Cell Surface Antigen Expression Research Articles

A detailed flow cytometric method for detection of low-level in vivo red blood cell–bound IgG, IgA, and IgM

Abstract Flow cytometric methods are commonly used to analyze white blood cell surface antigen expression. We developed a flow cytometric method to detect red blood cell (RBC)-bound immunoglobulin (Ig)G, IgA, and IgM. RBCs were washed; incubated with fluorescein isothiocyanate (FITC)-conjugated anti-IgG, -IgA, or -IgM; washed; and analyzed on the flow cytometer. The method was optimized by determining the dilution of FITC-conjugated anti-IgG, -IgA, and -IgM providing the greatest amount of fluorescence when tested with Ig-coated RBCs and the least amount of fluorescence when tested with naive RBCs. Tannic acid was used to prepare Ig-coated RBCs. Cross-reactivity of FITC-conjugated anti-IgG, -IgA, and -IgM with Ig-coated RBCs was evaluated, and a reference range was established. Use of this method may assist in clinical evaluation of patients who present with hemolysis and a negative direct antiglobulin test. Immunohematology 2016;32:161–169.

Heirarchical Clustering of Immunophenotypic Cell Surface Antigen Expression Identifies Clinically Meaningful Cohorts in Childhood AML: A Report from the Children's Oncology Group Protocol AAML0531

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse cell surface antigen expression. Multidimensional flow cytometry (MDF) quantifies the expression of such surface antigens on neoplastic cells, permitting an objective means for sub-classification of this disorder.Objective: To investigate the relationship between the presenting immunophenotype and response to therapy in a large, controlled study of pediatric AML patients.Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for MDF at diagnosis, (2) proper consent for specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs.Unsupervised hierarchical clustering analysis was performed to mathematically define groups of patients with similar diagnostic IEPs. Selection of the appropriate number of clusters was accomplished by minimizing within-cluster variation while maximizing between-cluster variation. The rate of measurable residual disease (MRD) by MDF at the end of initial induction therapy (EOI1) and event free survival (EFS) was determined for each group and compared to the mean MRD rate and EFS for the entire cohort. Of the 769 total patients, 643 (84%) had bone marrow specimens submitted for MRD by MDF at EOI1.Results:Of the 643 patients with bone marrows submitted for MRD by MDF at EOI1, 223 (35%) were positive. The 5 year EFS rate was 49% for the study (N=769). Hierarchical clustering analysis of the IEPs defined 11 groups of patients (A-K) with similar quantitative diagnostic immunophenotypes.Five immunophenotypic groups of patients were associated with comparableEOI1 MRD rates (15%-36%) and EFS (50%-69%) to the study average. These 5 groups (A-E) consisted of immunophenotypes close to the normal counterpart of myeloid progenitor cells (Groups A, B, C; N=266, 77 and 106) or normal monocytes (Groups D and E; N=41 and 68).Two immunophenotypic groups (Group F and G) of patients were associated with significantly higher rates of MRD (Group F: 54%, p<0.001; Group G: 88%, p<0.001) and lower EFS (Group F: 28%, p<0.001; Group G: 19%, p=0.006) than average for the study. The defining different-from-normal immunophenotypic features of Group F (N=81) were expression of CD34 with a lack of HLA-DR. The defining immunophenotypic features of Group G (N=16) were high intensity CD56 expression, a lack of HLA-DR, dim-to-negative CD38 expression and dim-to-negative CD45 expression. The majority of patients in both groups were stratified as low or standard risk (86% and 100%).Three immunophenotypic groups (H-J) had low rates of MRD (20%-24%) but poor EFS (27%-39%). The defining different-from-normal immunophenotypic features of Group H (N=52) were expression of CD56 and CD13 with a lack of CD34. The defining different-from-normal immunophenotypic features of Group I (N=11) were CD34 positive expression with a lack of CD117. The defining different-from-normal features of Group J (N=28) were bright CD11b expression with a lack of CD34 and CD117. The majority of patients in these groups were stratified as standard risk (94%, 100%, and 75% respectively).One immunophenotypic group (K) had a high rate of MRD (50%) and comparable EFS (52%) to the study average. The defining different-from-normal features of Group K (N=23) were expression of CD56 with a lack of CD34 and CD13.Conclusions: IEPs can provide a powerful tool to discriminate patient cohorts that have a high probability of relapse and subsequently elucidate when MRD by MDF at EOI1 is predictive of patient outcome. Notably 2 IEP groups (97 patients, 13% of total) had significantly higher MRD rates and were associated with a high probability of disease relapse. IEPs can identify these 2 groups of patients otherwise stratified as good or standard risk, which can provide clinicians with an additional analytical method to more appropriately stratify patients. DisclosuresLoken:Hematologics Inc.: Equity Ownership. Voigt:Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Pardo:Hematologics Inc.: Employment.

Thrombopoietin-Mimetic Romiplostim Confers the Complete Survival Rate to Mice Exposed to Lethal Ionizing Radiation

Radiation-related casualties following exposure to a lethal dose of ionizing radiation show severe acute radiation syndromes (ARS) involving bone marrow death and gastrointestinal death. ARS cause decreases in red blood cell count, white blood cell count, platelet count and gastrointestinal dysfunction, finally leading to death caused by systemic bleeding. Therefore, reconstitution and restoration of hematopoiesis is a top priority. Although bone marrow transplantation (BMT) is also available for recovery from radiation-induced bone marrow damage, BMT for victims in radiation accidents has many limitations, including histocompatibility, age constraints, HLA type and the fact that immunosuppression would be required to reduce the risk of graft versus host rejection. In contrast, pharmacological approaches can accommodate a large number of victims with few limitations. Our previous study showed that the combined administration of erythropoietin, granulocytecolony stimulating factor and nandrolone decanoate after lethal ionizing irradiation resulted in the survival of approximately 50% of irradiated mice at day 30. When a c-Mpl agonist (Romiplostim: RP) was added to this protocol, 100% survival was obtained. Finally, we found that RP play a key role in the survival of irradiated mice. In the present study, we examined the effects of RP alone on mice exposed to lethal radiation.RP was administered at a dosage of 50 μg/kg of body weight/day to 8-weekold female C57BL/6JJcl mice for 1, 3, or 5 days immediately following exposure to a lethal 7 Gy dose of 137Cs γ-rays. The condition of each animal was analyzed via morphological evaluations of the small intestine and various parameters such as the numbers of peripheral blood cells, bone marrow cells, and hematopoietic progenitor cells along with cell surface antigen expression. By day 30, all untreated irradiated control mice died, whereas RP administration for 3 or 5 consecutive days after irradiation led to a 100% survival rate among the irradiated mice. At this time, the numbers of peripheral blood cells, bone marrow cells and hematopoietic progenitor cells were not significantly different between RP-untreated non-irradiated and RP-treated irradiated mice. In addition, the expression of macrophages, granulocytes and erythroid progenitors-related cell surface antigens on the bone marrow cells was significantly recovered in RP-treated irradiated mice compared to RP-untreated irradiated mice until day 20 after γ-irradiation. And, to estimate the effects of RP on gastrointestinal tissues in each individual, morphological evaluation H&E stain of the small intestine was performed until day 20 after γ-irradiation. As a result, RP promoted the recovery of gastrointestinal tissues damages in RP-treated irradiated mice compared to RP-untreated irradiated mice.Regarding cell death, radiation-induced gamma-H2AX expression in the nuclear of bone marrow cell was significantly decreased in RP-treated irradiated mice compared to RP-untreated irradiated mice immediately and after a period of 24 hours following a lethal 7 Gy dose of X-irradiation, indicating that the rate of apoptotic bone marrow cells was significantly decreased by RP-treatment. Meanwhile, 53BP1, which is well known as non-homologous end joining (NHEJ) factor, was significantly increased, showing that RP promoted NHEJ DNA repair in bone marrow cells treated with RP. These results demonstrate that c-Mpl agonist RP promotes the recovery of serious damages caused by lethal irradiation to the hematopoietic and gastrointestinal systems, and RP might be a useful radiomitigator in the case of ARS. DisclosuresNo relevant conflicts of interest to declare.

Recurrent Genetic Abnormalities Are Reflected in Phenotype at Diagnosis in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group Protocol AAML0531

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with high genomic and phenotypic complexity. Although a relationship between phenotype and t(15;17) is recognized, the effect of other genotypic abnormalities on phenotype remains to be elucidated.Objective: To explore the relationships between genotypic and phenotypic abnormalities in diagnostic specimens in pediatric AML.Methods: Of 1022 newly diagnosed pediatric patients with de novo AML enrolled on protocol AAML0531, 769 satisfied three criteria for this study: (1) submission of a blood or bone marrow sample for multidimensional flow cytometry (MDF) at diagnosis, (2) consent to blood bank specimen testing and (3) leukemia comprising >10% of non-erythroid cells by MDF. The diagnostic AML tumor population was identified by gating on CD45 vs log-SSC. A fifteen-dimensional immunophenotypic expression profile (IEP) was defined by computing mean fluorescent intensities, antigen intensity coefficient of variations, and light scatter characteristics of each leukemia without analyst imposed cut-offs.Unsupervised hierarchical clustering was performed to mathematically cluster patients with similar IEPs. The underlying karyotypic and mutational profile of each patient was compared to the IEPs to identify clusters of patients with relationships between phenotype and identifiable genetic abnormalities. Supervised bagged tree-based models were used to quantify the importance of each cell-surface antigen in identifying these clusters. Levene's test was used to assess the homogeneity of mean cell-surface antigen expression for important markers.Results: Analysis of hierarchical clustering results revealed 7 immunophenotypic clusters (A-G) of patients with relationships between IEP and 4 common genetic abnormalities (Figure 1).Cluster A and Cluster B were both strongly associated with the t(8;21) karyotype (Figure 1). 54/62 patients in Cluster A (87%) and 24/26 patients in Cluster B (92%) were positive for t(8;21). The most important cell-surface antigens in identifying patients with the Cluster A associated phenotype were dim CD33, expression of CD56, expression of CD34 and expression of HLA-DR. The mean antigen expression of these markers was significantly more homogenous for patients in Cluster A compared to all other patients for CD33 (p = 0.02), CD34 (p < 0.01) and HLA-DR (p<0.01). Mean expression of CD56 was notably more heterogeneous (p = 0.40) but positive in 91% of patients in this cluster. The most important cell-surface antigens in identifying the Cluster B associated phenotype were dim CD13, expression of CD34, expression of HLA-DR and dim CD33. The mean expression of these markers was significantly more homogeneous for patients in Cluster B compared to all other patients (all p values <0.01). In contrast to Cluster A, CD56 MFIs were negative for patients in Cluster B. Collectively, 72% of all patients with t(8;21) segregated into Cluster A (50%) or Cluster B (22%).Cluster C was strongly associated with chromosome 16 abnormalities (Figure 1). Of patients in this cluster, 50/55 (91%) were positive for inv(16)/t(16;16). The mean high-expression of CD13 was the most important antigen in identifying patients with this phenotype, and patients in Cluster C had more consistent mean CD13 expression compared to all other patients (p<0.01). 53% of all patients with chromosome 16 abnormalities comprised this cluster.Cluster D was strongly associated with 11q23 abnormalities (Figure 1). Of patients in this cluster, 105/135 (78%) were positive for 11q23 translocations. The most discriminating markers in identifying these patients were dim-to-negative CD34 and dim-to-negative CD117. 69% of all patients with 11q23 abnormalities comprised this cluster.Clusters E, F and G were associated with FLT3 ITD mutations (Figure 1). Of patients in these clusters, 44%, 49% and 53% were positive for FLT3 ITD mutations. Cumulatively, 63% of all patients with FLT3 ITD mutations were grouped into one of these three clusters.Conclusion: Hierarchical clustering algorithms identify IEPs associated with common genetic abnormalities in pediatric AML, providing insight into the consistent dysregulation of cell-surface gene product expression in patients with common genetic abnormalities. Further analysis may reveal that IEPs can identify patients otherwise classified as good or standard risk that will have a poor outcome. [Display omitted] DisclosuresVoigt:Hematologics Inc.: Employment. Brodersen:Hematologics Inc.: Employment. Menssen:Hematologics Inc.: Employment. Loken:Hematologics Inc.: Equity Ownership.

Mitigative Effects of a Combination of Multiple Pharmaceutical Drugs on the Survival of Mice Exposed to Lethal Ionizing Radiation.

It is important to establish an easy-to-use therapeutic protocol for the emergency medical care of patients involved in radiation accidents to reduce the radiation-related casualties. The present study aimed to establish an optimum therapeutic protocol using currently approved pharmaceutical drugs to increase the survival of victims exposed to lethal radiation. Different combinations of four drugs-recombinant human erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), c-mpl receptor agonist romiplostim (RP) and nandrolone decanoate (ND)-were administered to mice within 2 h after exposure to a lethal 7 Gy dose of γ-irradiation. On day 30 after irradiation, the condition of the mice was analyzed using various hematological parameters, such as the number of peripheral blood cells, bone marrow cells, hematopoietic progenitor cells and the expression of cell surface antigens. Approximately 10% of the untreated irradiated control mice survived for 21 days, but all of the control mice died by day 30. The combined administration of G-CSF, EPO and RP for five days immediately after irradiation led to a complete survival of the irradiated mice until day 30. However, the treatment with G-CSF, EPO and RP with ND led to only 75% survival at day 30. The hematological analyses showed that the numbers of almost all of hematopoietic cells in the surviving mice treated with effective medications recovered to the levels of non-irradiated mice. The present findings show that the combination of G-CSF, EPO and RP may be a useful countermeasure for victims exposed to accidental lethal irradiation.

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody

The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.

Effect of low temperature on Schwann-like cell differentiation of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types. The potential of MSCs for differentiation and proliferation depends on the control of various processes. In this study, we have investigated the effects of low temperature on the Schwann-like cell differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). The growth of BM-MSCs decreased at low temperature, but increased Schwann-like cell differentiation. The expression of cell surface antigen decreased CD73, CD105, and CD90, which are known markers of MSCs at 35°C. Also, the levels of matrix RNA [S100, glial fibrillary acidic protein (GFAP), p75, myelin basic protein, P0, and others] at 35°C in the differentiation culture were higher than those at 37°C in the differentiation culture, and S100, p75, and GFAP proteins were expressed more than at 37°C. GFAP and S100 were stained strongly at 35°C by immunohistochemical examination. These data demonstrate that culturing BM-MSCs at reduced temperature can improve differentiation into Schwann-like cells.

Abstract 380: Capture of EpCAM-negative circulating tumor cells (CTCs) with a “Universal CTC-Chip”

Abstract Background: Circulating tumor cell (CTC) can be a potentially useful clinical marker in early diagnosis and monitoring therapeutic effects for patients with malignant tumors. Our previous study showed that CTC detected by an EpCAM (epithelial cell adhesion molecule) based immuno-magnetic separation system “CellSearch” was useful in the diagnosis of malignant pleural mesothelioma (MPM), and also useful in the prognosis of epithelioid type MPM (Yoneda K, et al. Ann Surg Oncol. 2013). However, EpCAM-negative tumor cells cannot be principally captured with the system. Therefore, we have developed EpCAM-independent “Universal CTC-Chip System”, and assessed its capture capability. Methods: PC-9 (lung cancer cell line) was employed as EpCAM-positive cells, ACC-MESO-1 and ACC-MESO-4 (mesothelioma cell lines) were employed as EpCAM-negative cells. The expression of cell surface antigen was confirmed by flow cytometry. A microfluidic devices made of resin was coated in two steps with bridging antibody and capture antibody. It is possible to use any antibody to capture. In this study, we use anti-EpCAM antibody, anti-podoplain antibody, anti-mesothelin antibody, and isotype control. First, we verified capture capability of this system using samples which were spiked tumor cells labeled with CFSE in PBS containing 5% BSA. Then, we measured in the same manner by adding the cells to the blood of healthy donor, and calculated capture rate. Results: The expression analysis of cell surface antigens, PC-9 was EpCAM-positive, podoplanin, mesothelin were negative. On the other hand, MESO-1, 4 was EpCAM negative, podoplanin, mesothelin were positive. The capture efficiency in PBS containing 5% BSA with each cell using anti-EpCAM antibody, anti-podoplanin antibody and anti-mesothelin were PC-9: 101.1% / 2.3% / 2.9%, MESO-1: 3.5% / 52.7% / 4.3%, MESO-4: 3.0% / 78.3% / 5.4%, respectively. In the study of blood, each of the capture rate using anti-EpCAM antibody and anti-podoplanin antibody were PC-9: 88.0/ 6.9%, MESO-1: 4.0%/ 12.7%, MESO-4: 2.2%/ 38.4%, respectively. Conclusions: The capture efficiency using this device depends on the expression intensity of cell surface antigens. In experiments with PBS samples, it was possible to capture targeting specific cell surface antigen. Mesothelioma cells in the blood ware captured with combination of this CTC-Chip system and anti-podoplanin antibody. Citation Format: Kazue Yoneda, Yasuhiro Chikaishi, Eri Kawashima, Tomoko So, Hidetaka Uramoto, Takashi Ohnaga, Fumihiro Tanaka. Capture of EpCAM-negative circulating tumor cells (CTCs) with a “Universal CTC-Chip”. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2015-380

Comparative analysis of human mesenchymal stem cells from fetal-bone marrow, adipose tissue, and Warton's jelly as sources of cell immunomodulatory therapy

To characterize different tissue MSCs as sources of cell immunomodulatory therapy. Examined the effects of IFN-γ on WJ-MSC and their immunomodulatory function characteristics. We compared human fetal bone marrow (F-BM), adipose tissue (AT), and Warton's Jelly-derived MSCs (WJ-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, gene expression, and whether IFN-γ affected WJ-MSC gene expression, as determined by real time quantitative PCR. Fifteen geneswere examined. We further assess WJ-MSCs-mediated immunomodulatory on peripheral blood mononuclear, stimulated by PHA, IL-2 and CD3Ab after 5 days of co-cultured in a 5:1 ratio (PBMC:MSCs). Examined the effect of WJ-MSCs on the Th1, Th2, Th17 cytokines production and Treg augument. MSCs from different tissues have similar levels of cell surface antigen expression and differentiation ability, while F-BM-MSCs and WJ-MSC had higher rates of cell proliferation and clonality than AD-MSCs. All 15 genes were expressed at similar levels in WJ-MSCs and AD-MSCs (P > 0.05). 9 genes were upregulated in WJ-MSCFor F-MSC, including IL-6, CXCL9, CXCL10, CXCL11, ICAM-1, IDO1, HLA-G5, SDF1A, and NOTCH were down expression, but VCAM-1 was lower expressionin WJ-MSCS. After IFN-γ treatment, 7 genes were upregulated in WJ-MSC, including chemokine ligands CXCL9, CXCL10 and CXCL11, and the adhesion protein VCAM1and ICAM1. Additionally, immunosuppressive factors, such as HLA-G and IDO were both increased. When cocultured with peripheral blood mononuclear, WJ-MSCs showed an immunosuppressive function by inhibit the proliferative response of Th1 and Th17 but augment Th2 and Treg. Primed WJ-MSCs by IFNγ caused a greater reduction in IFNγ and TNFα than untreated WJ-MSCs, also the effect on augument in Treg and inhibit Th17 (P < 0.01). Our results demonstrate that primitive F-BM-MSCs and WJ-MSCs have biological advantages as compared to adult cells, WJ-MSCs have a gene expression pattern similar to AT-MSCs but not F-BM MSCs, and that inflammatory stimuli regulate gene expression in WJ-MSCs. WJ-MSC showed the immunosuppressive function in co-cultured system with PBMC, and IFNγ can promoted the immunosuppressive function.

Effect of umbilical cord blood dendritic cells induced by gastric cancer antigen combined with CIK cells in gastric cancer cell lines SGC-7901

Objective To investigate the effect of umbilical cord blood dendritic cells (DCs) induced by gastric cancer antigen combined with cytokine induced killer (CIK) cells in gastric cancer cell lines SGC-7901 in vitro. Methods Mononuclear cells from umbilical cord blood were used to create DCs and CIKs. The cell surface antigen expression of the mature DCs such as CD83, CD86, CD11c and the cell surface antigen of CIKs such as CD3, CD56, CD4, CD8, CD16 were detected using flow cytometry. Sensitized DCs-CIKs, DCs-CIKs, CIKs as effective cells, and SGC-7901 as target cells, the killing activities of these effective cells were tested with LDH release, which the number ratio of cells between effective cells and SGC-7901 cells were 10∶1, 20∶1, 40∶1, respectively. Results The cell surface antigen expressions of the mature DCs, such as CD83+ CD86+ , CD11c+ CD83+ , CD86+ CD11c+ were (75.4±2.1)%, (79.3±1.4)%, (80.2±2.6)%, respectively. The mature sensitive-DCs surface antigen expressions, such as CD83+ CD86+ , CD11c+ CD83+ , CD86+ CD11c+ , were (77.7±1.5)%, (82.6±1.9)%, (76.9±2.6)%, respectively. There was no statistical significance about the surface antigen expression between DCs and sensitive-DCs (t=1.526, P>0.05; t=0.958, P>0.05; t=1.049, P>0.05). The CIKs surface antigen expressions, such as CD4+ , CD8+ , CD3+ CD56+ CD16+ , were (22.8±1.3)%, (77.3±1.8)%, (24.5±2.1)%, respectively. The results suggested that the killing effect of the three kinds of combination cells on gastric cancer cells was different. The number ratio of cells between sensitive-DCs and SGC-7901 cells were 10∶1, 20∶1, 40∶1, which the killing activities of sensitive-DCs-CIKs against SGC-7901 were (37.68±1.49)%, (41.67±0.90)%, (42.71±0.98)%, respectively. The killing activity of sensitive-DCs-CIKs was the highest when the ratio of cells between sensitive-DCs and SGC-7901 cells were 40∶1. The killing activities of DC-CIKs were (36.77±0.46)%, (38.94±0.95)%, (41.15±0.89)%, respectively. The killing activities of CIKs were (34.74±1.01)%, (37.76±0.43)%, (39.65±0.79)%, respectively. There were statistically significant differences among the three groups (F=5.92, P<0.05; F=19.13, P<0.05; F=8.88, P<0.05). Conclusion The tumor killing activity of CIK is enhanced obviously by umbilical cord blood DCs which is sensitized by gastric cancer tumor antigen. There is the highest killing activity when the number ratio of cells between sensitive-DC-CIK and SGC-7901 cells is 40∶1. Key words: Stomach neoplasms; Dendritic cells; Killer cells, lymphokine-activated

Human B-cell and progenitor stages as determined by probability state modeling of multidimensional cytometry data.

Human progenitor and B-cell development is a highly regulated process characterized by the ordered differential expression of numerous cell-surface and intracytoplasmic antigens. This study investigates the underlying coordination of these modulations by examining a series of normal bone marrow samples with the method of probability state modeling or PSM. The study is divided into two sections. The first section examines B-cell stages subsequent to CD19 up-regulation. The second section assesses an earlier differentiation stage before and including CD19 up-regulation. POST-CD19 ANTIGENIC UP-REGULATION: Statistical analyses of cytometry data derived from sixteen normal bone marrow specimens revealed that B cells have at least three distinct coordinated changes, forming four stages labeled as B1, B2, B3, and B4. At the end of B1; CD34 antigen expression down-regulates with TdT while CD45, CD81, and CD20 slightly up-regulate. At the end of B2, CD45 and CD20 up-regulate. At the end of B3 and beginning of B4; CD10, CD38, and CD81 down-regulate while CD22 and CD44 up-regulate. PRE-CD19 ANTIGENIC UP-REGULATION: Statistical analysis of ten normal bone marrows revealed that there are at least two measurable coordinated changes with progenitors, forming three stages labeled as P1, P2, and P3. At the end of P1, CD38 up-regulates. At the end of P2; CD19, CD10, CD81, CD22, and CD9 up-regulate while CD44 down-regulates slightly. These objective results yield a clearer immunophenotypic picture of the underlying cellular mechanisms that are operating in these important developmental processes. Also, unambiguously determined stages define what is meant by "normal" B-cell development and may serve as a preliminary step for the development of highly sensitive minimum residual disease detection systems. A companion article is simultaneously being published in Cytometry Part A that will explain in further detail the theory behind PSM. Three short relevant videos are available in the online supporting information for both of these papers.

Isolation, characterization and cardiac differentiation of human thymus tissue derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are promising candidate donor cells for replacement of cardiomyocyte loss during ischemia and in vitro generation of myocardial tissue. We have successfully isolated MSCs from the discarded neonatal thymus gland during cardiac surgery. The thymus MSCs were characterized by cell-surface antigen expression. These cells have high ability for proliferation and are able to differentiate into osteoblasts and adipocytes in vitro. For cardiac differentiation, the cells were divided into 3 groups: untreated control; 5-azacytidine group and sequential exposure to 5-azacytidine, bone morphogenetic protein 4, and basic fibroblast growth factor. Thymus MSCs showed a fibrolast-like morphology and some differentiated cells increased in size, formed a ball-like appearance over time and spontaneously contracting cells were observed in sequential exposure group. Immunostaining studies, cardiac specific genes/protein expression confirmed the cardiomyocyte phenotype of the differentiated cells. These results demonstrate that thymus MSCs can be a promising cellular source for cardiac cell therapy and tissue engineering.

The biological characteristics of different generation mesenchymal stem cells cultured in vitro and cocultured with vascular scaffold

Mesenchymal stem cells (MSCs) were used widely as seed cells in tissue engineering blood vessel construction. However, the biological characteristics difference of different generation MSCs in vitro culture is unknown, which laid a foundation for appropriate generation seeded cells selection for tissue engineering blood vessel construction. In this report, MSCs were isolated from SD rat bone marrow and identified by flow cytometry; cell growth curve test, cell surface antigen expression rate detection, cryopreservation resuscitation rate test, CD31 expression rate test, cell cycle analysis, and adhesion difference on vascular scaffold test were performed. The research results indicated that the MSCs shape was spindle and uniform with vigorous growth. CD105 and CD90 factor expression rate reached 82.5 and 84.9%, respectively, and the expression rate of CD45 was only 7.3%. The proliferation capacity of the fourth generation MSCs were more exuberant, with proliferation index as 20.3%; the cell proliferation index of the eighth generation decreased to only 9.1%. The cryopreservation resuscitation rate of the second generation and fourth generation MSCs were both higher than 80%, and the cryopreservation resuscitation rate of the eighth generation MSCs was only about 60%. After the induction for 5 days, MSCs had weak CD31 expression, and with the prolonged induction time, expression increased. All generation MSCs expressed CD31 after being induced for 10 days; however, the CD31 positive expression rate of the second generation, fourth generation, and sixth generation MSCs had significant difference with the eighth generation MSCs. Adhesion rate of MSCs before sixth generation was around 40%, but the adhesion rate of eighth generation MSCs was only about 27%. In all, biological characteristics of different generation MSCs existed certain differences, and especially the eighth generation MSCs aged seriously, whose cell activity decreased significantly. The researchers believed that the MSCs before the sixth generation can maintain excellent properties of MSCs, and can be used as seed cells for vascular tissue engineering.

Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential.

Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

Mucosal-associated invariant T cell is a potential marker to distinguish fibromyalgia syndrome from arthritis.

BackgroundFibromyalgia (FM) is defined as a widely distributed pain. While many rheumatologists and pain physicians have considered it to be a pain disorder, psychiatry, psychology, and general medicine have deemed it to be a syndrome (FMS) or psychosomatic disorder. The lack of concrete structural and/or pathological evidence has made patients suffer prejudice that FMS is a medically unexplained symptom, implying inauthenticity. Furthermore, FMS often exhibits comorbidity with rheumatoid arthritis (RA) or spondyloarthritis (SpA), both of which show similar indications. In this study, disease specific biomarkers were sought in blood samples from patients to facilitate objective diagnoses of FMS, and distinguish it from RA and SpA.MethodsPeripheral blood mononuclear cells (PBMCs) from patients and healthy donors (HD) were subjected to multicolor flow cytometric analysis. The percentage of mucosal-associated invariant T (MAIT) cells in PBMCs and the mean fluorescent intensity (MFI) of cell surface antigen expression in MAIT cells were analyzed.ResultsThere was a decrease in the MAIT cell population in FMS, RA, and SpA compared with HD. Among the cell surface antigens in MAIT cells, three chemokine receptors, CCR4, CCR7, and CXCR1, a natural killer (NK) receptor, NKp80, a signaling lymphocyte associated molecule (SLAM) family, CD150, a degrunulation marker, CD107a, and a coreceptor, CD8β emerged as potential biomarkers for FMS to distinguish from HD. Additionally, a memory marker, CD44 and an inflammatory chemokine receptor, CXCR1 appeared possible markers for RA, while a homeostatic chemokine receptor, CXCR4 deserved for SpA to differentiate from FMS. Furthermore, the drug treatment interruption resulted in alternation of the expression of CCR4, CCR5, CXCR4, CD27, CD28, inducible costimulatory molecule (ICOS), CD127 (IL-7 receptor α), CD94, NKp80, an activation marker, CD69, an integrin family member, CD49d, and a dipeptidase, CD26, in FMS.ConclusionsCombined with the currently available diagnostic procedures and criteria, analysis of MAIT cells offers a more objective standard for the diagnosis of FMS, RA, and SpA, which exhibit multifaceted and confusingly similar clinical manifestations.

Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

Research progress on multiple myeloma immunophenotyping and minimal residual disease detected by flow cytometry

Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and remained incurable. Flow cytometry has been widely used in the detection of immunophenotype and minimal residual disease, diagnosis, monitoring and prognosis of MM. Normal plasma cells and malignant plasma cells can be distinguished according to different cell surface antigen expression. The clinical significane of many immune markes has been elucidated. However, the clinical significance of some phenotype remains controversial, the detection scheme and gating strategy are not unified. This review discusses the recent research progress on detection of MM immunophenotype and minimal residual disease by flow cytovetry.

Role of novel retroviruses in chronic liver disease: assessing the link of human betaretrovirus with primary biliary cirrhosis.

A human betaretrovirus resembling mouse mammary tumor virus has been characterized in patients with primary biliary cirrhosis. The agent triggers a disease-specific phenotype in vitro with aberrant cell-surface expression of mitochondrial antigens. The presentation of a usually sequestered self-protein is thought to lead to the loss of tolerance and the production of anti-mitochondrial antibodies associated with the disease. Similar observations have been made in mouse models, where mouse mammary tumor virus infection has been linked with the development of cholangitis and production of anti-mitochondrial antibodies. The use of combination antiretroviral therapy has been shown to impact on histological and biochemical disease in mouse models of autoimmune biliary disease and in clinical trials of patients with primary biliary cirrhosis. However, the HIV protease inhibitors are not well tolerated in patients with primary biliary cirrhosis, and more efficacious regimens will be required to clearly link reduction of viral load with improvement of cholangitis.

The growth of dental pulp stem cells in portland cement micro-environment

Background: Portland cement (PC), the base material of mineral trioxide aggregate has been applied in vivo and in vitro studies, and showed promising physical and mechanical properties on several cell lineages but human dental pulp stem cells (DPSCs), known as mesenchymal stem cells with their multipotency. Our study aims to evaluate the cytotoxicity of PC mixing with distilled water (Td) or normal saline (Tn) on DPSCs. Materials and Methods: DPSCs were isolated from pulp tissue and identified before viability assay. DPSCs were treated with the concentration of 100%, 50%, 25%, 12.5% and 6.25% extracts in Td and Tn. Cell viability was evaluate after 24 h, 48 h, 72 h and 7 days treating. Cells were analyzed for cell surface antigen expression by flow cytometry. The pH levels of extracts were detected by pH meter. Cell viability was determined with cell counting kit-8 assay. Results: Viability of Td and Tn showed the general trend that dropped slightly at 24 h, increased at 48 h then showed no statistical differences to control at 72 h. The viability of the concentration of 100% groups dropped gradually from 24 h to 72 h. Cell proliferation was improved in low concentration groups on 48 h and 72 h. On day 7, cell viability showed no statistical difference to control except Tn 50% (110.9%) and Tn 100% (84.0%). Conclusions: PC is probably a potential candidate for the use of pulp therapy, or further, a budding material for pulp regeneration.

Comparison of mesenchymal stem cell markers in multiple human adult stem cells.

Objectives:Mesenchymal stem cells (MSCs) are adult stem cells which identified by adherence to plastic, expression of cell surface markers including CD44, CD90, CD105, CD106, CD166, and Stro-1, lack of the expression of hematopoietic markers, no immunogenic effect and replacement of damaged tissues. These properties led to development of progressive methods to isolation and characterization of MSCs from various sources for therapeutic applications in regenerative medicine.Methods:We isolated MSC-like cells from testis biopsies, ovary, hair follicle and umbilical cord Wharton’s jelly and investigated the expression of specific cell surface antigens using flow cytometry in order to verify stemness properties of these cells.Results:All four cell types adhered to plastic culture flask a few days after primary culture. All our cells positively expressed common MSC- specific cell surface markers. Moreover, our results revealed the expression of CD19and CD45 antigens in these cells.Conclusion:According to our results, high expression of CD44 in spermatogonial stem cells (SSCs), hair follicle stem cells (HFSCs),granulosa cells (GCs)and Wharton’s jelly- MSCs (WJ-MSCs)may help them to maintain stemness properties. Furthermore, we suggest that CD105+SSCs, HFSCs and WJ-MSCs revealed the osteogenic potential of these cells. Moreover, high expression of CD90 in SSCs and HFSCs may associate to higher growth and differentiation potential of these cells. Further, the presence of CD19 on SSCs and GCs may help them to efficiency in response to trans-membrane signals. Thus, these four types of MSCs may be useful in clinical applications and cell therapy.