Relationships between the gene polymorphisms of drug metabolizing enzymes and the outcome of the first induction chemotherapy in patients with de novo acute myeloid leukemia / 中国实验血液学杂志

The objective of this study was to investigate the correlation between the gene polymorphisms of drug metabolizing enzymes and the outcome of the first induction chemotherapy in patients with de novo acute myeloid leukemia (AML). 113 de novo AML patients were enrolled in this study. The genotypes of 11 single nucleotide polymorphisms (SNP) in drug metabolizing enzymes were detected by the SNPstream(®) Genotyping System. The correlation between the distribution of genotypes and the complete remission rate of first induction chemotherapy was analyzed by logical regression. The results showed that patients with variant genotype of CYP2D6 (rs16947) had a lower complete remission (CR) rate, as compared to those with wild type (p = 0.033, OR = 0.32, 95%CI 0.112 - 0.915); meanwhile the patients with variant genotype of GSTO2 (rs156697) had a higher CR rate as compared to those with wild type (p = 0.011, OR = 3.023, 95%CI 1.289 - 7.089). Combined analysis of the above polymorphisms, showed that patients with variant genotype of CYP2D6 and wild genotype of GSTO2 (V + W) had lower CR rates in comparison to patients with wild genotypes of both polymorphisms (p = 0.017, OR = 0.183, 95%CI 0.045 - 0.735). It is concluded that CYP2D6 (rs16947) and GSTO2 (rs156697) polymorphisms are independent factors influencing CR rates of the first induction chemotherapy in de novo AML patients.

Quality control for ABO blood group typing of neonatal umbilical cord blood / 中国实验血液学杂志

This study was aimed to investigate a quality control method for ABO typing of neonatal umbilical cord blood(UCB). The routine serology method was used to identify the ABO type of UCB samples. These samples with questions were further detected by sequence specific primer PCR (PCR-SSP). The results showed that among total of 76120 UCB samples identified by positive ABO typing, there were 78 samples (1 per thousand) which could not be determined. Of these 78 samples, 30 (56.92%) samples with a weak agglutination reaction were excluded by reverse ABO typing. Out of 260 samples in reverse ABO typing, 148 samples were consistent with positive ABO typing, 112 samples (43.08%) were inconsistent with the positive ABO typing. 58 undetermined samples were detected by PCR-SSP. Out of them the genotyping results of 45 samples confirmed the serological typing, the phenotyping results in 3 cases were inconsistent to that of genotyping. 10 cases showed the unconformity between positive and reverse typing, but the genotyping results were fully consistent with the positive typing. In conclusion, positive typing for red cell antigens combined with PCR-SSP is efficient and sensitive for quality control of ABO typing for neonatal UCB.

Frequencies of high-resolution HLA-Cw* alleles in China Northern Han population / 中国实验血液学杂志

This study was purposed to investigate the frequencies of HLA-Cw* loci in China Northern Han population at gene level and to analyze the population genetic characteristics of HLA-Cw* alleles and distribution difference of gene frequency in regions. The high resolution genotyping for HLA-Cw* loci of 420 cases in China Northern Han population was performed by using PCR-SSP typing technique and their distribution regularity was analyzed statistically. The results showed that 30 HLA-Cw* alleles were detected, among which the frequency of Cw* 0102 (0.1776), 0702 (0.1217), 0602 (0.1150) were highest; other alleles with higher frequency were as follow in proper order: Cw* 0304, 0801, 0303, 0302, 0401, 1402. The rare observed HLA-Cw* 0506, 0810, 1510, 1601 and 1701 were detected firstly in this population. The statistical analysis indicated that the genotype distribution of HLA-Cw* loci coincides with the Hardy-Weinberg test. In conclusion, application of high resolution allele typing can accurately understand the distribution regularity and characteristics of HLA-Cw* alleles in China Northern Han population which provides the basis for research related with HLA-Cw* loci.

Investigation of uninterpretative HLA typing in 311 umbilical cord blood samples / 中国实验血液学杂志

The aim of this study was to investigate the factors which affect HLA typing in 311 umbilical cord blood (UCB) samples. The HLA low resolution typing of UCB samples with misinterpreted HLA types from 311 UCB samples analyzed by PCR-SSO and PCR-SSP was performed. 7 samples difficult to determine their HLA genotype were sequenced directly and the reason leading to misinterpret HLA typing was analyzed. The results indicated that 99.4% of misinterpreted samples resulted from the restriction of HLA typing method itself and 0.6% of misinterpreted samples were suspected to be contaminated with maternal blood in UCB. It is concluded that HLA typing is mainly affected by the shortcomings of oligonucleotide probe design for PCR-SSO and lack of allele specific primers of PCR-SSP.

Ex vivo expansion of megakaryocyte progenitors from human umbilical cord blood CD34(+) cells / 中国实验血液学杂志

This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.

Differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood and bone marrow / 中国实验血液学杂志

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.

Isolation and purification of human islet cells with semi-automated digestion / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an semi-automated effective method for large-scale purification of islet cells from human pancreas.</p><p><b>METHODS</b>Human pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro.</p><p><b>RESULTS</b>The number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001).</p><p><b>CONCLUSION</b>The established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.</p>

Correlation of HLA-alleles with aplastic anemia / 中国实验血液学杂志

To investigate the correlation between the HLA genes and pathogenesis of aplastic anemia (AA), polymerase chain reaction with specific sequence primers (PCR-SSP) method was used to HLA typing in 82 patients with AA and 400 normal healthy individuals as control. The results showed that A*2301 (1.84%), B*5501 (4.36%) and DRB1*0901 (23.48%) gene frequency in AA patients were significantly higher than those in controls (relative risk: RR=5.0253, 3.3645, 2.1269, chi2=4.6634, 6.3120, 9.1511 respectively) (p<0.01). In contrast, DRB1*1301 (1.23%) gene frequency was significantly lower in AA than that in controls, RR=0.2257, chi2=6.6629 (p<0.01). It is concluded that A*2301, B*5501 and DRB1*0901 genes may be considered as the risk markers while DRB1*1301 gene as a protective marker of AA.

Effects of stromal cell-derived factor 1 and platelet factor 4 on the adhesion characteristics and chemotactic function of ex vivo expanded umbilical cord blood CD34+ cells / 中国实验血液学杂志

To investigate the effects of stromal cell-derived factor 1 (SDF-1) and platelet factor 4 (PF4) on the homing-related function of expanded ex vivo umbilical cord blood CD34(+) cells, purified cord blood CD34(+) cells were cultured in serum-free medium containing a HGF combination of FL + SCF + TPO (FST) with either 100 ng/ml SDF-1 alone, 100 ng/ml PF4 alone, or both of these 2 cytokines. The expansion rate of CD34(+) cells, colony formation, homing-related functions including expression of homing-related adhesion molecules of expanded CD34(+) cell, adhesion activity and chemotactic function of the re-selected expanded CD34(+) cells were evaluated at different time points. The results showed that expansion rate of CD34(+) cells and expansion multiple of CFU in SDF-1 groups were higher than those in control. The expression of CD49e on the expanded CD34(+) cells was remarkable up-regulated, in contrast, expression of CXCR-4 on the expanded CD34(+) cells was remarkable down-regulated in SDF-1 groups. The expression of CD49e, CD54 and CXCR-4 on the expanded CD34(+) cells were remarkably up-regulated in the PF4 groups. In all the SDF-1 group, PF4 group and SDF-1 plus PF4 group, the ability of expanded CD34(+) cells adhering to fibronectin layer were higher than those in the control on day 10. Spontaneous migration rate of expanded CD34(+) cells in SDF-1 groups were higher than those in control, while SDF-1-induced migration rate were lower than those in control on day 10. SDF-1-induced migration rate in PF4 groups were higher than those in control on day 10. Spontaneous and SDF-1-induced migration rate of expanded CD34(+) cells in the SDF-1 plus PF4 groups were higher than those in control on day 10. It is concluded that, SDF-1 and PF4 can up-regulate expression of adhesion molecules on expanded CD34(+) cells, and retain the adherent and migration ability of expanded CD34(+) cells, which is helpful for the homing of expanded CD34(+) cells. In short, SDF-1 and PF4 are helpful for the homing-related function of the expanded UCB HSPC.

The expression of CD133 in acute leukemia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the expression of CD133 and its clinical significance in acute leukemia (AL) patients.</p><p><b>METHODS</b>The expression of CD133 and CD133 mRNA in leukemic blasts from 76 AL patients were detected by three-color flow-cytometry and hemi-quantitative RT-PCR respectively.</p><p><b>RESULTS</b>(1) CD133 mRNA expression was highly correlated with CD133 expression in both of normal donors and AL patients groups. The expression of CD133 in AL patients was significantly higher than that in control group (P < 0.01). (2) The positive rates of CD133 and CD133 mRNA in AL group were 42.1% (32/76) and 46.1% (35/76) respectively. There was no significant difference in CD133 expression between AML-M(3) and normal control, AML and ALL, as well as T-ALL and B-ALL. The expression of CD133 in AML-M(4) were significantly higher than those in other AML subtypes (81.8% vs 43.7% and 81.8% vs 46.9% at CD133 and CD133 mRNA level, respectively, P < 0.01). (3) The expression of CD133 in AML was significantly correlated with the expression of CD34 and HLA-DR (P < 0.001). (4) The expression of CD133 had no relationship with the clinical prognostic factors such as cytogenetic or molecular aberrations, WBC counts, LDH, mdr1 expression and age. (5) There was a trend toward lower CR rate in CD133(+) cases, but only CD34/CD133(+) double positive cases had significant lower CR rate than that of negative ones (44.4% vs 71.4%, P < 0.05).</p><p><b>CONCLUSIONS</b>AL had significantly higher CD133 expression compared to normal control. The detection of CD133 expression might help to identify AL type and predict therapeutic outcomes. Co-expression of CD133/CD34 might convey adverse prognosis of AL.</p>

Studies on the homing-related adhesion activities of UCB HSPC in short-term ex vivo expansion / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of ex vivo expansion on the adhesion activities of umbilical cord blood hematopoietic stem and progenitor cells (HSPC).</p><p><b>METHODS</b>Fresh UCB CD(34)(+) cells were cultured in a serum and stroma-free culture system. At day 7, day 10 and day 14, CD(34)(+) cells were re-selected from the expanded products. The expression of adhesion molecules (CAMs) such as VLA-4, VLA-5, LFA-1, ICAM-1, HCAM, L-selectin and PECAM-1, and the adhesion activity of the expanded CD(34)(+) cells were evaluated and compared with those of precultured fresh CD(34)(+) cells.</p><p><b>RESULTS</b>(1) The CD(34)(+) cells expressing homing-related CAMs were increased (from 15-fold increase for CD(34)(+) CD(54)(+) subset to 72-fold increase for CD(34)(+) CD(49e)(+) subset at day 14). (2) The expressions of CD(49d), CD(44), CD(11a) and CD(49e) on the expanded CD(34)(+) cells were increased or sustained the same levels as those on fresh UCB CD(34)(+) cells, while the expression of CD(62L), CD(54) and CD(31) on expanded CD(34)(+) cells declined with the cultivating. (3) Spontaneous adhesion and SDF-1-induced adhesion tended to be increased in the course of the first 10 day's culture.</p><p><b>CONCLUSIONS</b>The culture system used in this study could substantially support the expansion of HSPCs expressing the above CAMs, and the expanded HSPCs would sustain their intrinsic adhesion potentials.</p>

The effects of cytokines mediated ex vivo expansion on the cell adhesion molecule expression of cord blood hematopoietic stem and progenitor cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the expression of cell adhesion molecules (CAMs) among VLA-4 (CD49 d), VLA-5 (CD49e), LFA-1 (CD11a), L-selectin (CD62L), and PECAM-1 (CD31) which are more related to the homing of hematopoietic stem and progenitor cells (HSPC) on the ex vivo expanded CD34+ subset with that of fresh isolated AC133+ cells.</p><p><b>METHODS</b>AC133+ cells selected from fresh cord blood (CB) samples were cultured in QBSF-60 serum-free media in the presence of Flt-3 ligand + SCF + TPO (FST), with initial addition of IL-3 for up to 2 week. Expansion potential and the expression of above CAMs were evaluated at day 0, day 7, day 10 and day 14.</p><p><b>RESULTS</b>(1) Simultaneously numerical expansion of various HSPC was constantly observed during the culture, and the fold expansion of AC133+ cells and CD34+ cells on day 14 were 33.50 and 64.56 respectively; (2) The number of CD34+ subsets expressing the above adhesions were all increased at different degrees (from 20 fold to 160 fold). (3) The expressions of CD11a, CD49d, and CD49e on ex vivo expanded CD34+ cells were increased as compared to their baseline levels, but the percentage of CD62L+ and CD31+ subpopulations in CD34+ cells were decreased.</p><p><b>CONCLUSIONS</b>Our short-term culture system can not merely support the simultaneous expansion of CB derived AC133+ cells, but the expanded hematopoietic progenitors may well sustained the expression of homing-related adhesion molecules.</p>

Antitumor Activities of KRN7000 in NS-1 Myeloma-Bearing Mice / 中国实验血液学杂志

To explore the antitumor activities of KRN7000 in a NS-1 myeloma-bearing mice and the underlying mechanisms, cytotoxic activities of the spleen cells treated with KRN7000 in vivo and in vitro were examined with Yac-1 (a NK-sensitive cell line) and NS-1 (a NK-insensitive cell line) as the targeted cells. The life span of NS-1 myeloma-bearing mice was observed after treating with KRN7000 and combined with cyclophosphamide (CTX). Furthermore, toxicities of KRN7000 administration on liver and kidney were evaluated. The results showed that KRN7000 enhanced NK cytotoxic activities of spleen cells. Around 20% of NS-1-bearing mice survived after KRN7000 administration and the survival rate reached up to 80% of NS-1-bearing mice when KRN7000 was used in combination with CTX at a dose of 100 mg/kg (P < 0.005). KRN7000, at a dose of 100 micro g/kg, had no toxic effects on liver and kidney. These findings suggest that KRN7000 might be a promosing agent in tumor immunotherapy.