CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.

Comparison of the clinical value of MRI classification system of deep gray matter injury in children with cerebral palsy / 西安交通大学学报(医学版)

Objective To investigate the reliability of MRI classification and clinical significance of deep gray matter injury(DGMI)in children with cerebral palsy(CP).Methods We made a retrospective assessment of 14 children with gross motor function classification system(GMFCS),manual ability classification system(MACS)and MRI classification system of deep gray matter injury.Based on T2WI,two radiologists worked independently and graded MRI pictures according to three-grading system and four-grading system.To evaluate the reliability of different grading systems,intra-observer and inter-observer agreements were tested by Kappa test.Spearman correlation analysis was performed to analyze the MRI classification system with GMFCS and MACS.Results The Kappa value of the intro-observer and inter-observe agreement of three-grading system was 0.873 and 0.873,respectively (P＜0.001).The Kappa value of the intro-observer and inter-observe agreement of four-grading system was 0.901 and 0.611(P＜0.001).Three-grading system had no significant correlation with GMFCS(r=0.053,P＞0.05)or MACS(r=0.128,P＞0.05).Four-grading system had a significant positive correlation with GMFCS(r=0.605, P＜0.05)and MACS(r=0.779,P＜0.05).Conclusion In the two grading systems,four-grading system is a more repeatable approach for detecting deep gray matter,gross motor function and manual function injuries in children with cerebral palsy.

Manual Ability Classification System (MACS): reliability between therapists and parents in Brazil

BACKGROUND: The Manual Ability Classification System (MACS) has been widely used to describe the manual ability of children with cerebral palsy (CP); however its reliability has not been verified in Brazil. OBJECTIVE: To establish the inter- and intra-rater reliability of the Portuguese-Brazil version of the MACS by comparing the classifications given by therapists and parents of children with CP. METHOD: Data were obtained from 90 children with CP between the ages of 4 and 18 years, who were treated at the neurology and rehabilitation clinics of a Brazilian hospital. Therapists (an occupational therapist and a student) classified manual ability (MACS) through direct observation and information provided by parents. Therapists and parents used the Portuguese-Brazil version of the MACS. Intra- and inter-rater reliability was obtained using unweighted Kappa coefficient (k) and intra-class correlation coefficient (ICC). The Chi-square test was used to identify the predominance of disagreements in the classification of parents and therapists. RESULTS: An almost perfect agreement resulted among therapists [K=0.90 (95% CI 0.83-0.97); ICC=0.97 (95%CI 0.96-0.98)], as well as with intra-rater (therapists), with Kappa ranging between 0.83 and 0.95 and ICC between 0.96 and 0.99 for the evaluator with more and less experience in rehabilitation, respectively. The agreement between therapists and parents was fair [K=0.36 (95% CI 0.22-0.50); ICC=0.79 (95% CI 0.70-0.86)]. CONCLUSIONS: The Portuguese version of the MACS is a reliable instrument to be used jointly by parents and therapists. .

Foetal stem cell derivation & characterization for osteogenic lineage.

Background & objectives: Mesencymal stem cells (MSCs) derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR) analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC) cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45-/CD14- was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP) activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.

Pluripotent lineage of CD133 stem cells isolated from human skin samples.

Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.

Desempeño funcional y asociación entre función motora gruesa y habilidad manual en niños con parálisis cerebral del Hogar Clínica San Juan de Dios, Lima-Perú / Functional performance and association between gross motor function and manual skills in children with cerebral palsy from the Hogar Clínica San Juan de Dios, Lima-Perú

Introduction: Children with cerebral palsy (CP) are a heterogeneous and difficult group to classify, therefore, we recommend using a functional approach based on the gross motor function and manual dexterity. We propose a transversal and descriptive study to 1) classify a population of children with CP and to determine the degree of association between Gross Motor Function Classification System (GMFCS) and Manual Ability Classification System (MACS) and 2) establish the relationship of GMFCS with age, gender, topographic distribution, predominant motor disorder and gross motor function. Patients and Methods: We evaluated 122 children (1-12 years) according to GMFCS, in the subgroup (81/122) of children over 4 years, MACS was applied. The relationship between variables was evaluated using the association test x2, and the association between GMFCS-MACS using Kappa statics with p < 0.05. Results: According to GMFCS, the level V predominates in the different groups: 44.4 percent in < 2 years, 34.8 percent in 2-4 years, and 40 percent in 6-12 years. 88.5 percent of the hemiplegic had level I or II and 75 percent of the quadriplegics had level IV or V (p < 0.01). In regards of manual dexterity, 38.3 percent had MACS I or II. In only 6.1 percent (5/81) we observed GMFCS and MACS level I. There is a relationship between the two systems, but the degree of agreement was low (weighted Kappa = 0.40). Conclusion: The use of both systems helps to functionally characterize our patients and therefore to establish impact measures in the clinical practice, reinforcing the interventions to improve the activities and participation.

Introducción: Los niños con parálisis cerebral (PC) constituyen un grupo heterogéneo de difícil clasificación, por tanto, se recomienda emplear un enfoque funcional basado en la función motora gruesa y la habilidad manual. Se plantea un estudio descriptivo de corte transversal para 1) clasificar una población de niños con PC y determinar el grado de asociación entre los sistemas Gross Motor Function Clasification System (GMFCS) y Manual Ability Classification System (MACS) y, 2) establecer la relación de GMFCS con edad, género, distribución topográfica, trastorno motor predominante y función motora gruesa. Pacientes y Métodos: Se evaluaron 122 niños (1-12 años) según GMFCS; en subgrupo (81/122) de niños mayores de 4 años, se aplicó MACS. La relación entre las variables fue evaluada con prueba de asociación basada en x2 y, la asociación GMFCS-MACS mediante estadística Kappa con p < 0,05. Resultados: Según GMFCS, el nivel V predomina en los distintos grupos: 44,4 por ciento en < 2 años; 34,8 por ciento de 2-4 años y 40 por ciento en 6-12 años. 88,5 por ciento de los hemipléjicos tenían nivel I o II y el 75 por ciento de los cuadripléjicos nivel IV o V (p <0,01). Según habilidad manual 38,3 por ciento tuvieron MACS I o II. En sólo el 6,1 por ciento (5/81) se observó un GMFCS y MACS nivel I. Existe una relación entre ambos sistemas, pero el grado de concordancia fue bajo (Kappa ponderado =0,40). Conclusión: El uso de ambos sistemas permite caracterizar funcionalmente a nuestros pacientes para establecer medidas de impacto en la práctica clínica, reforzando las intervenciones que mejoren las actividades y la participación.

Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133+Enriched Cells / 华中科技大学学报（医学）（英德文版）

Two isolation methods for sorting of endothelial progenitor cells(EPCs): from peripheral blood mononuclear cells(PBMCs)and CD133+ enriched cells were compared,by defining the cell morphology,phenotype,reproductive activities and function in vitro,to provide a reference for clinical application of EPCs.PBMCs from healthy subjects were used either directly for cell culture or for CD133+ sorting.The two groups of cells were cultured in complete medium 199(M199)for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS.The proliferation of differentiated EPCs was studied by MTT assay,and the VEGF concentration was measured using an ELISA kit.ECM gel experiment and migration assay were performed in vivo.The results showed that PBMCs produced more colony-forming units(CFU)than CD133+enriched cells from the same volume of blood(P<0.01).From day 7 to 14,the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers,but CD144+cells in CD133+ group were less than in PBMCs group(P<0.01).PBMCs group secreted more VEGF than CD133+group on the day 7(P<0.01).As compared with CD133+ group,PBMCs group had more potent potential of proliferation and vascularization in vitro.It was concluded that CD133+sorted cells showed a lower capacity of differentiation,secretion,proliferation and vascularization in vitro,suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.

Comparation of magnetic activated cell sorting and chemotherapy agent DDP in enriching cancer stem cell of laryngeal carcinoma cell line Hep-2 / 肿瘤研究与临床

Objective To compare effect of chemotherapy agent DDP to MACS in sorting cancer stemcells (CSC) of laryngeal carcinoma cell line Hep-2. Methods CD133 magnetic beads were applied to sort Hep-2 cells. Different dosages of DDP were used to treat Hep-2 cells for 48 hours. Enrichment rate of CD133+ cells by MACS and after DDP treatment was detected by Flow Cytometer (FCM). Morphologic change was observed under inverse-phase microscope. Results FCM showed that the sorting rate of CD133+ cells through MACS was 64.33 %, while after DDP treatment for 48 hours, the rate of CD133+ cells was enriched significantly in each dosage of DDP, with the maximal rate was 50.7 %, in the dosage of 4 μg/ml. There was a significantly difference between MACS and each of DDP group (P <0.01). Cells treated with DDP were abnormal in morphology. Conclusion MACS and DDP sorting has respective advantages in enriching CSC in Hep-2 cell lines.

Characterization of MACS Isolated Cells from Differentiated Human ES Cells / 대한불임학회지

OBJECTIVE: Human embryonic stem (ES) cells have a great potential in regenerative medicine and tissue engineering. The human ES cells could be differentiated into specific cell types by treatments of growth factors and alterations of gene expressions. However, the efficacy of guided differentiation and isolation of specific cells are still low. In this study, we characterized isolated cells from differentiated human ES cells by magnetic activated cell sorting (MACS) system using specific antibodies to cell surface markers. METHODS: The undifferentiated hES cells (Miz-hESC4) were sub-cultured by mechanical isolation of colonies and embryoid bodies were spontaneously differentiated with DMEM containing 10% FBS for 2 weeks. The differentiated cells were isolated to positive and negative cells with MACS system using CD34, human epithelial antigen (HEA) and human fibroblast (HFB) antibodies, respectively. Observation of morphological changes and analysis of marker genes expression were performed during further culture of MACS isolated cells for 4 weeks. RESULTS: Morphology of the CD34 positive cells was firstly round, and then it was changed to small polygonal shape after further culture. The HEA positive cells showed large polygonal, and the HFB positive spindle shape. In RT-PCR analysis of marker genes, the CD34 and HFB positive cells expressed endodermal and mesodermal genes, and HEA positive cells expressed ectodermal genes such as NESTIN and NF68KD. The marker genes expression pattern of CD34 positive cells changed during the extension of culture time. CONCLUSION: Our results showed the possibility of successful isolation of specific cells by MACS system from undirected differentiated human ES cells. Thus, MACS system and marker antibodies for specific cell types might be useful for guided differentiation and isolation of specific cells from human ES cells.

Removing Murine Embryonic Stem Cells From the Differentiating Cell Culture By Using Magnetic Activated Cell Sorting / 中国生物工程杂志

Objective:To remove murine embryonic stem cells(mESC)from the differentiating cell culture and purify the differentiated cells by Magnetic Activated Cell Sorting(MACS).Methods:Neural differentiation of mESC was induced by a 5-stage method.The specific cell surface marker,SSEA-1,was used to identify ES cells in the differentiating cells.The optimal dilutions of mouse anti mouse SSEA-1 IgM primary antibody and FITC conjugated goat anti mouse secondary antibody were determined before the flow cytometry test.The incubation time and incubation temperature of primary antibody were all optimized to make the cytometry test accurate.After the optimization,stage 4 cells were dissociated into single cell suspension,incubated with antibody of SSEA-1 and microbeads conjugated goat anti mouse IgM,and then sorted through the magnetic field.The rate of SSEA-1 positive cells in pre-and post-separation groups was assessed by flow cytometry,and the viability of cells was evaluated by trypan blue staining counting under light microscopy.Results:The proportion of SSEA-1 positive cells in the separated cells can be reduced from(7.19?1.36)% to(1.34?0.80)%.The survival rate of sorted cells was more than 92%,similar to that of pre-separation cells.Conclusions:The MACS system we used can effectively remove mESC from the differentiated cells.The sorted cells will be well provided for the subsequent studies about transplantation therapy.

Prenatal Diagnosis of Fetal Trisomy 21 from Maternal Peripheral Blood

This study was undertaken to establish a noninvasive prenatal genetic diagnostic method for trisomy 21 using the fetal nRBCs that is rarely present in maternal circulation. Peripheral venous blood samples were collected from 30 women with an advanced maternal age, abnormal triple marker test results, or abnormal ultrasound findings such as an increased nuchal translucency. The blood samples were treated with heparin. The triple density gradient centrifugation, and MACS using CD45 and CD71 were used to isolate the fetal cells. FISH analysis using probe 21 was performed with GPA-immunostaining. The study population consisted of 30 patients from 13 to 25 weeks of gestation, and nRBCs were separated in all cases. In GPA-immuno FISH analysis using probe 21, 3 cases of trisomy 21 were diagnosed and these results were confirmed by the amniocentesis. In conclusion, a prenatal diagnosis of trisomy 21 through GPA- immuno fluorescence in situ hybridization (FISH) analysis using separated fetal nRBCs is a useful, innovative, accurate, rapid and non-invasive diagnostic method.

Isolation and identification of tumor-like stem cells from human nasopharyngeal carcinoma cell line / 解放军医学杂志

Objective To investigate the expression of CD133 in human nasopharyngeal carcinoma cell line SUNE,and to detect proliferative and differential ability of CD133+ tumor cells in vitro,and to identify tumor-like stem cells in SUNE.Methods Immunocytochemical staining method and flow cytometry were used to detect the expression of putative tumor-initiated cell marker CD133 in nasopharyngeal carcinoma cell line SUNE,and the isolation technique with immunomagnetic beads was applied to purify CD133+ cells.The proliferative ability of CD133+ tumor cells in vitro was estimated by MTT assay,and the proliferative ability of CD133+,CD133-and control SUNE cells were statistically compared.Finally,the immunocytochemical staining method and flow cytometry were used to detect differential ability of CD133+ tumor cells in vitro.Results CD133 was positively expressed only in 0.35% of the cells in nasopharyngeal carcinoma cell line SUNE.In the serum-free medium RPM I1640,the UV optical density of the CD133+ tumor cells enriched with immunomagnetic beads was 0.317?0.007,0.370?0.002 and 0.558?0.004 at 3,5 and 7 days,respectively.Compared with CD133-cells and control SUNE cells,CD133+ cells showed increased proliferative capacity(P

Isolation of Fetal Cells in Maternal Peripheral Blood and Genetic Analysis of Trisomy 21 by GPA-immuno FISH(Fluorescence in situ hybridization / 대한산부인과학회잡지

INTRODUCTION: Down's syndrome is the most common congenital chromosomal anomalies which occurs 1 out of 700-1000 births. Until now, for prenatal diagnosis of Trisomy 21, invasive techniques such as amniocentesis, chorionic villus sampling(CVS) and cordocentesis were used, but they encompass the rare possibility of morbidity to the mother and fetus. Triple marker using maternal serum is a currently used noninvasive method, but it only shows the accuracy of 60%. Accordingly, a noninvasive method, using fetal cells from maternal blood is under extensive investigation. This study was undertaken to establish a noninvasive prenatal genetic diagnostic method of trisomy 21 using fetal nRBCs rarely present in maternal circulation. MATERIALS AND METHODS: Peripheral venous blood samples were collected from 76 women and treated by heparin. For the isolation of fetal cells, we used a triple density gradient centrifugation, and Vario-MACS and Mini-MACS using CD45 and CD71, and then, the morphological differentiation of the fetal nRBC was performed by Kleihaur-Betke stain. With GPA immunostain, nRBCs were identified by cytoplasm and GPA attatchment, and after marking the site, a FISH was performed. RESULTS: This study population included 76 patients from 8 to 41 weeks of gestation, and nRBC was separated from all cases. The morphological differentiation was achieved by K-B stain. The mean number of nRBC collected from 20 ml of maternal peripheral blood was 15. The number of nRBCs retrieved reached its peak in 12-18 gestational weeks(18.9 6.0) which decreased from 20 gestational weeks and thereafter. Fetal sex was determined by FISH analysis using probe X, Y with GPA-immunostained cells. GPA-immuno FISH analysis using probe 21 in 30 cases of advanced maternal age or positive triple markers, we confirmed 3 cases of Down's syndrome. These results were also confirmed using the CVS or amniocentesis. CONCLUSIONS: Fetal nRBCs were separated from all cases after 8 gestational weeks. Prenatal diagnosis of trisomy 21 through GPA-immuno FISH analysis of chromosome 21 using separated fetal nRBCs is an useful, innovative, accurate, rapid and non-invasive diagnostic method. But for clinical use, more cases of experiments will be needed.

Characteristics of B Cell proliferation by polysaccharide fraction of Paeonia japonica miyabe

BACKGROUND: Paeonia j ap onica Miyabe is a medicinal plant which has been widely used as a component of blood-building decoctions (Chinese medicinal concept : Bu-Xie). The immunopharmacological characteristics of the extract of Paeonia j ap onica (PJ) were investigated. METHODS: The effects of fractions of PJ extract on lymphocyte proliferation were measured by H3 -thymidine incorporation assay . The proliferated lymphocyte subsets were analyzed in flow cytometry . The subset cell populations of spleen cells were separated by magnetic cell separation system, and their proliferation by the extract were investigated. The effect of the extract on antibody production was determined in mice challenged with sheep red blood cells (SRBC) using hemolytic plaque forming cell assay. RESULTS: Spleen cells were proliferated by water extract of PJ. Polysaccharide fraction (PJ-P) of the extract was most active in the proliferation. It was found in flow cytometry that the lymphocyte subset proliferated by PJ-P was B cell population. Among the separated subset cell populations, T cell-depleted cell population and macrophage-depleted cell population were most proliferated by PJ-P. However, positively selected populations of B cells and T cells were not proliferated by PJ-P. These result s indicate that B cell proliferation by PJ-P may require the assistance of macrophages or T cells. These result s suggest that firstly PJ-P may stimulate macrophages or T cells, and then B cells are activated. The number of antibody-secreting cells was increased by administration of PJ-P in mice immunized with SRBC as a T-dependent antigen. CONCLUSION: These result s suggest that macrophages and accessory cells are directly activated by PJ-P and then helper T cells and B cells are indirectly activated. As the results, immune responses might be coordinately improved. In conclusion, PJ-P, a polysaccharide of P. j ap onica, may be a characteristic immunostimulator, which is analogous to polysaccharides such as lentinan, PSK and ginsan.

Prenatal genetic diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative and positive magnetic activated cell sorting

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.

Study of positive and negative magnetic activated cell sorting separation of platelet purification / 中国输血杂志

Objective To gain purified platelets for further research of the immune function of platelet.Methods Platelets were purified by positive(MS) and negative(LD) magnetic cell sorting(MACS) separation.The percentage of activated platelets was detected by flow cytometry and nucleated cell clearance was evaluated by white cell count.Results The percentage of activated platelets before separation was(2.39?1.10)%,and increased to(2.56?1.08)% and(16.76?4.04)%,respectively,after MS and LD MACS.The clearances of nucleated cells after MACS MS and LD were(98.44?0.24)% and(98.47?0.18)%,respectively.The recovery rate of purified platelet after MS and LD was(76.50?1.49)% and(79.20?2.61)%.Conclusion MACS LD method was more suitable for the platelet purification.

The formula of tonifying kidney of vital essence modulates secretion of IL-4 and IFN-? by CD4 positive T cells of human decidual / 中国免疫学杂志

Objective:To explore whether the Formula of Tonifying Kidney of Vital Essence can modulate secretion of IL-4 and IFN-? by the CD4+T cells from human decidua during first trimester,which will lead to further investigate the anti-abortional effect mechanisms of the Formula of Tonifying Kidney of Vital Essence.Methods:40 female adult SD rats were divided randomly into two groups:one group was gavaged with the Formula of Tonifying Kidney of Vital Essence;another was gavaged with normal saline,twice a day for 3 days.The two groups of rats were gavaged with the same volume of the decoction or saline.Thereafter the blood was collected from carotid artery.Human decidua tissues were collected by artificial abortion at 6-10 weeks of gestation.The CD4+ T cells were separated by MACS.CD4+ T cells were divided into four groups during cell culture.Group A was treated with 10% fetal calf serum,which was the control group.Group B was treated with 10% of the rat serum from the herbal medicine;Group C was treated with 20% of the rat serum from the herbal medicin.Group D was treated with the rat serum of the saline control.The supernatants were harvested at 24,48 and 72 h of the culture,respectively.The amounts of IL-4 and IFN-? in the culture supernatants were measured by ELISA.Results:In both group B and C of the culture the amount of IL-4 was evidently elevated when compared with the control,group A(P