Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

OBJECTIVETo explore the clinical application value of transurethral ureteroscopy in the diagnosis and treatment of hemospermia.

METHODSWe summed up and analyzed the experience in the diagnosis and treatment of 43 hemospermia patients by transurethral ureteroscopy and douching therapy.

RESULTSThe disease causes were clearly determined and the therapeutic procedures successfully accomplished in all the cases. The mean surgical time was 20 (18 -45) minutes. No significant complications developed either intraoperatively or postoperatively. The patients were followed up for 1 -24 months, during which hemospermia symptoms completely disappeared in 35 and were relieved in 6 of the cases, but the other 2 remained unimproved. All the patients had normal sexual life and none experienced retroinfection postoperatively.

CONCLUSIONTransurethral ureteroscopy, advantageous for its safety, high rate of detection, good effect of treatment and fewer complications, deserves to be popularized in the clinical diagnosis and treatment of hemospermia.

Adult ; Aged ; Hemospermia ; diagnosis ; surgery ; Humans ; Middle Aged ; Ureteroscopy ; methods

BACKGROUNDThe transradial approach is regarded as a useful vascular site for coronary procedures. The aim of this study was to test whether 4Fr catheters assisted by ACIST variable rate injector system can produce comparable angiographic quality and reduce the risk of radial artery injury compared to hand manifold 6 Fr catheters.

METHODSA total of 1816 patients were studied consecutively, among whom 856 patients received coronary angiography by 4 Fr catheters (4Fr group) and 960 patients by 6 Fr catheters (6Fr group). Angiographic and procedural characteristics were observed and recorded. The luminal inner radial arterial diameter before and after the procedure were collected.

RESULTSThe baseline clinical characteristics were similar in both groups. There were no significant differences in procedure time, radiation dose and quality scores in both groups (P > 0.05), but more contrast media was delivered in the 6Fr group (P < 0.001). The mean radial arterial diameter six months after the procedure in the 6Fr group reduced significantly compared to that measured one day prior to the procedure (P < 0.001).

CONCLUSIONSCoronary angiography using the 4Fr catheters with Acist power injection system can achieve an acceptable diagnostic quality while at the same time minimizing radial artery injury and contrast media consumption.

Aged ; Cardiac Catheterization ; instrumentation ; Coronary Angiography ; instrumentation ; methods ; Female ; Humans ; Male ; Middle Aged ; Radial Artery ; diagnostic imaging ; Ultrasonography

Purpose: This study aimed to compare the ability of B-mode ultrasonography and magnetic resonance imaging (MRI) to predict the repairability of large-to-massive rotator cuff tears (RCTs). Methods: This cross-sectional study included participants with large-to-massive RCTs who underwent arthroscopic repair. B-mode ultrasonography and MRI were conducted prior to arthroscopic repair. B-mode ultrasonography was used to evaluate the echogenicity of the rotator cuff muscle using the Heckmatt scale. Intra-rater and inter-rater reliabilities were examined for two independent physicians. MRI was used to evaluate the degrees of tendon retraction, fatty infiltration of rotator cuff muscles, and muscle atrophy. Finally, two experienced orthopedic surgeons performed surgery and decided whether the torn stump could be completely repaired intraoperatively. Results: Fifty participants were included, and 32 complete repairs and 18 partial repairs were performed. B-mode ultrasonography showed good intra-rater reliability and inter-rater reliability for assessment of the muscle echogenicity of the supraspinatus and infraspinatus muscles. The correlation coefficients between B-mode ultrasound findings and MRI findings showed medium to large effect sizes (r=0.4-0.8). The Goutallier classification of the infraspinatus muscles was the MRI predictor with the best discriminative power for surgical reparability (area under the curve [AUC], 0.89; 95% confidence interval [CI], 0.81 to 0.98), while the Heckmatt scale for infraspinatus muscles was the most accurate ultrasound predictor (AUC, 0.85; 95% CI, 0.74 to 0.96). No significant differences in AUCs among the MRI and ultrasound predictors were found. Conclusion B-mode ultrasonography was a reliable examination tool and had a similar ability to predict surgical reparability to that of MRI among patients with large-to-massive RCTs.

This study was aimed to explore the clinical significance of monitoring level of minimal residual disease (MRD) at different time point in B-lineage childhood acute lymphoblastic leukemia (B-ALL). Two hundred and six children with B-ALL were enrolled in this study from Augest 2008 to September 2011 in our hospital. MRD levels were detected by flow cytometry at day 15, 33 and week 12 after initial chemotherapy. The event-free survival (EFS) for patients based on MRD levels measured at different stages of chemotherapy were compared by Kaplan Meier analyses. The results showed that out of 206 cases 196 cases achieved complete remission (CR) after induction therapy (CR rate 95.1%), the 1- and 3-year EFS rate were (92.7 ± 1.8)% and (78.7 ± 3.7)%, respectively, and the 3-year EFS rate was (85.6 ± 4.9)% in standard risk group, (82.1 ± 5.8)% in intermediate risk group and (58.1 ± 9.2)% in high risk group, there was significant statistical difference between above mentioned 3 groups (P < 0.001). The MRD analysis at different time points showed that the higher the MRD level, the lower the 3-year EFS rate of children with ALL, in which the 3-year EFS rate of MRD ≥ 10(-2) at day 15, MRD ≥ 10(-3) at day 33 and MRD ≥ 10(-3) at week 12 were significantly lower. The MRD ≥ 10(-3) at week 12 was proven to be an independent predictor by multivariate Cox proportional-hazards regression model. The 3-year EFS rate for patients with MRD < 10(-3) and MRD ≥ 10(-3) at week 12 were (86.3 ± 4.1)% vs (55.8 ± 9.1)% (P < 0.05); 8 relapsed among 98 cases with negative MRD (MRD < 10(-4)) at day 33, 19 relapsed among 108 cases with positive MRD at day 33 between the two groups for recurrence rate has significant difference (P < 0.05). It is concluded that dynamically monitoring MRD by multi-parameter flow cytometry can precisely evaluate treatment response, judge treatment outcome and predict relapse in childhood B-ALL. The MRD 10(-2) at day 15, MRD 10(-3) at day 33 and MRD 10(-3) at week 12 should be considered as the best cut-off. MRD ≥ 10(-3) at week 12 was proven to be an independent factor of poor prognosis.
Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Male ; Neoplasm, Residual ; diagnosis ; therapy ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; therapy ; Prognosis ; Retrospective Studies

OBJECTIVETo investigate the effects of peroxisome proliferator-activated receptor (PPAR) α/γ agonist on atherosclerotic plaque stabilization in diabetic LDL receptor knockout (LDLr-/-) mice.

METHODSFemale 4-week-old LDLr-/- mice fed with high-glucose and high-fat diet for 4 weeks were randomly divided into three groups (n = 15 each): control group (only fed with high-glucose and high-fat diet), diabetic group [induced by high-glucose and high-fat diet combined with a low-dose of streptozotocin (STZ)] without tesaglitazar and with tesaglitazar (20 µg/kg oral treatment). After 6 weeks, the mice were sacrificed, body weight, fasting blood glucose (Glu), total cholesterol (TC), triglyceride (TG) levels were measured. The expression of ICAM-1, VCAM-1, MCP-1 in the brachiocephalic atherosclerotic lesions were determined by Western blot and immunohistochemistry, respectively. Brachiocephalic artery was prepared for morphologic study (HE, oil red O, Sirius red staining) and immunohistochemical analysis (macrophage surface molecule-3, α-smooth muscle actin), respectively.

RESULTSSerum TC [(32.34 ± 3.26) mmol/L vs. (16.17 ± 1.91) mmol/L], TG [(3.57 ± 0.99) mmol/L vs. (2.21 ± 0.11) mmol/L] and Glu [(15.21 ± 4.67) mmol/L vs. (6.89 ± 0.83) mmol/L] levels were significantly higher in diabetic group than in the control group (all P < 0.01). The expression of ICAM-1 (2.31 ± 0.35 vs.1.34 ± 0.21), VCAM-1 (1.65 ± 0.14 vs.0.82 ± 0.26), MCP-1 (2.27 ± 0.16 vs.1.56 ± 0.23) were significantly upregulated in diabetic group compared with control group (all P < 0.01). Brachiocephalic atherosclerotic plaque area [(4.597 ± 1.260)×10(3) µm(2) vs. (0.075 ± 0.030)×10(3) µm(2)], lipid deposition [(47.23 ± 2.64)% vs. (9.67 ± 1.75)%], Mac-3 positive area [(19.15 ± 3.51)% vs. (1.72 ± 0.16)%], α-smooth muscle actin [(5.54 ± 1.17)% vs. (2.13 ± 0.41)%] and collagen content [(4.27 ± 0.74)% vs. (0.43 ± 0.09)%] were all significantly larger/higher in diabetic LDLr-/- mice than in the control group (all P < 0.01). While tesaglitazar treatment significantly reduced serum TC [(30.47 ± 3.18) mmol/L], TG [(3.14 ± 0.71) mmol/L] and Glu [(7.92 ± 1.28) mmol/L] levels (all P < 0.01). Similarly, the expression of ICAM-1 [(1.84 ± 0.22)], VCAM-1 [(1.27 ± 0.11)], MCP-1 [(1.83 ± 0.24)], brachiocephalic atherosclerotic lesion area[(1.283 ± 0.410)×10(3) µm(2)], lipid deposition[(23.52 ± 1.39)%] were also significantly reduced by tesaglitazar (all P < 0.05). Moreover, tesaglitazar increased α-smooth muscle actin [(9.46 ± 1.47)%] and collagen content [(6.32 ± 1.15)%] in diabetic LDLr-/- mice (all P < 0.05). In addition, lipid deposition and Mac-3 positive areas [(10.67 ± 0.88)% vs. (15.83 ± 1.01)%] in the aortic root were also reduced in tesaglitazar treated diabetic LDLr-/- mice (P < 0.01).

CONCLUSIONSTesaglitazar has anti-inflammatory effects in the diabetic LDLr-/- mice. Tesaglitazar could reduce lipid deposition, increase collagen and α-SMA content in the brachiocephalic atherosclerotic lesions, thus, stabilize atherosclerotic plaque in this model.

Actins ; metabolism ; Alkanesulfonates ; pharmacology ; Animals ; Collagen ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Diet, High-Fat ; adverse effects ; Female ; Intercellular Adhesion Molecule-1 ; metabolism ; Lipid Metabolism ; Mice ; Mice, Knockout ; PPAR alpha ; agonists ; PPAR gamma ; agonists ; Phenylpropionates ; pharmacology ; Plaque, Atherosclerotic ; metabolism ; pathology ; Receptors, LDL ; genetics ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo investigate the role of tumor necrosis factor (TNF) alpha on the homing efficiency of hematopoietic stem/progenitor cells (HS/PC) into bone marrow and its mechanism.

METHODSCFSE-labeled umbilical cord blood (UCB) CD34+ cells were transplanted into irradiated (control group) or combined with TNF alpha prepared (experimental group) BALB/c recipient mice. The distribution in peripheral blood, liver, lung and homing characteristics in bone marrow and spleen of UCB CD34+ cells, in BALB/c recipient mice were determined 20 hours after xenotransplantation by flow cytometry (FACS) and their homing efficiency was calculated. ELISA was used to measure serum SDF-1 alpha level. CXCR4 expression levels of on UCB CD34+ cells were assessed by FACS pre-/post-manipulation with TNF alpha. SDF-1 alpha expression level in bone marrow and spleen was tested by immunohistochemistry.

RESULTSUCB CD34+ cells mainly home into recipient mice bone marrow and spleen; The homing efficiency in experimental group bone marrow [(0.65 +/- 0.13)%] was significantly higher than that in control ones [(0.30 +/- 0.09)%, P < 0.01], whereas the homing efficiency in experimental group spleen was dramatically lower than that in control ones (P < 0.01); Treatment with TNF alpha did not affect recipient serum SDF-1 alpha level; After 18 hours co-cultured with TNF alpha, the CXCR4e expression level on UCB CD34+ cells was similar to that on fresh ones; TNF alpha treatment induced significantly higher SDF-1 alpha expression on osteoblastic and stromal cells in bone marrow, and reversed spleen SDF-1 alpha gradient that was originally favorable for CD34+ cells homing.

CONCLUSIONTNF alpha enhances the homing efficiency of HS/PC via up-regulating SDF-1 alpha gradient in bone marrow, and might be an useful enhancer for HS/PC homing in clinical practice.

Animals ; Antigens, CD34 ; Bone Marrow ; Cell Movement ; Cell Separation ; Chemokine CXCL12 ; metabolism ; Female ; Fetal Blood ; cytology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; drug effects ; immunology ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C ; Receptors, CXCR4 ; metabolism ; Transplantation Conditioning ; Transplantation, Heterologous ; Tumor Necrosis Factor-alpha ; pharmacology

Aspergillus fumigatus ; Cytokines ; Humans ; Interleukin-1beta ; Keratitis ; Receptors, IgE ; Tumor Necrosis Factor-alpha ; Tumor Necrosis Factors

Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
Animals ; Carcinogenesis/pathology* ; Cattle ; Cell Cycle Proteins/metabolism* ; Cell Proliferation/drug effects* ; Cell Transformation, Neoplastic/pathology* ; Cells, Cultured ; Epithelial Cells/pathology* ; Female ; Histone Deacetylase 2/metabolism* ; Imatinib Mesylate/pharmacology* ; Interferon-gamma/pharmacology* ; Mammary Glands, Animal/pathology* ; Mammary Neoplasms, Experimental/pathology* ; Proto-Oncogene Proteins c-abl/metabolism* ; Signal Transduction ; Valproic Acid/pharmacology*

Recent studies have shown that diet can affect the body's immunity. Roughage of dairy cows consists of a variety of plant materials which make different contributions to health. This study investigated the effect of different roughages on the immunity of dairy cows. Serum, peripheral blood mononuclear cells (PBMCs), and milk samples were collected from 20 multiparous mid-lactation cows fed mixed forage (MF)- or corn straw (CS)-based diets. Expression profile analysis was used to detect the differentially expressed genes (DEGs) from PBMCs. The results showed that milk protein in the MF group increased to 3.22 g/100 ml, while that of the CS group milk was 2.96 g/100 ml; by RNA sequencing, it was found that 1615 genes were differentially expressed between the CS group and the MF group among the 24 027 analyzed probes. Gene ontology (GO) and pathway analysis of DEGs suggested that these genes (especially genes coding cytokines, chemokine and its receptors) are involved in the immune response. Results were confirmed at the protein level via detecting the levels of interleukin-2 (IL-2), IL-6, IL-10, IL-12, leptin (LEP), interferon-γ (IFN-γ), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) in peripheral blood by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay analysis. Our data supported the conclusions that the protein content in milk of the MF group was higher than that of the CS group, the CS-based diets induced more release of cytokines than the MF-based diets in dairy cows' PBMCs, and milk protein content may be affected by cytokines.
Animals ; Cattle/immunology* ; Cytokines/physiology* ; Diet ; Female ; Gene Ontology ; Leukocytes, Mononuclear/immunology* ; Milk/chemistry* ; Transforming Growth Factor beta/physiology* ; Zea mays