Full-length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template. The amplified fragment (about 1.34 kb) was cloned into plasmid pQE32, and the recombinant plasmid pQENS5A was expressed in JM109 strain. The NS5A protein was purified by NiSO4 metal chelating resin, and characterized by Western-blot. Its antigenecity was determined by ELISA. The positive detection rate of anti-NS5A was 75% (69/92) in ninety-two clinic sera. The positive rate of anti-NS5A was 82.5% (33/40) in fourty positive standand sera, and the negative rate of anti-NS5A was 100% (40/40) in fourty negative standand sera. The results showed that the Full-length NS5A proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera, we suggested that NS5A protein was a useful antigen for blood screening.

OBJECTIVETo evaluate the clinical efficacy of intradermal injection of methylene blue for treatment of moderate to severe acute thoracic herpes zoster and prevention of postherpetica neuralgia in elderly patients.

METHODSSixty-four elderly patients with herpes zoster were randomized to receive a 10-day course of intradermal injection of methylene blue and lidocaine plus oral valaciclovir (group A, 32 cases) and intradermal injection of lidocaine plus oral valaciclovir (group B).Herpes evaluation index, pain rating index, incidence of postherpetic neuralgia, and comprehensive therapeutic effect were compared between the two groups at 11, 30 and 60 days after the treatment.

RESULTSThe baseline characteristics were comparable between the two groups (all P>0.05). Compared with that in group B, the time for no new blister formation, blister incrustation and decrustation, and pain relief was significantly shortened in group A (P<0.05) with also obviously lower pain intensity after the treatment. The incidence of postherpetic neuralgia was significantly lower in group A than in group B at 30 days (P<0.05), but not at 60 and 90 days after the treatment. The total clinical response rate was 93.8% in group A, much higher than that in group B (62.5%, P<0.05).

CONCLUSIONIntradermal injection of methylene blue can effectively shorten the disease course, reduce the pain intensity and prevent the development of postherpetic neuralgia in elderly patients with herpes zoster.

Acyclovir ; administration & dosage ; analogs & derivatives ; therapeutic use ; Aged ; Herpes Zoster ; complications ; Humans ; Incidence ; Injections, Intradermal ; Lidocaine ; administration & dosage ; therapeutic use ; Methylene Blue ; administration & dosage ; therapeutic use ; Neuralgia, Postherpetic ; therapy ; Pain Measurement ; Valine ; administration & dosage ; analogs & derivatives ; therapeutic use

OBJECTIVETo construct a GFP/Puro double-labeled lentiviral expression vector for CK8 silencing and assess the effects of CK8 silencing on cell apoptosis.

METHODSThe siRNA sequences of CK8 were inserted into the lentiviral expression vector GV248 and transfected into 293T cells with the packaging plasmids PMD and SPA. The lentivirus was collected at 24 and 36 h post-transfection. Flow cytometry was used to detect the virus titer and the positive cells were selected with puromycin. The knockdown of CK8 was examined by Western blotting. The effect of CK8 down-regulation on cell apoptosis induced by cisplatin was detected with Annexin V/PI staining.

RESULTS AND CONCLUSIONWe successfully constructed CK8 interference lentiviral vector and obtained a stable cell line with CK8 knock-down that was sensitive to cisplatin-induced apoptosis.

Apoptosis ; Cell Line ; Down-Regulation ; Genetic Vectors ; Humans ; Keratin-8 ; genetics ; Lentivirus ; Plasmids ; RNA Interference ; RNA, Small Interfering ; Transfection

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.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo study the relationship between myc gene rearrangement and myc protein expression in diffuse large B cell lymphoma (DLBCL), and their correlation with prognosis.

METHODSOne hundred and six cases of DLBCLs with follow-up data were analyzed using interphase fluorescence in situ hybridization (FISH) technique. Immunophenotyping analysis for CD20, CD3, myc, Mum-1, CD10, bcl-6 was also performed using EnVision immunohistochemistry.

RESULTSThe percentages of tumor cells expressing myc, Mum-1, CD10 and bcl-6 were 70.8%, 56.6%, 21.7% and 26.4%, respectively. Twenty six cases (24.5%) were of GCB type and the rest (75.5%) were of non-GCB (non germinal center) type. The myc rearrangement was identified in 13 (12.3%) of 106 cases. 13 cases showed to be of non-GCB type. There was no correlation between myc rearrangement and myc protein expression. DLBCLs (n = 13) with myc rearrangement showed significantly poorer overall survival (OS) and progression free survival (PFS), with a median OS and PFS time of 4.7 and 3.2 months, respectively (for OS and PFS, P < 0.001). Multivariate analysis using Cox proportional hazard model confirmed that myc rearrangement, ECOG performance status of 2-4, immunophenotyping subgroup and myc protein were independent factors affecting the prognosis and significantly associated with the survival. However, myc rearrangement was the strongest prognostic factor.

CONCLUSIONSDLBCL with myc gene rearrangement is a subgroup of non-GCB DLBCL with poor outcome. It is an independent and useful factor for prognosis in DLBCL. Expression of myc is influenced by many factors and myc rearrangement may be one of these factors.

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Disease-Free Survival ; Doxorubicin ; therapeutic use ; Female ; Follow-Up Studies ; Gene Rearrangement, B-Lymphocyte ; Genes, myc ; Humans ; In Situ Hybridization, Fluorescence ; Interferon Regulatory Factors ; metabolism ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Neprilysin ; metabolism ; Prednisone ; therapeutic use ; Proportional Hazards Models ; Proto-Oncogene Proteins c-bcl-6 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Survival Rate ; Vincristine ; therapeutic use

OBJECTIVETo 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).

METHODSPLA 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.

RESULTSThe 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.

CONCLUSIONSThese findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.

Animals ; Brain ; blood supply ; Capillaries ; cytology ; Cells, Cultured ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; Lactic Acid ; NF-kappa B ; genetics ; Nanoparticles ; Oligonucleotides ; genetics ; Polyesters ; Polymers ; Rats ; Thromboplastin ; genetics ; metabolism ; Transfection

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.
ADP-ribosyl Cyclase 1 ; analysis ; Adult ; Antigens, CD34 ; analysis ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; immunology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans

OBJECTIVETo investigate the clinical and computed tomography (CT) appearances of pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma.

METHODSThe CT findings and clinical data of 13 patients with pathologically proven pulmonary MALT lymphoma were retrospectively reviewed.

RESULTSAmong these 13 patients, seven presented no notable abnormalities, six manifested respiratory symptoms including cough, expectoration, and dyspnea; one of these six patients experienced fever. Chest CT showed solitary nodule in 2 patients and multiple nodules in 3 patients; meanwhile, it showed solitary consolidation in 3 patients and multiple consolidations in 5 patients. Other CT findings included air bronchogram (n = 13), airway dilatation (n = 4), ground glass opacities (n = 5), and interstitial changes (n = 5). One patient had mediastinal lymphoadenopathy and 2 had pleural effusion. Pathology showed massive lymphocyte infiltration; cells with notable nuclear atypia were also seen, which were generated from B cells.

CONCLUSIONSThe main CT findings of pulmonary MALT lymphoma include nodules, mass or patchy consolidations with air brochogram; hilar and mediastinal lymphadenopathies are rare. Clinical diagnosis should also be based on pathological findings and immunohistochemical results.

Adult ; Aged ; Female ; Humans ; Lung Neoplasms ; diagnosis ; diagnostic imaging ; pathology ; Lymphoma, B-Cell, Marginal Zone ; diagnosis ; diagnostic imaging ; pathology ; Male ; Middle Aged ; Radiography ; Retrospective Studies

OBJECTIVETo distinguish the edema, injury, or rupture in the traumatic skeletal muscle fiber in vivo using diffusion tensor imaging (DTI) and tractography on magnetic resonance imaging (MRI).

METHODSThe skeletal muscle trauma models were made in 4 rabbits (eight hindlimbs) by iron discus (weight 1.0 kg, diameter 6 cm) falling down vertically from 45 cm height to rabbits' thighs. Conventional sequences and two-dimensional (2D) diffusion-weighted (DW) spin-echo (SE) echo planar imaging (EPI) sequence with fat suppression (b = 600 s/mm2) were performed on 1. 5T MRI scanner. The grading of edema, injury, and fiber rupture in the damaged muscle were made according to their histopathological views, which was consistent with the images. The mean apparent diffusion coefficient (ADC) values and fractional anisotropy (FA) values were measured from the region of interests (ROIs) of all groups on 2D DW images used for tractography. Analysis of variance test was performed to analyze all data.

RESULTSADC values of the areas in normal muscle, edema muscle, injury muscle, and ruptured muscle were (6.12 +/- 1.34) x 10(-3), (6.38 +/- 1.30) x 10(-3), (8.06 +/- 0.97) x 10(-3), and (9.57 +/- 0.93) x 10(-3) mm2/s, respectively. There was significant difference among groups (P < 0.001), but no difference between edema muscle and normal muscle group (P > 0.05). The FA values of normal muscle, edema muscle, injury muscle, and ruptured muscle were 0.42 +/- 0.12, 0.36 +/- 0.12, 0.26 +/- 0.09, 0.12 +/- 0.08, respectively, with a significant difference among groups (P < 0.001). In the edema muscle, the tracking cross-fiber could be seen but it decreased slightly. In the injury muscle, the tracking fiber decreased markedly. In the ruptured muscle, the transverse-orientation tracking fiber vanished, yet some interrupted longitudinal-orientation tracking fiber could be found.

CONCLUSIONThe edema, injury, and rupture of muscle fiber in rabbit damaged skeletal muscle can be verified according to the ADC and the FA on DTI and tractography.

Animals ; Anisotropy ; Diffusion Magnetic Resonance Imaging ; methods ; Echo-Planar Imaging ; Edema ; diagnosis ; pathology ; Male ; Muscle, Skeletal ; injuries ; pathology ; Rabbits ; Rupture ; diagnosis ; pathology ; Thigh ; injuries ; pathology

OBJECTIVETo investigate whether vitamin C can promote the proliferation ability of bone marrow mesenchymal stem cells (BMMSCs) derived from aging mice.

METHODSThe senescence-accelerated mouse prone 6 (SAMP6) mice and senescence-accelerated mouse resistant 1 (SAMR1) mice were used as the test group and the control group, respectively, and the SAMP6 mice were examined by micro-CT to verify the senescent phenotype. BMMSCs were harvested from the two mouse lines and cultured in vitro, and the cells from SAMP6 mice were subjected to treatment with different concentrations of vitamin C. The proliferation ability of the cells from the two mouse lines was tested using MTT assay and growth curves, and TeloTAGGG Telomerase PCR ELISA was used to measure the telomerase activity; PCR and Western blotting were performed to detect the expression level of telomerase reverse transcriptase (TERT) in the cells.

RESULTSThe SAMP6 mice displayed a bone senescent phenotype. The proliferation ability of BMMSCs derived from SAMP6 mice and their telomerase activity were significantly lower than those derived from SAMR1 mice (P<0.05). Vitamin C treatment significantly enhanced the proliferation ability of BMMSCs derived from SAMP6 mice in a dose-dependent manner (P<0.05) and increased telomerase activity and TERT expression in the cells (P<0.05). At the concentration of 100 µg/mL, vitamin C produced the strongest effect in promoting the proliferation of BMMSCs from SAMP6 mice, while at the concentration of 1000 µg/ml, growth suppression occurred in the cells.

CONCLUSIONVitamin C can promote the proliferation of BMMSCs from aging mice possibly by increasing the cellular telomerase activity.

Aging ; Animals ; Ascorbic Acid ; chemistry ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; chemistry ; Hematopoietic Stem Cells ; Mesenchymal Stromal Cells ; cytology ; Mice ; Telomerase ; metabolism