Objective To establish a method for studying molecular mechanism of Rhubarb inhibiting anti-Yersinia pesti based on DNA microarray.Methods A whole genome DN A microarray containing 4005 annotated genes of Yersiniapesti Was used.The minimal inhibitory concentration(MIC)of Rhubarb to Yersiniapestiwas determined by liquid dilution method.The gene expression profile of Yersinia pesti was performed after the exposure to Rhubarb at a concentration of 10×MIC for 30 minutes.The total RNA extracted and purified from Yersinia pesti Was reversely transfected to cDNA and labeled by Cy3-Cy5 dye.The labeled probes were hybridized to the microarray anti the results were obtained by a laser scanner and the microarray data was confirmed by real-time quantitative RT-PCR.Results The platform of the DNA microarray-based bacteria transcriptional profile was established.A total of 498 genes of Yersinia pesti changed significantly in response to Rhubarb.Among them.358 genes were up-regulated,140 down-reguated.Conclusions The whole genome DNA microarray can be used in the studying of molecular anti-Yersinia pesti mechanism of Rhubarb.

To elucidate the expression of WT1 in all types of leukemias and its implications for monitoring minimal residual disease in patients with acute leukemia, the peripheral blood from 55 leukemia patients and 10 normal voluteer was detected by using FQ-RT-PCR. Follow-up monitoring of WT1 expression of peripheral blood was performed for 20 patients with acute leukemia. The results showed that the expression of WT1 gene in all types of leukemias was significantly higher than that in normal control (P < 0.001). For ANLL and ALL patients, the survival time in the group of WT1 6.8 x 10(-3), (P = 0.027). Follow-up detection of the expression of WT1 in peripheral blood samples from 20 acute leukemia patients, 7 cases relapsed after complete remission has been done. In 5 of 7 relapsed patients, the expression of WT1 had obviously increased about 2 - 3 months before clinical relapse became apparent. It is concluded that the established FQ-RT-PCR method is accurate and specific. The expression of WT1 gene is relatively high in all types of leukemias compared with normal peripheral blood cells, the higher WT1 expression may associate with poor prognosis in acute leukemia, and the dynamics of WT1 level correlate with the disease status. The quantitative assessment of WT1 expression in peripheral blood samples by FQ-RT-PCR may be a useful tool for monitoring minimal residual disease.
Acute Disease ; Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Child ; Female ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia ; blood ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm, Residual ; blood ; diagnosis ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; WT1 Proteins ; genetics

AIM:To verify the safety application of MIL60 in the treatment of corneal neovascularization both in vivo and in vitro.METHODS:We observed the biological characteristics of human corneal epithelial cells.The cell proliferation was analyzed using CCK-8 assay,which also used to test the toxicity of MIL60 and the solvent on cultured human corneal epithelial (HCE).FACs was used to analyze the apoptosis of HCE after treated with MIL60.Also we evaluated the effect of subconjunctival injection of MIL60 on corneal epithelial healing model in normal rat and rats with epithelium defect through slit lamp-microscopy,Draize scores and histopathology way.RESULTS:The proliferation speed of HCE in three groups was the same.MIL60 did no harm on the proliferation of HCE and the apoptosis of HCE,and has no effect on corneal epithelial healing and other parts of the ocular in rats without inflammation cells infiltration.CONCLUSION:When given subconjunctival injection,Mil60 does no harm to the proliferation and apoptosis of HCE,and is safe with ocular application.

Objective To investigate the antibacterial molecular mechanism of Traditional Chinese Medicine Coptis rhizome against Yersinia pestis(Y.pestis).Methods The method based on whole genome DNA micrnarray of Y.pestis was used.The minimal inhibition concentration(MIC)of berberine to Y.pestis was determined with liquid dilution method.Then gene expression profile of Y.pestis was performed after exposed to berberine at the concentration of 10×MIC for 30 minutes.Total RNA extracted and purified from Y.pestis and reverse-transcribed to cDNA,then labeled by Cy-dye.Finally,the labeled probes were hybridized to the microarray and the results were obtained by a laser scanner and analyzed by the SAM software.Results The gene expression profile data revealed that the response of Y.pestis to berberine was a global phenomenon.A total of 360 genes changed significantly.Among them,333 genes were up-regulated,27 down-regulated.These differentially expressed genes were further classified into 24 different functional categories based on the genomie annotation of Y.pestis CO92,in which the number of mainly related genes were 83,75 and 48,including cell envelop,unkown,transport/binding proteins functions.The 40 genes related to the metabolism were upregulated,which was a remarkable change.Conclusion Our results have revealed the general gene expression changes of Y.pestis in response to berberine and demonstrated the antibacterial molecular mechanism of the Coptis rhizome.The major mechanism of Y.pestis in response to berberine is the upregulation of genes related to the metabolism.

This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.
14-3-3 Proteins ; metabolism ; ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Apoptosis ; Cell Proliferation ; HL-60 Cells ; metabolism ; Humans ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

OBJECTIVETo observe the incidence of elimination delay in high dose methotrexate (HDMTX) therapy, its side effects and influence to next course of chemotherapy and analyze the relationship between the dosage, the duration of MTX infusion and the morbidity of the elimination delay.

METHODSA total of 121 childhood acute lymphoblastic leukemia (ALL) (497 infusions of HDMTX) were analysed in this study. The elimination delay rate and the adverse effects in different dose groups (3 g/m2 vs 5 g/m2) and different infusion duration groups (7 h vs 24 h) were compared. The adverse effect evaluation was based on the World Health Organization (WHO) Toxicity Grading Criteria. The rescue dosages of calcium folinate (CF) among these groups were compared through CF/MTX index.

RESULTSThe overall morbidity of elimination delay was 12.1% with a relative risk of 30.6% for the first time. The relative risk for the second time of occurrence was increased to 45.9% (P < 0.01) and it was not significantly increased for the third time (35.3%). Children with elimination delay had lower platelet count (P < 0.01) and higher CF rescue dosage (P < 0.01), while the damage of oral mucous membrane was more severe (P < 0.05) and the next course of chemotherapy would be postponed for a median of 4 days in 3 g group. There was no significant difference in elimination delay rates between 3 g and 5 g groups (12.1% vs 12.0%, P > 0.05), and between 7 h and 24 h MTX infusion groups (13.6% vs 11.9%, P > 0.05). The only side effect occurred in 5 g group was gastrointestinal morbidity. The CF/MTX index of 5 g group without elimination delay was less than that of 3 g group (P < 0.01).

CONCLUSIONElimination delay in HDMTX therapy accompanies the suppression of bone marrow and damage of oral mucous membrane, which need more CF rescues and will postpone the following course of chemotherapy. Elimination delay is not associated with the duration of the infusion and the dosage of MTX within the range of 3 approximately 5 g/m2 but there are individual differences.

Adolescent ; Antimetabolites, Antineoplastic ; adverse effects ; pharmacokinetics ; therapeutic use ; Child ; Child, Preschool ; Dose-Response Relationship, Drug ; Female ; Humans ; Infant ; Male ; Metabolic Clearance Rate ; Methotrexate ; adverse effects ; pharmacokinetics ; therapeutic use ; Nausea ; chemically induced ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; Retrospective Studies ; Treatment Outcome ; Vomiting ; chemically induced ; Young Adult

The aim of this study was to evaluate the value of CD117/CD11b phenotypic analysis to diagnosis and prognosis of acute promyelocytic leukemia (APL). Three- or four-color flow cytometry with a series of 22 monoclonal antibodies and CD45/Side Scatter (SSC) gating strategy were used to identify immunophenotypic characteristics of APL as compared to CML in chronic phase (CML-CP). PML/RAR alpha fusion gene was detected by using reverse-transcription polymerase chain reaction (RT-PCR) technique. The results showed that MPO, CD13 and CD33 were almost expressed in all patients with APL and CML-CP whereas HLA-DR and CD34, the hematopoietic progenitor cell markers, were rarely expressed. The positive rate of CD15 in APL was significantly lower than those in CML-CP (P < 0.01). CD117 was positive in 78.3% of the APL cases and in none of the cases of CML-CP. On the other hand, CD11b was almost positive in all cases of CML-CP, but only 16.9% of the APL cases were found positive for this antigen. The CD117+ CD11b- phenotype was present in 72.3% of APL cases while none of cases with CML-CP with this phenotype. Almost all of the cases with CML-CP had the phenotype of CD117- CD11b+. CD117- CD11b+ phenotype was detected in all patients recovering from APL with CD117+ CD11b- phenotype at diagnosis and after treatment with all-trans-retinoic acid (ARTA) for 2 months. PML/RAR alpha fusion gene was positive in 80.6% (25/31) of the APL cases, of which, 64% of the cases belonged to the type L while only 36% of the cases were showed type S for this fusion gene. The positive rates of CD117 were 87.5%, 44.4% and 33.3% in type L group, S group and negative group respectively. It is concluded that analysis of both CD117 and CD11b phenotype may be helpful to the diagnosis, therapy and prognosis of APL in children and adults and to differentiation of APL from recovering benign myeloid proliferation.
Adolescent ; Adult ; CD11b Antigen ; analysis ; Child ; Female ; Humans ; Immunophenotyping ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; immunology ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; immunology ; Male ; Oncogene Proteins, Fusion ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; analysis

OBJECTIVETo investigate the expression of CD19 on childhood acute leukemia (AL) and its significance, and to provide evidence for the diagnosis and differential diagnosis as well as monoclonal antibody-targeting treatment of leukemia.

METHODSThere were 210 cases of childhood AL, of which 130 cases were male and 80 were female with a mean age of 9 years old. Using a panel of 27 fluorochrome directly labeled monoclonal antibodies, 210 samples from the patients were analyzed with CD45/SSC double parameters and multi-color flow cytometry to determine the expression of CD19.

RESULTSIn the 93 cases of B lineage acute lymphoblastic leukemia (ALL), the positive rate (98.9%, 92/93) of CD19 was significantly higher than that of the other B cell related antigens, such as CD10 (88.2%, 82/93, P = 0.003), CD20 (24.7%, 23/93, P = 0.001) and CD22 (60.2%, 56/93, P = 0.001). CD19 was expressed on all 8 cases of B/myeloid (My) hybrid acute leukemia (HAL) and 1 case of B/T HAL, but was not expressed on all 24 cases of T lineage leukemia and 5 cases of T/My HAL. In the 79 cases of acute myeloid leukemia (AML), only 5 (6.3%) cases expressed CD19. The positive rate (6.3%) of CD19 on AML was significantly lower than that on B lineage ALL (98.9%, P = 0.001). The percentage of CD19 positive cells in B/My HAL (41.6% - 88.7% with a mean of 73.8%) was significant higher than that in CD19(+)-AML (21.4% - 50.4% with a mean of 24.2%; Run Sum test, P = 0.0084). Of the 210 cases, 102 were B lineage related AL including B lineage ALL, B/My HAL and B/T HAL. In B lineage related AL, the sensitivity and the specificity of CD19 was 99.0% (101/102) and 95.4% (13/108) while the positive predictive and the negative predictive values to B lineage were 95.3% (101/106) and 99.0% (103/104), respectively. Using CD19(+) as a single reagent to diagnose B lineage, the false positive rate was 4.6% (5/108) and the false negative rate was 1.0% (1/102) with a general diagnosis index (GDI) of 94.4% [GDI = 1-(false positive rate + false negative rate)].

CONCLUSIONCD19 is continuously and stably expressed on all stages of B lineage differentiation. It is a reliable cell membrane marker for diagnosing B lineage ALL and an ideal target for antibody-targeting treatment of leukemia as well; the expression degree of CD19 can be used to distinguish B/My HAL from CD19(+)-AML; CD19 didn't express on normal myeloid cells but did on some AML cells. Therefore it can be used to detect the minimal residual disease.

Adolescent ; Antigens, CD19 ; analysis ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; classification ; immunology

OBJECTIVELeukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.

METHODS2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.

RESULTSAfter 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.

CONCLUSION2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD19 ; Apoptosis ; drug effects ; Cell Line, Tumor ; Flow Cytometry ; Genistein ; immunology ; pharmacology ; Humans ; Immunotoxins ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology

OBJECTIVETo prepare fluorescein isothiocyanate (FITC) directly conjugated to monoclonal antibody (McAb) anti-human CD14, ZCH-7-2F9 (2F9-FITC).

METHODSAfter generation and purification, the purity and the murine immunoglobulin subtype of the antibody were evaluated with SDS-PAGE and multicolor flow cytometry (FCM). 2F9 McAb was directly labeled with FITC through modified Marsshall's method and the positive rate of the 2F9-FITC on different types of leukemic cells were compared with the standard CD14-FITC by FCM.

RESULTA large quantity of purified 2F9 McAb was prepared. The subtype of 2F9 was murine IgG1kappa. 2F9-FITC was successfully manufactured with A295/A280 ratio of 0.44. The positive cell percentages of 2F9-FITC and CD14-FITC on the monocytes were 84.50% and 90.08%, respectively, while those on lymphocytes were only 0.52% and 1.01%. There was no significant difference between the CD14 expressions with 2F9-FITC and CD14-FITC on each type of leukemia (n=23, t=0.922, P=0.367).

CONCLUSION2F9-FITC has been successfully prepared and it can be applied in diagnosis and differentiation of monoblastic leukemias.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Cells, Cultured ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; analysis ; chemical synthesis ; Fluorescent Antibody Technique ; Humans ; Leukemia, Lymphoid ; pathology ; Leukemia, Myeloid, Acute ; pathology ; Lipopolysaccharide Receptors ; immunology ; Mice ; Mice, Inbred BALB C ; Monocytes ; cytology