OBJECTIVETo examine the expression of Fas and Fas ligand (FasL) in T lymphocytes of oral lichen planus (OLP) and the effects of Fas, FasL and activation-induced cell death (AICD) on OLP.

METHODSThe oral mucosa samples from patients with OLP and normal oral mucosa were assessed by in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine-5'-triphosphate (dUTP)-biotin nick end-labelling (TUNEL) assay for nucleosomal DNA fragmentation of apoptotic cells in infiltrating lymphocytes. Immunohistochemical technique was used for detection of expression level of Fas and FasL. Immunohistochemical double labeling was performed to examine the expression of Fas and FasL in CD8+ T cells and CD4+ T cells.

RESULTSThe rate of apoptosis in infiltrating lymphocytes of OLP was (1.9 +/- 1.8)%, which was lower than that of normal oral mucosa (P = 0.013). The expression of Fas and FasL were increased in lymphocytes of OLP (P = 0.005 and 0.000 respectively). The positive rate of double labeled cells of CD8+ and Fas+ was not increased in OLP (P = 0.313), and of CD8+ and FasL+ in OLP was higher than that of normal oral mucosa (P = 0.002). The expression of double labeled cells of CD4+ and Fas+ in OLP was higher than that of control group (P = 0.031). The expression of CD4+ and Fas+ T cells in reticular OLP were increased (P = 0.019), and of CD4+ and FasL+ did not show remarkable change (P = 0.097).

CONCLUSIONSThere was a low frequency of lymphocytic apoptosis. T cells, especially CD8+ T cells in OLP and CD4+T cells in atrophic-erosive OLP appeared to escape from AICD, which may account for the persistence of inflammation. Some CD4+ T cells in reticular OLP may go through AICD.

Adult ; Apoptosis ; CD4-Positive T-Lymphocytes ; metabolism ; CD8-Positive T-Lymphocytes ; metabolism ; Fas Ligand Protein ; metabolism ; Female ; Humans ; In Situ Nick-End Labeling ; Lichen Planus, Oral ; metabolism ; pathology ; Male ; Middle Aged ; Mouth Mucosa ; metabolism ; pathology ; fas Receptor ; metabolism

OBJECTIVEThis study was to assess the influence of interaction of combination of immobilized nitrogen cycling bacteria (INCB) with aquatic macrophytes on nitrogen removal from the eutrophic waterbody, and to get insight into different mechanisms involved in nitrogen removal.

METHODSThe aquatic macrophytes used include Eichhornia crassipes (summer-autumn floating macrophyte), Elodea nuttallii (winter-growing submerged macrophyte), and nitrogen cycling bacteria including ammonifying, nitrosating, nitrifying and denitrifying bacteria isolated from Taihu Lake. The immobilization carriers materials were made from hydrophilic monomers 2-hydroxyethyl acrylate (HEA) and hydrophobic 2-hydroxyethyl methylacrylate (HEMA). Two experiments were conducted to evaluate the roles of macrophytes combined with INCB on nitrogen removal from eutrophic water during different seasons.

RESULTSEichhornia crassipes and Elodea nuttallii had different potentials in purification of eutrophic water. Floating macrophyte+bacteria (INCB) performed best in improving water quality (during the first experiment) and decreased total nitrogen (TN) by 70.2%, nitrite and ammonium by 92.2% and 50.9%, respectively, during the experimental period, when water transparency increased from 0.5 m to 1.8 m. When INCB was inoculated into the floating macrophyte system, the populations of nitrosating, nitrifying, and denitrifying bacteria increased by 1 to 2 orders of magnitude compared to the un-inoculated treatments, but ammonifying bacteria showed no obvious difference between different treatments. Lower values of chlorophyll a, COD(Mn), and pH were found in the microbial-plant integrated system, as compared to the control. Highest reduction in N was noted during the treatment with submerged macrophyte+INCB, being 26.1% for TN, 85.2% for nitrite, and 85.2% for ammonium at the end of 2nd experiment. And in the treatment, the populations of ammonifying, nitrosating, nitrifying, and denitrifying bacteria increased by 1 to 3 orders of magnitude, as compared to the un-inoculated treatments. Similar to the first experiment, higher water transparency and lower values of chlorophyll a, COD(Mn) and pH were observed in the plant+ INCB integrated system, as compared to other treatments. These results indicated that plant-microbe interaction showed beneficial effects on N removal from the eutrophic waterbody.

Biodegradation, Environmental ; Eutrophication ; physiology ; Magnoliopsida ; metabolism ; Nitrogen ; isolation & purification ; pharmacokinetics ; Systems Integration ; Water Microbiology ; Water Pollutants, Chemical ; isolation & purification ; pharmacokinetics ; Water Purification ; methods

Female ; Humans ; Hydatidiform Mole ; diagnosis ; Pregnancy ; Uterine Neoplasms ; diagnosis

OBJECTIVETo observe the inhibition of NK4 protein in the proliferation of human Raji lymphoma xenografts in nude mice, and to explore its molecular mechanism.

METHODSModels of human Raji lymphoma xenograft transfected with HGF gene were established by subcutaneous inoculation in nude mice. After establishment of the models, the mice received continuous NK4 protein via tail vein for 4 weeks, and the weight and tumor growth were monitored every week. After 8 weeks, the expression of HGF mRNA and c-Met mRNA of tumor tissues was measured by real-time fluorescent quantitation PCR. The apoptotic index (AI) and microvessel density (MVD) were evaluated by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and immunohistochemistry, respectively.

RESULTSThe models of human Raji lymphoma xenograft were successfully established. Although the animal weights of all groups declined, especially in the groups with NK4 protein injection, there was no statistical significance (P > 0.05). The tumor volume in HGF gene transfected group was larger than those of the control groups (P < 0.01), and there was no statistical significance among the control groups (P > 0.05). However, the tumor volume of the NK4 protein injection group decreased significantly (P < 0.01). Expression of HGF mRNA and c-Met mRNA in HGF gene transfected group increased significantly after injection of NK4 protein (P < 0.01). AI in HGF gene transfected group (33.5% ± 12.3%) was significantly lower than that of control groups (89.1% ± 22.3% vs. 81.9% ± 27.0%, P < 0.05), but became significantly higher (119.1% ± 18.9%) after NK4 protein injection (P < 0.01). MVD in HGF gene transfected group (28.5 ± 2.0) was higher than that of control groups (12.2 ± 1.4, 13.8 ± 1.3, P < 0.01), although declined (15.5 ± 2.5) after NK4 protein injection (P < 0.01).

CONCLUSIONSNK4 protein suppresses significantly the growth of human Raji lymphoma xenografts transfected with HGF gene. The pathogenesis may be involved in promoting tumor cell apoptosis and restraining tumor angiogenesis through competitive interrupting HGF/Met signal pathway.

Animals ; Apoptosis ; Hepatocyte Growth Factor ; genetics ; metabolism ; Heterografts ; Humans ; Lymphoma ; genetics ; metabolism ; therapy ; Mice ; Mice, Nude ; Microvessels ; pathology ; Neovascularization, Pathologic ; Proto-Oncogene Proteins c-met ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; T-Box Domain Proteins ; administration & dosage ; Transfection ; Transplantation, Heterologous

OBJECTIVE: To investigate the relationship of miR-140 expression level with the therapeutic effect of decitabine, and to explore whether the molecular mechanism is dependent on the regulation of TLR4 expression. METHODS: Forty-seven patients with acute myeloid leukaemia (AML) were enrolled in our study and divided into decitabine combination treatment group (22 cases) and traditional treatment group (25 cases). The clinical efficacy was compared between these two groups. Real-time PCR was used to determine the plasma level of miR-140 in AML patients. Decitabine, miR-140 mimic and miR140 inhibitor were used to treat AML HL-60 cells in vitro, the real-time PCR and Western blot were used to detect the expressions of miR-140, TLR4 and NF-κB at both mRNA and protein levels. RESULTS: Compared with traditional treatment group, decitabine combination treatment group showed more significant clinical efficacy. Plasma miR-140 level in both 2 treatment groups both decreased, but the plasma miR-140 level was higher in decitabine combination treatment group as compared with traditional treatment group. Experiment in vitro showed that 0.3 μmol/L decitabine significantly inhibited the HL-60 cell proliferation accompanied by up-regulation of miR-140 expression and down-regulation of expression of TLR4 and NF-κB. These effects induced by decitabine were partly reversed by pretreating the cells with 200 nmol/L miR-140 inhibitor. CONCLUSION Decitabine-induced up-regulation of miR-140 expression may be related with its chemotherapeutic effects, and miR-140/TLR4/NF-κB pathway may partly mediate the pharmacologic action of decitabine.
Decitabine ; therapeutic use ; Down-Regulation ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; MicroRNAs ; NF-kappa B

OBJECTIVETo investigate the alteration and clinical significance of IFN-γ, IL-4, IL-17 and TGF-β levels in serum of patients with chronic lymphocytic leukemia treated with FCR.

METHODSForty-seven CLL patients treated with FCR regimen were enrolled in CLL group, meanwhile 30 healthy persons were selected in control group. The serum levels of IFN-γ, IL-4, IL-17 and TGF-β were detected by ELISA in CLL group before and after treatment and in control group, then the differences of IFN-γ, IL-4, IL-17 and TGF-β levels as well as IFN-γ/IL-4 ratio and TGF-β/IL-17 ratio were compared between 2 groups.

RESULTSBefore treatment with PCR regimen, the IL-4, IL-17 and TGF-β levels as well as TGF-β/IL-17 in CLL group were higher than those in control group (P<0.05), while the IFN-γ level and IFN-γ/IL-4 ratio in CLL group were lower than those in control group (P<0.05); after treatment with PCR regimen, the IL-4, IL-17 and TGF-β levels as well as TGF-β/IL-17 ratio all significantly decreased (P<0.05), while IFN-γ level and IFN-γ/IL-4 ratio significantly increased (P<0.05) as compared with those before treatment, moreover, the IL-4 and IL-17 levels as well as TGF-β/ IL-17 and IFN-γ /IL-4 ratio were no significantly different from those in control group (P>0.05), only the IFN-γ and TGF-β levels were significantly diffrent from control group (P<0.05). The analysis of Binet staging (stage A, B, C) showed that along with pregression of Binet stages, the TGF-γ/IL-17 levels as well as the IFN-γ/IL-4 ratio in CLL group negatively correlated with Binet staging (r=-0.53), while the TGF-β/IL-17 ratio positively correlated with Binet staging (r=0.46). The analysis of grouping accoraing to therapentic efficacy fonnd that the IL-4 and IL-17 levels and IFN-γ/IL-4 and TGF-β/IL-17 ratios in CR and PR groups were significantly different before and after treatment (P<0.05), while those in SD and PD groups did not showed statistical difference before and after treatment (P>0.05).

CONCLUSIONAlong with the progression of disease, the IFN-γ/ IL-4 ratio gradually decreases, and the TGF-β / L-17 ratio gradually increases. The treatment with FCR regimen can overcome this tread, therefore dynamically monitoring the chages of IFN-γ/ IL-4 and TGF-β / L-17 ratios may contribute to guide the clinical treatment.

OBJECTIVE: Two sgRNAs transfected FLT3-ITD⁺AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC₅₀ and higher apoptosis rate. CONCLUSION The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.
CRISPR-Cas Systems ; Doxorubicin/pharmacology* ; Drug Resistance ; Gene Editing ; Humans ; Leukemia, Myeloid, Acute/genetics* ; MicroRNAs/genetics* ; RNA, Guide/genetics* ; fms-Like Tyrosine Kinase 3/genetics*

OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G/G phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.
Apoptosis ; Cell Proliferation ; Gene Knockdown Techniques ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Nuclear Proteins

OBJECTIVETo evaluate the safety and effectiveness of a novel therapeutic regimen for bronchiolitis obliterans sydrome (BOS) affter hematopoietic stem cell transplantation (HSCT).

METHODSSeven patients who had received HSCT and had been diagnosed as BOS were enrolled in this study. They received weekly intravenous injection of umbilical cord-derived mesenchymal stem cells (MSC) at a dose of 1 × 10(6)/kg for 4 weeks. Budesonide was given orally at a daily dose of 0.25 g, and salmeterol was inhaled at a dose of 4.5 µg for 3 times per day. Methylprednisolone was given at a dose of 1 mg/(kg·d) for 2 weeks when respiratory failure occured. The dose of methylprednisolone was tapered to 0.25 mg/(kg·d) after 4 weeks and was adjusted according to the occurrence and severity of chronic graft-versus-host disease (cGVHD).

RESULTSThe therapy was generally safe and no severe acute toxicity was observed. One patient died of heart failure during the treatment, the other 6 patients were alive and the pulmonary function parameters including FEV1, FEV1/FVC, PaO2 and AaDO2 were significantly improved after 6 months as compared with the baseline parameters (P < 0.05).

CONCLUSIONMSC combined with budesonide, almeterol and azithromycin has been confirmed to be generally safe and can reduce the dose of glucocorticoid in treatment of BOS after HSCT.

Azithromycin ; therapeutic use ; Bronchiolitis Obliterans ; therapy ; Budesonide ; therapeutic use ; Combined Modality Therapy ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Humans ; Mesenchymal Stem Cell Transplantation ; Methylprednisolone ; administration & dosage ; therapeutic use ; Salmeterol Xinafoate ; therapeutic use

OBJECTIVETo explore the clinical value of detecting adenosine triphosphate (ATP) level in CD4T lymphocytes (Immuknow ATP) of patients on early stage after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

METHODSThe base-line ATP value in CD4T lymphocytes in cases of hematological malignancies and the ATP level in CD4T lymphocytes of acute leukemia patients before allo-HSCT were detected. Allo-HSCT recipients were devided into 3 groups with different level of immunereactivity according to ATP concentraiton in month 3 (day 90±5) after allo-HSCT. The clinical characteristics of patients in 3 groups were analyzed.

RESULTSThe mass concentration of Immuknow ATP in 15 cases of hematological malignancies before allo-HSCT ranged from 56.21-435.71 ng/ml, with a mean of 203.98±112.72 ng/ml. The ATP level in 46 cases after allo-HSCT ranged from 1.69-333.09 ng/ml, with a median of 41.96 ng/ml. Both 91.26 ng/ml (mean-SD) and 316.70 ng/ml (mean+SD) were used as cutoff, and 36 allo-HSCT recipients (78.3%) were assigned to low immunereactivity group, 8 recipients (17.4%) to middle group and 2 recipients (4.3%) to high group. The incidence of infection in low immunereactivity group was significantly higher than that in middle immunereactivity group (86.1% vs 50.0%)(P=0.022), and also significantly higher than that in high immunereactivity group (86.1% vs 0%)(P=0.002). There were no statistical differences in the incidences of severe infection among 3 groups. The incidence of grade II or higher acute graft versus host disease (aGVHD) in high immunereactivity group was superior to that in low immunereactivity group statistically (100% vs 13.9%)(P=0.002). Immune-mediated organ injury occurred more frequently in high immunereactivity group as compared with low and middle immunereactivity groups (100% vs 0% and vs 0%)(P=0.000; P=0.002). There were no significant differences in relapse rates of leukemia among 3 groups. The percentage of patients with increased trough blood concentration of cyclosporine A(CsA) was not significantly different among 3 groups (P=0.720).

CONCLUSIONDetection of ATP level in CD4T lymphocytes on early stage after allo-HSCT possesses clinical significance for predicting infection, severity at aGVHD and immune-mediated organ injury.