available.     

Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major induced Cutaneous Leishmaniasis

Background

Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.

Methods

Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.

Results

The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).

Conclusion

IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.     

Screening with monoclonal anti-Fy3 to provide blood for phenotype- matched transfusions for patients with sickle cell disease

BACKGROUND: In the United States, there is a shortage of blood group phenotype-matched red cells (RBCs) for patients with sickle cell disease (SCD). A protocol designed to supply phenotype-matched RBCs for these patients by combining the recruitment of African American blood donors and automated testing of RBCs for these patients for the presumptive Fy(a-b-) phenotype using monoclonal anti-Fy3 was evaluated. STUDY DESIGN AND METHODS: African American donors were recruited, to increase the likelihood of phenotype matches in the donor population. Samples of RBCs were tested for the presumptive Fy(a-b-) phenotype by using monoclonal anti-Fy3 and an automated blood typing analyzer. RBCs confirmed to be Fy(a-b-) were retyped for selected Rh, MNS, Kell, Duffy, and Kidd blood system antigens. The extended phenotypes were matched with those of 41 SCD patients requiring transfusions. RESULTS: Of 8323 blood donations during the study, approximately 40 percent (3329) were made by African Americans. Approximately 22 percent (737) of African Americans were identified as Fy(a-b-) by this protocol and 12 percent (410) were phenotype matches for the 41 SCD patients. CONCLUSION: Combining the recruitment of African American blood donors and automated phenotyping using monoclonal anti-Fy3 offers a practical, relatively low-cost strategy for supplying phenotype-matched RBCs for SCD patients. This protocol increases the options for addressing the shortage of phenotype-matched RBCs for SCD patients.