OBJECTIVETo investigate the levels of CD4+CD25+CD127(low) regulatory T cells (Tregs) and the expression of Foxp3 gene in peripheral blood of children with aplastic anemia (AA) and to study their roles in the pathogenesis of AA.

METHODSTwenty-one children with chronic AA, 9 with acute AA and 15 healthy children were enrolled. The proportion of CD4+CD25+ CD127low Tregs in CD4+ T cells was evaluated by flow cytometric analysis. The level of Foxp3 mRNA was ascertained by RT-PCR.

RESULTSThe percentage of peripheral blood CD4+T cells and CD4+CD25+ and CD4+CD25+CD127(low) Tregs in CD4+T cells in both the acute and chronic AA groups was significantly lower than that in the normal control group (P<0.05).The acute AA group had more decreased CD4+ T cells and CD4+CD25+ and CD4+CD25+CD127(low) Tregs percentage compared with the CAA group (P<0.05). The expression of Foxp3 mRNA in peripheral blood decreased obviously in the acute AA group (0.47 + or - 0.08%) compared with that in the normal control (0.71 + or - 0.12%) and the CAA groups (0.68 + or - 0.14%) (P<0.05).

CONCLUSIONSThe low expression of Tregs and Foxp3 mRNA in peripheral blood may be involved in pathogenesis of AA.The more decreased Tregs and Foxp3 mRNA expression in acute AA than chronic AA suggests their possible roles in the assessment of the severity of AA.

Adolescent ; Anemia, Aplastic ; etiology ; genetics ; immunology ; Child ; Child, Preschool ; Female ; Forkhead Transcription Factors ; genetics ; Humans ; Male ; RNA, Messenger ; blood ; T-Lymphocytes, Regulatory ; immunology

Objective To investigate the expression of cyclin dependent kinases 5(CDK5)in the temporal lobes of the epilepsy patients and to explore the possible roles of CDK5 in the pathogenesis of refractory epilepsy.Methods The brain tissues of intractable epilepsy(IE)were studied by fluorescence quantative polymerase chain reaction(FQ-PCR)for CDK5 mRNA,while immunohistochemistry and Western blot were used to study the protein expression.Nonepileptogenic control brain tissues were used for comparison.Results FQ-PCR analysis showed that the expression of CDK5 mRNA in epilepsy patients was significant higher than those in the control group.And immunohistochemistry showed that the protein mainly existed in the neuron and glial.At the 35000 relative molecular mass,Western blot could been seen that there is a limpid strap.The optical density of CDK5 in IE(temporal lobe 1.4293?0.1839,hippocampus 2.0733?0.4738)was significantly higher than that in the control(temporal lobe 0.9680?0.4147, hippocampus 1.4030?0.6160,P

Objective To study the cytotoxicity of multiple gliomaassociated antigens sensitized dentritic cell activated cytotoxic T lymphocytes (GDC-CTL) on the human glioma cell line U87 in vitro and the anti-tumor effect of GDC-CTL on the BALB/c nude mouse model of malignant glioma in vivo.Methods Multiple glioma-associated antigens sensitized dentritic cell (GDC) and GDC-CTL were prepared and then analyzed with the phenotypes by flow cytometry.Cytotoxicity of GDC-CTL on U87 cells was determined by CCK8 assay and the level of interferon-γ (IFN-γ) secreted from GDC-CTL co-culturing with U87 cells for 48 h was detected by ELISA at different effect/target ratios (5∶ 1,10∶1,20∶1).The T lymphocytes without activation with GDC were evaluated as the control group.The BALB/c Nude mice tumor model established by the subcutaneous injection of U87 cells was adopted to assess the anti-tumor effect.The mice were randomly divided into four groups:the control group receiving subcutaneous injection with 0.9％ NaCl 0.2 mL,the model,intravenous treatment and local treatment groups receiving subcutaneous injection with 1 × 107 U87 cells in 0.2 mL Dulbecco's Modified Eagle Medium (DMEM).When the diameter of tumor tissue reached 3 mm,the model group was subcutaneously injected with 0.9％ NaC1 0.2 mL surrounding the tumor,while the intravenous treatment group and local treatment group were injected with 0.2 × 107 GDC-CTL in 0.2 mL phosphate buffer saline (PBS) through the tail vein and subcutaneous injection into the surrounding area of the tumor respectively,3 times a week for 2 weeks.The tumor volume was calculated and the pathological changes in the tumor tissues were observed for comparison.Results Matured GDC expressing the high levels of CD83,CD1a and HLA-DR successfully activated GDC-CTL in which 93.00％ of CD3 + T lymphocytes and 69.00％ of CD3 + CD8 + T lymphocytes were detected.In vitro experiments proved that the killing rates of GDC-CTL and T lymphocytes on U87 cells were (24.35 ±1.12)％ vs (15.21 ±0.91)％,(38.57±2.10)％ vs (23.35 ±1.30)％,(59.44±3.79)％ vs (35.23 ± 2.33) ％,and the IFN-γlevels secreted from GDC-CTL and T lymphocytes co-culturing with U87 cells were (405.36±27.65) vs (371.11 ±23.23) pg · mL-1,(1509.22 ±97.16) vs (913.54 ±48.35) pg · mL-1,(2429.57 ±183.18) vs (1814.97 ± 123.24) pg · mL-1,at the different effect/target ratios of 5∶1,10∶1 and 20∶1 respectively.There were significant differences between this two groups at the effect/target ratios of 10∶1 and 20∶1 (P ＜0.05).The results obtained from the in vivo experiments showed that the tumor volumes in the intravenous treatment group and local treatment group shrank 34.83％ and 45.37％ respectively,when comparing with the model group (100.00％,P ＜ 0.05).The pathological changes of tumor tissues showed that the tumor cells in the local treatment group and intravenous treatment group were significandy decreased.Conclusion The experimental results that GDC-CTL can significantly inhibit the growth of ghoma provide more evidences to further study the effective targeting therapy on glioma.

Objective The lentiviral vector was recombined with metal-lothionein ( MT) gene to identify the MT overexpression in human adi-pose-derived mesenchymal stem cells ( hADSCs) after transfection and then to study the lead tolerance of genetically modified hADSCs with MT (MT-hADSCs).Methods The recombinant plenti-CMV-MT2A-EYFP vector was constructed with pLenti -CMV -hChR 2 ( E123 T -H134R)-EYFP and MT2A gene for transfecting hADSCs to obtain the MT-hADSCs.The overexpression of MT in hADSCs was identified by immunofluorescence assay.The MTT method was used to assess the cell viability of hADSCs, hADSCs transfected with empty vector, and MT-hADSCs, all of which were treated with lead acetate.Results The re-combinant plenti -CMV -MT2A -EYFP was successfully constructed and transfected into hADSCs.The overexpression of MT was positively detected in the MT -hADSCs.The tolerance of MT-hADSCs to lead was significantly higher than the hADSCs and hADSCs transfected with empty vector.Conclusion MT can significantly increase the tolerance of hADSCs to lead, indicating that MT can reduce the cytotoxicity of lead.The experimental results from this study provide more evidences for further study of using MT-hADSCs to treat lead poisoning.

OBJECTIVETo investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells.

METHODSThe cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 μmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively.

RESULTSSen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05).

CONCLUSIONSen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Hypoxia ; drug effects ; Cell Nucleus ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Fluorescence ; Intracellular Space ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; NADPH Oxidases ; metabolism ; Neuroprotective Agents ; pharmacology ; Oxygen ; pharmacology ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; Staining and Labeling

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipe-line and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to gen-erate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.