Bispecific CS1-BCMA CAR-T cells are clinically active in relapsed or refractory multiple myeloma.

Multiple myeloma (MM) bears heterogeneous cells that poses a challenge for single-target immunotherapies. Here we constructed bispecific CS1-BCMA CAR-T cells aiming to augment BCMA targeting with CS1. Sixteen patients with relapsed or refractory (RR) MM received CS1-BCMA CAR-T infusion. Six patients (38%) had cytokine release syndrome, which was of grade 1-2 in 31%. No neurological toxicities were observed. The most common severe adverse events were hematological, including leukopenia (100%), neutropenia (94%), lymphopenia (100%) and thrombocytopenia (31%). Three patients with solitary extramedullary disease (sEMD) did not respond. At a median follow-up of 246 days, 13 patients (81%) had an overall response and attained minimal residual disease-negativity, and six (38%) reached a stringent complete response (sCR). Among the 13 responders, 1-year overall survival and progression-free survival were 72.73% and 56.26%, respectively. Four patients maintained sCR with a median duration of 17 months. Four patients experienced BCMA+ and CS1+ relapse or progression. One patient responded after anti-BCMA CAR-T treatment failure. Lenalidomide maintenance after CAR-T infusion and the resistance mechanism of sEMD were preliminarily explored in three patients. CAR-T cells persisted at a median of 406 days. Soluble BCMA could serve as an ideal biomarker for efficacy monitoring. CS1-BCMA CAR-T cells were clinically active with good safety profiles in patients with RRMM. Clinical trial registration: This study was registered on ClinicalTrials.gov, number NCT04662099.

A Six-Gene Risk Model Based on the Immune Score Reveals Prognosis in Intermediate-Risk Acute Myeloid Leukemia.

Tumor microenvironment (TME) has been revealed as an important determinant of diagnosis and treatment response in AML patients. The scores of immune and stromal cell scores of AML in the intermediate-risk group from The Cancer Genome Atlas (TCGA) database were calculated using the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data algorithm. Differentially expressed genes were identified between high and low scores. Gene set enrichment and pathway analyses were performed. A risk score model based on TME for six immune-related genes was established and validated. Patients with a lower immune score had a longer overall survival than those with a higher score (P = 0.044). A total of 805 intersected genes as differentially expressed genes were identified and selected according to the comparison of both immune and stromal scores. The functional enrichment analysis shows that these genes are mainly associated with the immune/inflammatory response. The risk score model based on TME for six immune-related genes (including MEF2C, ENPP2, FAM107A, CD37, TNFAIP8L2, and CASS4) was established and validated in the TCGA database and well validated in the TARGET database (P = 0.005). A key microenvironment-related gene signature was identified that affects the outcomes of AML patients in the intermediate-risk group and might serve as therapeutic targets.

Optimal predictor for 6-mercaptopurine intolerance in Chinese children with acute lymphoblastic leukemia: NUDT15, TPMT, or ITPA genetic variants?

BACKGROUND: 6-mercaptopurine (6-MP) contributes substantially to remarkable improvement in the survival of childhood acute lymphoblastic leukemia (ALL) patients. However, 6-MP also has dose-limiting toxicities, particularly life-threatening myelosuppression, due to genetic polymorphisms in enzymes that metabolize 6-MP. Promising biomarkers for predicting 6-MP-induced leukopenia is still unclear in Chinese population. Here, we evaluated the associations of NUDT15, TPMT and ITPA genotypes with 6-MP intolerance in our cohort of childhood ALL patients. METHODS: A total of 105 Chinese pediatric patients with a confirmed diagnosis of ALL were enrolled. We identified the NUDT15 coding variant rs116855232 (c.415C > T), a newly discovered 6-MP toxicity-related locus in Asians, and polymorphisms in TPMT rs1142345 and ITPA rs11273540. Associations between genotypes and 6-MP dose sensitivity, leukopenia, hepatotoxicity, and therapy interruption were evaluated. RESULTS: The minor allele frequencies (MAFs) of NUDT15 rs116855232, TPMT rs1142345 and ITPA rs11273540 were 15.7, 2.9, and 18.1%, respectively. NUDT15 and TPMT genetic variants were strongly associated with 6-MP dose intensity. Patients with NUDT15 homogenous genotype (TT) were highly sensitive to 6-MP (dose intensity of 60.27%) compared to these with heterozygous genotype (TC) or wild type (CC), who tolerated an average dose intensity of 83.83 and 94.24%, respectively. The NUDT15 variant was a predictor for leukopenia (OR: 3.62, 95% CI 1.377-9.501, P = 0.009) and early-onset leukopenia (OR: 9.63, 95% CI 2.764-33.514, P = 3.75 × 10- 4). No differences were found between 6-MP dose intensity and ITPA polymorphisms. CONCLUSION: NUDT15 variant is an optimal predictor for 6-MP intolerance in Chinese pediatric ALL patients and may have greatly clinical implications for individualized therapy.

Effects of dendritic cell-activated and cytokine-induced killer cell therapy on 22 children with acute myeloid leukemia after chemotherapy.

The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.

Cluster of differentiation 96 as a leukemia stem cell-specific marker and a factor for prognosis evaluation in leukemia.

Resistance to chemotherapy is a major challenge for leukemia treatment. It has been suggested that leukemia stem cells (LSCs), a small pool of self-renewing leukemic cells, play important roles in development of chemotherapy resistance. The expression of cluster of differentiation 96 (CD96), a potential marker for LSCs, was investigated in CD34+CD38- cells of 105 acute leukemia (AL) patients by flow cytometry. The data showed that all the CD34+, CD34+CD38- and CD34+CD38-CD96+ proportions were much higher in AL compared to the normal control (P<0.01), while a clear difference was identified in the CD34+CD38- and CD34+CD38-CD96+ proportions between acute lymphoid leukemia and acute myeloid leukemia (AML). However, all the AML patients with >15% CD34+CD38- cells achieved complete remission (CR), suggesting that as an LSC-rich population, the amount of CD34+CD38- cells may not be positively associated with the proportion of refractory LSCs. The mean percentage of the co-presence of CD96 expression itself was similar in AML patients with CR and non-CR (P>0.05). However, the CR rate was significantly higher in the AML population with <10% CD96 expressed, which indicated that a distinct sub-group of CD34+CD38-CD96+ cells may still contribute to the drug resistance or poor prognosis.

[Biological Effects of the SARI Over-expression on K562 Cell Line].

OBJECTIVE: To construct a lentivirus vector carrying SARI gene and to investigate its biological effects on K562 cells. METHODS: SARI was amplified from the plasmid containing SARI cDNA and subcloned into pLOV.CMV.eGFP virus vector. After sequencing, lentivirus packaging, titering, the viruses of SARI-pLOV.CMV.eGFP were harvested and tansfected into the K562 cells. Real-time quantitive PCR and Western blot were performed to validate the SARI expression at the level of mRNA and protein respectively. Simultaneously, the proliferation, apoptosis and cell cycle of K562 cells were detected by CCK-8 and flow cytometry respectively. RESULTS: The SARI overexpressed lentivirus vector was successfully constructed. The mRNA and protein levels of SARI increased significantly in the pLOV.CMV.eGFP-SARI group, which was confirmed by Q-PCR and Western blot; as compared with blank and mock groups, SARI over-expression leaded to significant proliferation inhibition and increased apoptosis of K562 cells, without visible effects on cell cycle. CONCLUSION: the over-expression of SARI gene obviously suppresses the cell proliferation of the K562 cells as well as promotes the apoptosis. The results implied that the induction of the SARI gene expression may be an important candidate therapeutic method for the CML.

Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation.

A female patient diagnosed with acute myelocytic leukemia M5a (AML-M5a) relapsed 986 days after her allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an unrelated male donor with matched human leukocyte antigen (HLA). Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer (CIK) cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion (DLI) and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.

Detection of circulating tumor cells and tumor stem cells in patients with breast cancer by using flow cytometry: a valuable tool for diagnosis and prognosis evaluation.

Circulating tumor stem cells (CTSC), a subpopulation of circulating tumor cells (CTC), may lead to recurrent diseases. The aim of this study was to detect CTC (CD45(-)EpCAM(+)) and CTSC (CD45(-)EpCAM(+)CD44(+)CD24(-)) of breast cancer (BC) patients, as well as to explore their clinical relevance. CTC and CTSC in peripheral blood (PB) of 45 female BC patients were detected by using flow cytometry (FCM). SKBR-3 cells were mixed with MNC of four healthy volunteers at different ratios in order to evaluate the sensitivity of FCM. Real-time quantitative polymerase chain reaction (QRT-PCR) was conducted and compared with FCM. The expression of EPCAM between CTC < 50 and CTC ≥ 50 groups (19.98 ± 23.93 versus 29.46 ± 29.27 × 10(-5)), and the expression of CD44 between CTSC negative and positive groups (0.85 ± 0.91 versus 0.81 ± 0.75) were statistically the same. FCM had higher specificity than QRT-PCR. Statistical differences were obtained between CTC < 50 and CTC ≥ 50 groups among different TNM stages, histology stages, estrogen receptor (ER) status and progesterone receptor (PR) status (P < 0.05). Statistical differences between CTSC negative and positive groups within different TNM stages and regional lymph node metastasis (RLNM) status (P < 0.05) were also obtained. Moreover, the percentage of CTC on CD45 negative cells (CD45(-)C) among different clinical pathology was statistically different, P = 0.000. Additionally, the percentage of CTSC on CD45(-)C with TNM stage was rising (0: 0.00 ± 0.00‰, I: 0.03 ± 0.05‰, II: 0.06 ± 0.14‰, III: 0.10 ± 0.09‰, IV: 0.29 ± 0.35‰, P = 0.034). Statistical difference in the percentage of CTSC on CD45(-)C among different RLNM status (P = 0.001) was also obtained. FCM to detect CTC and CTSC may be used to diagnose disease at early stage, to guide clinical therapy or to predict prognosis.

Detection of circulating tumor cells in breast cancer patients utilizing multiparameter flow cytometry and assessment of the prognosis of patients in different CTCs levels.

We wanted to demonstrate the value of multiparameter flow cytometry in detecting human tumor cells of breast cancer (BC) (SKBR-3) in normal peripheral blood. In addition, we investigated a cluster of patients to compare the overall survival (OS) between advanced BC patients [circulating tumor cells (CTCs) >or=5 group] and limited BC patients (CTCs <5 group). SKBR-3 human BC cells were serially diluted in normal whole blood to demonstrate the sensitivity of multiparameter flow cytometry for detecting CTCs, and we also compared the specificity with reverse transcriptase polymerase chain reaction (RT-PCR) method. On the other hand, we detected CTCs among 45 patients by multiparameter flow cytometry. OS was calculated by the Kaplan-Meier product limit method, and compared it between CTCs <5 and CTCs >or=5 groups with the log-rank test. Cox regression models were fitted to determine the associated factors on survival. Human BC cells (SKBR-3) could be differentiated from normal blood based on the multiple light scatter and cell surface marker expression by multiparameter flow cytometry. The method was found to have a sensitivity limit of 10(-5) and was effective for detecting human BC cells in vivo. It also found that this method had a higher specificity compared with RT-PCR. For the retrospective study, the median OS was 95 weeks and 65.5 weeks (P < 0.05, 2-tailed) for patients with CTCs <5 and CTCs >or=5, respectively. Kaplan-Meier was used to analyze the patients' survival with Log Rank P = 0.004 and Breslow P = 0.003, which showed that these two groups had statistically significant difference. Cox regression analysis was performed, and we found CTCslevels, metastasis and age (P < 0.05) were three relative factors for patients' survival. Multiparameter flow cytometry can detect CTCs effectively and has the potential to be a valuable tool for prognosis assessment among BC patients in clinical situations in China.

Cross-lineage expression in 505 patients with acute lymphoblastic leukemia by multiparametric flow cytometry analysis.

The expression of immunological markers of one hematopoietic lineage on the abnormal cells of another lineage (cross-lineage expression) is a known feature of leukemia. The present study was aimed to investigate the cross-lineage expression in ALL cells. The cross-lineage expression in ALL cells from 505 patients was detected by flow cytometry using 23 monoclonal antibodies (McAbs) in triple staining combinations. The results showed that in whole ALL, the expression of myeloid antigens occurred in 56.4% of the cases, and CD13 was the most frequently expressed myeloid marker (32.7%) followed by CD33 (29.5%), CD15 (19.2%) and CD11b (7.7%). CD13/CD33 expressions were more frequent in CD34(+) cases than in CD34(-) cases. In B-ALL, T-cell antigen CD4, CD5, CD7 and CD2 were found in 27 (6.3%), 12 (2.8%), 8 (1.9%), and 6 (1.4%) cases respectively, and CD7(+), CD2(+) and CD4(+) cases commonly expressed CD13/CD33. In T-ALL, B-cell antigen cCD79a, CD19 and CD22 were found in 6 (8.1%), 5 (6.8%), and 2 (2.8%) cases respectively, and all of CD19(+) and CD22(+) cases were all accompanied with CD13/CD33. It is concluded that cross-lineage expression in ALL mostly exists in the immature stages, ALL cells more frequently express phenotypes B(+)M(+), T(+)M(+) and occasionally B(+)T(+)M(+), but B(+)T(+)M(-) phenotype is extremely rare.

[Peripheral T cell subsets in chronic tonsillitis patients].

OBJECTIVE: To investigate the characteristics of T lymphocytic subsets in chronic tonsillitis patients for evaluation of their clinical implication. METHOD: Fresh peripheral blood samples were obtained from 54 chronic tonsillitis patients and 52 healthy counterparts. CD4+ and CD8+ T lymphocyte subsets including the naive (CD45RA+), memory (CD45RO+), functional (CD28+), activated (HLA-DR+, CD25+) and apoptosis (CD95+) T lymphocytes, were analyzed by flow cytometry, respectively. The clinical data such as serum Cystatin C con centration, ASO and ESR were simultaneously recorded from each chronic tonsillitis patient. RESULT: The CD4+ T cells, the rate of CD4+/CD8+ and CD4+ CD45RA+ /CD4+ CD45RO+, the CD4+ CD45RA+ T cells and the CD4+ CD25+ T cells in chronic tonsillitis were significantly lower than those of the control group (P < 0.05) while an obviously increasing percentage of memory (CD45RO+) and apoptosis (CD95+) T lymphocytes in chronic tonsillitis patients, and there were significant differences between the patients with chronic tonsillitis and the healthy volunteers (P < 0.05). Furthermore, in the chronic tonsillitis patients, Serum Cystatin C level was negatively correlated with CD4+ CD45RA+ T(P < 0.05), and not significantly correlated with other T lymphocyte subtypes (P > 0.05). CONCLUSION: Immune disorder is present in the peripheral blood of chronic tonsillitis patients. Our data may provide valuable information for evaluation of disease progression of chronic tonsillitis patients.

[Effect of CCL23/myeloid progenitor inhibitory factor 1 (MPIF-1) on the proliferation, apoptosis and differentiation of U937 cells].

CCL23 is a human CC chemokine with potential suppression effects on both human and murine myeloid progenitor cells both in vitro and in vivo, and only expressed and released by dendritic cells differentiated from monocytes in blood cells. However, recent study has shown that CCL23 was over-expressed in bone marrow and peripheral blood cells from pediatric patients with acute myeloid leukemia (AML). In order to investigate the effects of CCL23 on the development, therapy and prognosis of leukemia, the U937 cells, a leukemic cell strain, were adopted and cultured with rhCCL23 for 72 hours. The cell proliferation and apoptosis rate were detected by Cell Counting Kit-8 and FITC-AnnexinV/PI respectively; the morphologic changes and the expression of CCR1 (the only receptor of CCL23 known by now) were observed during the differentiation process. The results showed that no obvious effect on the proliferation, apoptosis and differentiation of U937 was found by using CCL23 alone (P > 0.05), but cultured in combination with CCL23 and PMA, the differentiation of U937 cells were promoted remarkably, during which the CCR1 expression increased (P < 0.05). It is concluded that CCL23 alone did not inhibit the proliferation and differentiation of U937, while its use in combination with PMA may possess synergistic effect on inducting differentiation of U937 through the increase of receptor CCR1 expression.

[Effects of acute myeloid leukemia cell supernatant on the proliferation and apoptosis of CD4+ and CD8+ T cell subsets].

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.

[Karyotyping and immunophenotyping analyses of the CD34+ CD38- cells isolated from human umbilical cord blood].

OBJECTIVE: To cultivate hematopoietic stem/progenitor cells (CD34(+)CD38(-)) isolated from umbilical cord blood (UCB) long for the observation of cell growth and expansion in vitro, surface marker expression, and chromosomal complements. METHODS: By flow cytometry CD34-FITC and CD38-PE labeled CD34(+) and CD38(-) stem/progenitor cells were isolated from UCB. The cells were cultivated in vitro for 6 months in a stem cell culture system with addition of six kinds of cell growth factors (IL-3, IL-6, GM-CSF, Epo, SCF, IGF-1). One month after cultivation, cultured cells were investigated for surface marker expression by flow cytometry and karyotype by G banding method. RESULTS: After 7-12 days cultivation, the CD34(+)CD38(-) stem/progenitor cells began proliferation. The proliferation rate and the peak proliferation duration were greater in 1 cell/well cultivation conditions than in 10 cells/well. The cells remained CD34(+)CD38(-) and their karyotypic characteristics remained unchanged. CONCLUSION: CD34(+)CD38(-) stem/progenitor cells from UCB may provide a larger than original amount of stem/progenitor cells for transplantation after long-term cultivation in vitro.

[Correlation of immunophenotype to cytogenetics and clinical features of adult acute myeloid leukemia].

BACKGROUND & OBJECTIVE: New WHO classification has been rapidly used in diagnosis of leukemia. Based on coexpression and correlation of lineage-associated antigens, multiparameter high-resolution flow cytometry has been developed to precisely identify lineage characteristics of leukemia. Some immunophenotypes correlate with cytogenetic abnormality and prognosis. This study was to analyze immunophenotype of naive acute myeloid leukemia (AML), and explore its correlations to cytogenetics, clinical features, and FAB subtype of AML. METHODS: Multiparameter high-resolution flow cytometry with a panel of 25 different monoclonal antibodies was used to analyze the surface and cytoplasmic antigens expressions of 96 adults with AML; G-binding technique was used to analyze karyotype of 73 of the 96 patients. RESULTS: In these AML patients, some antigens were correlated with FAB subtypes:expression of CD2 was enhanced in AML-M3; HLA-DR, CD34, and CD56 were absent in AML-M3; expression of CD19 was increased in AML-M2; expressions of CD14 and CD56 were enhanced in AML-M5; MPO was absent in AML-M0. Karyotype abnormality was detected in 40(54.8%) patients. CD22, CD56, and TdT expressions were correlated with karyotype abnormality. t(8; 21) was only detected in 10 AML-M2 patients with high expressions of CD15, CD19, CD34, and CD56; no lymphoid lineage antigens were detected in 7 AML-M3 patients with t (15; 17). Expressions of CD4 and TdT were positively correlated with patient's age; expressions of CD7 and CD14 were positively correlated with high white blood cell count; expressions of CD4, CD14, and CD56 were positively correlated with high platelet count. CONCLUSIONS: The abnormal antigen expression of AML is tightly linked with karyotype abnormality. Detection of immunophenotype may help to diagnose and classify AML.

[Proliferation and apoptosis in vitro of umbilical cord blood CD34+CD38- hematopoietic early progenitor cells].

To cultivate CD34(+)CD38(-) cells isolated from umbilical cord blood of healthy puerperal women over a longer-period of time for observation of cell division, proliferation, apoptosis, and effects of stem cell factor on the growth of CD34(+)CD38(-) cells, with flow cytometry, CD34(+)CD38(-) cells were isolated from umbilical cord blood of 10 healthy puerperal women and cultivated in stem cell media with supplement of IL-3, IL-6, GM-CSF, EPO, IGF-1 and SCF 6 kinds cell growth stimulating factors for six months. The cell growth curves were established. The effects of stem cell factor on the growth of CD34(+)CD38(-) cells and cell apoptosis were investigated with the single cell gel electrophoresis technique and flow cytometry method, respectively. The results showed that CD34(+)CD38(-) cells isolated from umbilical cord blood were capable of proliferating after being cultivated in vitro over a longer-period of time with no evidence of the presence of excessive apoptosis. In conclusion, under appropriate culture conditions, CD34(+)CD38(-) hematopoietic early progenitor cells from umbilical cord blood can serve as a resource providing a large amount of primitive cells for transplantation therapy after a longer period of cultivation and proliferation in vitro.

[Immunophenotypes in 115 patients with acute myeloid leukemia by multi-color flow cytometry].

Immunophenotyping has become common in the diagnosis and classification of leukemia. To evaluate the immunophenotype of acute myeloid leukemia (AML), multiparameter flow cytometry and CD45/SSC gating were used to analyze the surface and cytoplasmic antigen expressions in 115 cases of AML. The results were compared with the French-American-British (FAB) Cooperative Group classification to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of AML. The results showed that CD38, CD38 and CD13 were the most commonly expressed antigen (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%) and CD2 (15%). Some immunophenotypes correlated with FAB type, including increased frequency of CD2 in M(3); lack of HLA-DR, CD34 and CD56 expression in M(3); increased frequency of CD19 in M(2), CD14 and CD56 in M(5) and lack of MPO in M(0). In conclusion, multiparameter flow cytometry is a reliable technique in the diagnosis of AML, and some immunophenotypes correlate with FAB type.

[Regulation of tissue factor expression in brain microvascular endothelial cells by PLA nanoparticles coating NF-kappaB decoy oligonucleotides].

OBJECTIVE: To investigate a new strategy of polylactic acid (PLA) nanoparticles delivery system coating nuclear factor-kappaB (NF-kappaB) decoy oligonucleotides (ODNs) for inhibiting TF expression in cultured brain microvascular endothelial cells(BMECs). METHODS: PLA nanoparticles coating FITC-labeled NF-kappaB decoy ODNs were formulated by nano-deposition method and the characteristics of nanoparticles were detected. BMECs were isolated and cultured in vitro. The cellular uptake and intracellular localization of nanoparticles in BMECs was detected by flow cytometry and confocal microscopy. Changes in the expressions of TF and nuclear protein P65 were examined by RT-PCR and Western blot in NF-kappaB decoy ODNs transfected BMECs by LPS stimulation. RESULTS: The decoy-nanoparticles obtained were uniform spherical particles with an effective diameter of 162.1 nm and a polydispersity index of 0.118. NF-kappaB decoy ODNs encapsulated in nanoparticles could be released in a controlled manner in phosphate-buffered saline for up to 28 days. It was observed that the cellular uptake of nanoparticles were increased with the time of incubation and the concentration of nanoparticles in the medium. Nanoparticles localized mainly in the BMECs cytoplasm. LPS-induced upregulation of TF transcription was inhibited by NF-kappaB decoy ODNs transfection but not by missense ODNs transfection. Furthermore, changes in the transcription level of TF were paralleled by a reduction of capacity of P65 in nuclear extract of NF-kappaB decoy ODNs transfected cells. CONCLUSIONS: These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.