Background: The reduction of erythrocytes from cord blood is very need for long - term storage of C034 cells for transplantation. Reduced erythrocyte will reduces preservative blood volume, preservatives and freely HST when defrosting, so stem cells are better protected. Objectives: To study selection of the best centrifugal procedure to reduce maximal erythrocytes and lose minimal C034 cells from cord blood. Subjects and methods: 20 blood samples selected from 60 cord blood units was used for this study. The study was carried out through two steps. In the first step, the centrifugal speed was fixed and the centrifugal time was changed.In the second step, the centrifugal time was fixed, the centrifugal speed was changed. From collected results the best appropriate procedure to reduce erythrocytes from cord blood have been selected. Results: The procedure of gradient centrifuge with speed of 500g in 6 minutes isolated> 50% of erythrocytes, kept > 84% of CD34 cells and then centrifuge of 1000 g in 10 minutes reduced about 40% of volume of nuclear cell - suspension. Conclusion: The procedure can use for preparation of stem cell suspension from cord blood to storage in nitrogen liquid. \r\n', u'\r\n', u'
Erythrocytes/ pathology ; Fetal Blood/ chemistry ; drug effects ; immunology

The study was purposed to explore the effect of panel reactive antibody (PRA) serum from patients with β-thalassemia on proliferation and apoptosis of the CD34(+)cells from cord blood and its mechanism. CD34(+) cells of umbilical cord blood were incubated with different sera and complement respectively. After incubation, the samples were centrifuged and the supernatants were collected for lactate dehydrogenase (LDH) detection, and the CD34(+) cells were harvested and measured for the apoptosis by flow cytometry with Annexin V/PI. The intracellular DNA synthesis were also quantified by [(3)H]TdR incorporation using liquid scintillation counter. The results showed that concentration of LDH in PRA positive groups was higher as compared with control group, and the DNA synthesis of CD34(+) cells in PRA positive groups were inhibited. There were no differences in the percentage of cell apoptosis and necrosis among different groups. It is concluded that thalassemic serum PRA impairs the cell membrane, inhibits the DNA synthesis, which can be increased by addition of the complement, but PRA had no significant effect on apoptosis of CD34(+) cells.
Antibodies ; blood ; immunology ; Antigens, CD34 ; Apoptosis ; immunology ; Cell Proliferation ; Child ; Fetal Blood ; cytology ; Humans ; beta-Thalassemia ; blood ; immunology ; pathology

OBJECTIVETo evaluate the variety of non-myeloperoxidase-mediated system activity of neutrophils in newborns during bacterial infection and the effect of cord plasma on the activation of non-myeloperoxidase-mediated system.

METHODSAn infection model with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and a non-infection model with phorbol-12-myristate-13-acetate (PMA) were established to investigate the activation of non-myeloperoxidase-mediated system in neutrophils. According to the intensity of fluorescence, the activation of non-myeloperoxidase-mediated system of neutrophils was detected by flow cytometry (FCM). The blood cells and plasma were separated from cord blood and adult blood and cross-mixed in order to investigate the opsonic activity.

RESULTSIn the non-infection model, the activation of non-myeloperoxidase-mediated system with PMA stimulation in cord blood was lower compared with that in adult blood, the statistical difference was significant (t = 3.378, P < 0.01). In the infection model, the activations of non-myeloperoxidase-mediated system in cord blood were also lower compared with those in adult blood, while the statistical difference could only be found in the model with E. coli stimulation (t = 12.150, P < 0.001). Furthermore the experiments demonstrated that cord plasma could deeply depress the non-myeloperoxidase-mediated system activity with E. coli stimulation. On the contrary, adult plasma could successfully recruit the potential of non-myeloperoxidase-mediated system activity of neutrophils in newborns.

CONCLUSIONThe function of neonatal neutrophils might not developed very well. As a stimulant, E. coli failed to induce the non-myeloperoxidase-mediated system activity in neonates, which might be related to the lower level of immunoglobulins in cord blood. This result indicated that immunoglobulins played a more important modulating role in bacterial killing during gram-negative bacterial infections.

Escherichia coli ; immunology ; Fetal Blood ; immunology ; Flow Cytometry ; Humans ; Infant, Newborn ; Neutrophils ; enzymology ; immunology ; Peroxidase ; metabolism ; Staphylococcus aureus ; immunology

The aim was to investigate the cytolytic activity of cytotoxic T lymphocytes (CTL) induced by dendritic cells (DC) derived from human cord blood in vitro. Human cord blood mononuclear cells (CBMNC) were cultured in vitro with addition of various cytokines. DC was confirmed by morphology, immune phenotype and capacity of stimulating MLR (mixed lymphocyte reaction). CTL were generated through the co-culture of autologous T lymphocytes and DC. (51)Cr-release assays were performed for the measurement of cytotoxicity of CTL. The results showed that distribution of the subgroups of T lymphocytes in CBMNC was similar to that in adult peripheral blood. The percentage of CD1a-expressing cells in CBMNC was very low, merely (0.41 +/- 0.09)%. During culture, DC became larger and more irregular in shape. Spiny dendrites or multiple cell processes in morphology emerged on the surface of DC. Among the cell populations at 15 days of culture, there were (28.4 +/- 3.55)% of CD1a-expressing cells, (63.67 +/- 23.33)% of CD86-positive, (8.7 +/- 1.49)% of CD83-positive and (32.5 +/- 1.53)% of HLA-DR-positive cells. DC derived from CBMNC is capable of stimulating the proliferation of allogeneic lymphocytes in MLR. CTL derived from autologous T lymphocytes induced by dendritic cells pulsed with lysates of HL-60 cells, possessed specific cytolytic effects against HL-60 cells. In conclusions, relatively high percentage of CD1a-expressing cells can be generated in culture system of this study. DC derived from cord blood is able to induce the production of anti-leukemic CTL in vitro.
Antigens, CD1 ; analysis ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Fetal Blood ; immunology ; Humans ; Immunophenotyping ; Leukemia ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVEThe low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA.

METHODSeven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10.

RESULTThe concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P > 0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P > 0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P > 0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P > 0.05).

CONCLUSIONPRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.

Antibodies ; immunology ; Antigens, CD34 ; Apoptosis ; immunology ; Cell Differentiation ; immunology ; Cell Proliferation ; Cells, Cultured ; Child ; Fetal Blood ; cytology ; immunology ; metabolism ; Flow Cytometry ; Humans ; Quorum Sensing ; immunology ; beta-Thalassemia ; immunology

Blood Group Incompatibility ; complications ; Erythroblastosis, Fetal ; etiology ; Humans ; Infant, Newborn ; MNSs Blood-Group System ; immunology ; Male

This study was aimed to investigate the specific anti-leukemia cell effect of cytotoxic T lymphocytes (CTLs) induced by HL-60 or K562 cell-sensitized dendritic cells (DCs) from umbilical cord blood. 12 units of human umbilical cord blood (UCB) were collected and the mononuclear cells (MNCs) were isolated from UCB, then cultured with granulocyte monocyte colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO for 3 - 4 weeks. Flow cytometry was used to determine the number of DCs and cell surface antigens before and after culture with monoclonal antibodies including CD83, CD1a, CD11c and CDw123. HL-60 and K562 were frozen-thawed, and released their tumor antigen peptides (TAP). The CTLs were produced by sensitizing T lymphocytes with DC-loaded HL-60 and K562 cell antigens. The test of (3)H-TdR incorporation was used to detect the immunostimulation activity of DCs. MTT assay was applied to evaluate specific cytotoxicity of CTL on leukaemia cells. The results indicated that the MNCs of UCBs cultured with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a(+) CD11c(+) CD83(+) CDw123(+) DCs. Numbers of DCs from UCBs remarkably increased in 2 - 4 weeks and then decreased. After culture with cytokines DCs increased (10.6 - 28.2) x 10(5)/ml in actual numbers. The CTL induced by DC pulsed with HL-60, K562 frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were (42.04 +/- 8.46)% and (31.25 +/- 11.07)% respectively. It is concluded that the MNCs of UCBs cultured with cytokines of GM-CSF, SCF, EPO and IL-3 can differentiate into CD1a(+), CD83(+), CD11c(+) and CDw123(+) DCs. The CTL induced by DCs pulsed with HL-60, K562 frozen-thawed lysates can effectively kill HL-60 and K562. These DCs as antigen presenting cells play an important role in cancer immunotherapy.
Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; HL-60 Cells ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo investigate the phenotypes and functions of cord blood dendritic cells of fetuses whose mothers are patients with chronic hepatitis B.

METHODSPeripheral blood and cord blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation with Ficoll-Hypaque. The adherent cells were cultured in AIM-V medium containing recombinant human IL-4, TNF-alpha and GM-CSF. On day 9, mature DCs (mDC) were harvested and used for phenotype analysis. The amounts of IL-12 which dendritic cells produced were measured. The dendritic cells that were studied and compared were from cord blood of fetuses of both CHB positive and negative mothers and from CHC adult peripheral blood.

RESULTSThe expression rate of CD80 and CD83 of chronic hepatitis B mother cord blood dendritic cells was low compared with that of the healthy cord blood, healthy adult peripheral blood, and chronic hepatitis B adult peripheral blood, P < 0.05. The amount of IL-12 produced by chronic hepatitis B mother cord blood dendritic cells was lower than that of healthy cord blood, healthy adult peripheral blood, chronic hepatitis B adult peripheral blood (P < 0.05). The T lymphocyte proliferation inducing ability of dendritic cells of healthy adult peripheral blood was higher in inducing cord blood T lymphocytes proliferation, which was greater than that of the healthy adult peripheral blood in inducing adult T lymphocytes and was greater than that of the healthy cord blood dendritic cells in inducing cord blood T lymphocytes, which was greater than that of the healthy cord blood in inducing adult T lymphocytes, which was greater than that of chronic hepatitis B mothers in inducing cord blood T lymphocytes, which was greater than that of chronic hepatitis B mother cord blood in inducing adult T lymphocytes.

CONCLUSIONThe maturation and functioning of CHB mother cord blood dendritic cells were lower than those of healthy cord blood, healthy adult peripheral blood and CHB adult peripheral blood.

Adult ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Female ; Fetal Blood ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Phenotype ; Pregnancy ; Pregnancy Complications, Infectious ; immunology ; T-Lymphocytes ; immunology

OBJECTIVEMesenchymal stem cells (MSCs) were adult stem cells which contribute to the regeneration of mesenchymal tissues such as bone cartilage, muscle, ligament, tendon, adipose and stroma. Due to the multipotential ability and self-renewal capacity, the mesenchymal stem cells can be applied in many fields, such as the seed cells in tissues engineering, cell therapy and gene therapy. To enhance the clinical use of MSCs, the investigators studied the isolation and expansion of MSCs from adult bone marrow, fetal bone marrow and human umbilical cord blood, and investigated their biological identities.

METHODSTwo kinds of incubation systems containing L-DMEM or MSC special culture medium were used to purify and expand MSCs. The growth, purification and proliferative abilities of 3 kinds of MSCs were observed and their immunophenotypes were determined by flow-cytometry.

RESULTS(1) The shapes of 3 kinds of cells were same. There was no difference in number and size. The colonies formed early in adult bone marrow MSCs. (2) There was no difference in the expansion speed of the 3 kinds of MSCs, but after the colonies confluenced there had no touching constrain in MSCs from umbilical cord blood and fetal bone marrow. When the colonies confluenced, the cells also had proliferation ability. But in adult bone marrow, the touching constrain was significant. (3) MSCs had strong self-renewal capacity. After primary culture approximately 5 - 6 x 10(5) MSCs were obtained from 8 x 10(6) MNC of bone marrow and 25 x 10(6) MNC of umbilical cord blood. After passage 3, passage 5 and passage 10, the investigators could get 10(7), 10(8) and 10(10) MSCs, respectively. (4) Along with the increase in the passage and prolonging of culture time, the ability of expansion decreased, but they maintained good puripotentiality. After passage 2, passage 3 and passage 5, the purity of MSCs was 90%, 95% and 99%, respectively. (5) Three kinds of MSCs were all positive for CD(29), CD(44), CD(59), CD(90), CD(105), CD(166) and all negative for the markers of hematopoietic cells such as CD(11a), CD(14), CD(33), CD(34), CD(28), CD(45). All the important GVHD correlation markers were negative, such as HLA-DR, B7-1 (CD(80)), B7-2 (CD(86)), CD(40) and CD(40L). There were no differences in the phenotype among the 3 kinds of MSCs cells. (6) The 2 kinds of culture mediums used did not markedly affect isolation and expansion of MSCs, and the biological properties of MSCs.

CONCLUSIONS(1) Human MSCs could be isolated from many kinds of human tissues, and they had no difference in their origin; (2) Human MSCs maintained good puripotentiality and self-renewal capacity. Therefore, they could meet with the need of clinical tissue engineering. (3) The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier but had broad clinical use.

Bone Marrow Cells ; cytology ; immunology ; Cell Culture Techniques ; Cell Differentiation ; immunology ; Cell Division ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; immunology ; Time Factors

The Kidd blood group is clinically significant since the Jk antibodies can cause acute and delayed transfusion reactions as well as hemolytic disease of newborn (HDN). In general, HDN due to anti-Jk(b) incompatibility is rare and it usually displays mild clinical symptoms with a favorable prognosis. Yet, we apparently experienced the second case of HDN due to anti-Jk(b) with severe clinical symptoms and a fatal outcome. A female patient having the AB, Rh(D)-positive boodtype was admitted for jaundice on the fourth day after birth. At the time of admission, the patient was lethargic and exhibited high pitched crying. The laboratory data indicated a hemoglobin value of 11.4 mg/dL, a reticulocyte count of 14.9% and a total bilirubin of 46.1 mg/dL, a direct bilirubin of 1.1 mg/dL and a strong positive result (+++) on the direct Coomb's test. As a result of the identification of irregular antibody from the maternal serum, anti-Jk(b) was detected, which was also found in the eluate made from infant's blood. Despite the aggressive treatment with exchange transfusion and intensive phototherapy, the patient died of intractable seizure and acute renal failure on the fourth day of admission. Therefore, pediatricians should be aware of the clinical courses of hemolytic jaundice due to anti-Jk(b), and they should be ready to treat this disease with active therapeutic interventions.
Bilirubin/blood ; Erythroblastosis, Fetal/*blood ; Fatal Outcome ; Female ; Humans ; Infant, Newborn ; Isoantibodies/blood ; Kidd Blood-Group System/*immunology