Background The characters of diabetic retinopathy (DR) include capillary occlusion,microcirculation disturbance and retina neovascularization near ischemic areas of the retina.The pathogenic mechanism is still incompletely clear,but the primary mechanism of DR is the change of permeability of retinal blood capillary.Objective The aim of this study was to investigate the effect of lycium bararum polysaccharides (LBP) on the blood glucose,blood fat and permeability of blood-retinal barrier in diabetic rats.Methods Fifty-four clean adult SD rats were randomized into 4 groups.The diabetes mellitus models were created in 36 SD rats by injection of 45 mg/kg streptozotocin(STZ) via caudal vein.The rats with the blood glucose ＞ 16.7 mol/L were defined as the diabetic mellitus in 72 hours after injection.LBP of 200 rmg/( kg · d) was intragastriccally administered in 18 models once a day in LBP group,and equal volume of normal sodium was used at the same way in 18 models in DM group,and 18 normal rats were as normal control group.The rats were killed in 4,8 and 12 weeks respectively for the detect of blood glucose level,triglyceride,total cholesterol.The retina tissue were isolated for the quantification of vascular permeability using Evans blue method.Results The blood glucose level was significantly elevated in the DM group and LBP group compared with normal control group,and that in LBP group was obviously declined in comparison with the DM group with a 57％,40％,36％ fall off in 4,8,12 weeks respectively,showing a statistically significant differences( P＜0.05 ).The triglyceride concentration was considerably raised in the DM group compared with normal control group in all of the time points( P＜0.01 ),however,the rise of triglyceride concentration was found only in 12 weeks in LBP group(P＜0.05).The cholesterol level was increased in 8 and 12 weeks in DM group compared with normal control group and that of LBP group went up only in 12 weeks (P＜0.0l ).The Evans blue contents in dry retina tissue were(26.23±2.00),(29.78± 1.78 ) and( 34.08±3.03 ) μg/g in 4,8,12 weeks in the DM group,and those of LBP were ( 13.27 ±0.77 ),( 18.01 ± 1.77 ),( 25.05 ±:1.50 ) μg/g,showing statistical differences between two groups ( P＜0.01 ).However,in comparison with normal control group ( 12.34 ±4.30,12.76 ± 2.11,12.45 ±4.40 μg/g),the Even blue content were elevated in various time points (P＜0.01 ).Conclusions LBP can suppress the increase of the blood-retinal barrier permeability in STZ-induced diabetic rat by lowing the levels of blood glucose,triglyceride and total cholesterol.These results suggest that LBP has a therapeutical effect on DR.

M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot.20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2 subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.

Rutaecarpine (Rut) is a type of indole quinazoline alkaloid exracted from Ruticarpum. Studies showed that Rut has a wide range of pharmacological effects, such as anti-hypertension, anticancer, anti-inflammation, anti-thrombus formation. Currently, many scholars are committed to developing it into a new antihypertensive and anti-inflammatory drug with all new mechanisms. But studies found that Rut is a highly fat-soluble drug with low water and oil solubility. Its high insolubility is the main obstacle in its oral absorption and application, which greatly reduced its bioavailability. Therefore, hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was used as the inclusion material to prepare Rut-HP-beta-CD inclusion complex in this experiment, in order to increase its water solubility and bioavailability. In this experiment, the inclusion complex was prepared by the stirring-freeze-dry method. The preparation process was optimized by the orthogonal test, with the inclusion rate as the index, and molar ratio between host and guest molecules, inclusion temperature, time and stirring speed as the impacting factors. Moreover, the inclusion complex was verified by detecting the apparent solubility, thin layer chromatography, microscopic identification, melting point detection and dissolution study. The results showed that under the conditions of the molar ratio between Rut and HP-beta-CD of 1: 1, temperature at 60 degrees C, inclusion time of 4h and stirring speed at 600 r x min(-1), the inclusion rate of Rut-HP-beta-CD reached 91.04%. Therefore, the preparation process of Rut-HP-beta-CD inclusion under the optimum conditions is simple and feasible, with a highest inclusion rate and reproducibility, and could significantly improve Rut's solubility and bioavailability, and provide a reliable experimental basis for its clinical application.
2-Hydroxypropyl-beta-cyclodextrin ; Alkaloids ; chemistry ; Chemistry, Pharmaceutical ; methods ; Drug Carriers ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Rutaceae ; chemistry ; Solubility ; beta-Cyclodextrins ; chemistry

Aim To investigate the protective effects of chrysophanol( Chry) on immune function of lead poi-soning mice. Methods The lead poisoning model was established by peritoneally injecting mice with 7 mg · kg-1 lead acetate every day for 8 days. After Chry was ip injected for 14 days,spleen and thymus index, the white blood cell count, T, B lymphocyte proliferation, phagocytic function of macrophages, natural killer cell ( NK cell) activity were detected. The concentrations of IFN-γ,IL-2,IL-4,IL-10 in the lead poisoning mice ser-um were determined by enzyme-linked immunosorbent assay ( ELISA) . Results Intraperitoneal injection of 7 mg · kg-1 lead acetate for 8 consecutive days could cause an immunity decline in lead poisoning mice, Chry could significantly improve the immunity of lead poisoning mice. Compared with model group, Chry sig-nificantly improved growth rate, viscera index and white blood cell count of lead poisoning mice at differ-ent extent ( P<0 . 05 or P<0. 01 ) . Chry ( 1 . 0 ,10 . 0 mg·kg-1 ) significantly increased the B,T lymphocyte proliferation ( P<0 . 01 ) and the phagocytosis of macro-phage and NK cell activity ( P <0 . 01 ) . Compared with the control group, the concentrations of IFN-γ, IL-2 , IL-4 , IL-10 of lead poisoning mice serum were significantly reduced ( P <0 . 01 ) . Compared with the model group, Chry (1. 0、10. 0 mg·kg-1 ) significant-ly increased the concentrations of IFN-γ, IL-2 , IL-4 , IL-10 ( P <0. 01 ) , while there were no significant changes in (0. 1 mg·kg-1 ) concentrations of IFN-γ, IL-2 , IL-4 in Chry ( 0 . 1 mg · kg-1 ) treatment group ( P>0. 05 ) , only IL-10 was significantly increased in Chry ( 0 . 1 mg · kg-1 ) treatment group ( P<0 . 05 ) . Conclusion Chry can significantly improve the im-mune function of lead poisoning mice.

OBJECTIVETo evaluate the impact of platelet membrane glycoprotein (GP)-specific autoantibodies on high-dose dexamethasone therapy in patients with idiopathic thrombocytopenic purpura (ITP).

METHODSModified direct monoclonal antibody immobilization of platelet antigen assay (MAIPA) was used to detect platelet GPIIb/IIIa and/or GPI b specific autoantibodies. All patients received oral dexamethasone 40 mg/d for 4 days.

RESULTSThe response rate of high-dose dexamethasone in GPIIb/IIIa and/or GPIb specific autoantibody-negative patients was significantly different from that of antibody-positive patients (P<0.05). The response rate of GPIIb/IIIa specific autoantibody-positive patients was lower than that of antibody-negative patients (P<0.05). GPIb specific autoantibody had no significant impact on the efficacy of high-dose dexamethasone (P>0.05).

CONCLUSIONPlatelet membrane GPIIb/IIIa-specific autoantibody can be a potential negative indicator for ITP patients'response to high-dose oral dexamethasone.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Autoantibodies ; blood ; Dexamethasone ; administration & dosage ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Platelet Glycoprotein GPIb-IX Complex ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; drug therapy ; immunology ; Treatment Outcome ; Young Adult

BACKGROUNDRemodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-beta1 (TGFbeta1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFbeta1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.

METHODSAdenoviral vector containing TGFbeta1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFbeta1, the expression of VEGF165 and TGFbeta1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFbeta1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.

RESULTSThe results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFbeta1 and VEGF165 genes. Co-expression of TGFbeta1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.

CONCLUSIONCo-expression of TGFbeta1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

Adenoviridae ; genetics ; Animals ; Anterior Cruciate Ligament ; cytology ; metabolism ; Cell Movement ; Cells, Cultured ; Collagen ; genetics ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibroblasts ; physiology ; Fibronectins ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; Wound Healing

OBJECTIVETo prepare ITP plasma IgG and its F(ab')2 fragments and investigate their immunoreactivity to platelet GPIIb/IIIa and/or GPIb/IX and their effects on platelet aggregation function.

METHODSThe ITP patients having inhibitory autoantibody to the platelet aggregation were selected by modified MAIPA and platelet aggregation test with turbidimetry. Plasma IgG and its F(ab')2 fragments were prepared by streptococcal protein A affinity column and pepsin digestion. The immunoreactivity and the effects on platelet aggregation function of the whole antibody and its fragments were detected by modified MAIPA and platelet aggregation test, respectively.

RESULTS(1) Anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were detected in 34 of 68 (53.6%) ITP patients' plasmas and that from 5 patients significantly inhibited the platelet aggregation induced by ADP or ristocetin. (2) By using protein A column combined with protease digestion, pure IgG and its F(ab')2 fragments were successfully obtained. (3) The purified IgG and its F(ab')2 fragments retained the ability to bind to their respective glycoproteins and inhibited the platelet aggregation function, whereas the IgG depleted plasma lost the ability of binding to the platelet GPs.

CONCLUSIONSF(ab')2 fragment of the IgG antibody is a functional fragment, which not only has the binding ability to the platelet GPs but also inhibits the platelet aggregation function in a dose-dependent manner.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Humans ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin G ; immunology ; Integrin beta3 ; immunology ; Male ; Middle Aged ; Platelet Aggregation ; Platelet Membrane Glycoprotein IIb ; immunology ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; physiopathology ; Young Adult

OBJECTIVETo investigate the change of B-cell activating factor of the TNF family (BAFF) and regulatory T-cells (Tregs) before and after high-dose dexamethasone(HD-DXM) therapy and assess the effect of BAFF on Treg cells in immune thrombocytopenic purpura (ITP).

METHODSThe plasma BAFF concentration was measured by ELISA, and Treg cell numbers by flow cytometry.

RESULTSThe plasma BAFF level \[(599.70 +/- 199.40) pg/ml\] was significantly increased (P < 0.05), and the percentage of Treg cells \[(1.56 +/- 0.73)%\] was significantly decreased (P < 0.01) in ITP patients before treatment as compared with that in controls \[(454.5 +/- 132.5) pg/ml and (4.08 +/- 1.08)%, respectively\]. After treatment with HD-DXM, the plasma BAFF level \[(296.9 +/- 119.7) pg/ml\] was significantly decreased (P < 0.01), and the percentage of Treg cells \[(5.94 +/- 2.22)%\] was significantly increased (P < 0.01). The BAFF level and Treg proportion had no significant correlation with platelets count (P > 0.05). In in vitro assays, no difference was found in the number of Treg cells between rhBAFF0 group and rhBAFF20 group \[(1.53 +/- 0.69)%, (1.49 +/- 0.67)%, P = 0.89)\].

CONCLUSIONBAFF level was increased and Treg cells decreased in ITP patients. HD-DXM might play a role in ITP treatment by down-regulating BAFF expression and up-regulating Treg cells number. BAFF had no influence on the number of Treg cells.

B-Cell Activating Factor ; Dexamethasone ; administration & dosage ; Humans ; Interleukin-4 ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo study the anti-platelet GPVI single chain Fv phage antibody which can inhibit the aggregation function of platelet by using phage antibody library technology.

METHODSITP patients with anti-platelet GPVI autoantibody that could inhibit the aggregation function of platelet were screened by MAIPA assay and platelet aggregation test. The gene fragments of heavy chain and light chain variable region (VH and VL) of immunoglobulin were amplified by RT-PCR from peripheral blood lymphocytes mRNA of the screened patients. The VH and and VL fragments were linked through a DNA linker encoding the peptide (Gly4Ser)3 to construct single chain Fv (ScFv) gene. The ScFv gene was digested with SfiI/NotI restriction enzymes and cloned into the pHEN2 phage display vector, then electrically transformed to E. coli TG1. The TG1 containing ScFv-pHEN2 was rescued by helper phage M13K07 to produce ScFv phage antibody. The anti-platelet GPVI phage ScFv antibody was enriched and purified. The effect of the phage antibody on platelet aggregation function was studied.

RESULTSOf 806 chronic ITP patients, 11 (1.36%) were positive for anti-platelet GPVI autoantibody and 2 (0.24%) patients'plasma significantly inhibited the collagen induced platelet aggregation. The length of VH and VL fragments was about 380 to 400 bp, and were successfully formed ScFv fragments of about 800 bp by DNA linker. After cloning ScFv to phagemid vector pHEN2 and transforming ScFv-pHEN2 to TG1, 4.1x10(7) clones were obtained. After M13K07 rescue, 2.62x10(10) cfu/ml ScFv phage antibodies were produced. The purified anti-platelet GPVI ScFv phage antibody inhibited the collagen induced platelet aggregation.

CONCLUSIONAnti-platelet GPVI ScFv phage antibody produced by phage antibody library technology can inhibit the aggregation function of platelet.

Adolescent ; Adult ; Aged ; Escherichia coli ; genetics ; Female ; Humans ; Male ; Middle Aged ; Peptide Library ; Platelet Aggregation ; immunology ; Platelet Aggregation Inhibitors ; immunology ; pharmacology ; Platelet Membrane Glycoproteins ; immunology ; Single-Chain Antibodies ; genetics ; immunology ; pharmacology ; Transformation, Bacterial ; Young Adult

OBJECTIVETo evaluate the role of interleukin (IL)-18 and IL-18 receptor (IL-18R) in the predominant Th1 type cytokine response in patients with immune thrombocytopenia (ITP).

METHODSFifteen patients with active phase ITP, eighteen in remission and thirteen healthy controls were enrolled in this study. T-bet and GATA-3 mRNA levels in peripheral blood mononucleated cells (PBMNC) were measured by reverse transcriptase polymerase chain reaction (RT-PCR); the plasma IL-18 level by enzyme linked immunosorbent assay (ELISA), the expression of IL-18R on CD3(+) lymphocytes and total lymphocytes by flow cytometry(FCM).

RESULTSThe T-bet mRNA levels in patients with active phase ITP was 3.572 fold as much as that in the controls (P < 0.05), while the GATA-3 mRNA levels were 0.378 fold of that in controls (P < 0.05). The levels of plasma IL-18 and IL-18R on CD3(+) lymphocytes were significantly increased in active phase ITP than in remission phase and controls. There was no difference in ratio of T-bet/GATA-3 between remitted ITP and controls and so was for T-bet mRNA, GATA-3 mRNA, plasma IL-18 and IL-18R on CD3(+) lymphocytes.

CONCLUSIONITP as a disease of Th1-dominant response there is an unbalance between T-bet and GATA-3 in its active phase; IL-18 and IL-18R being upregulated.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Female ; GATA3 Transcription Factor ; metabolism ; Humans ; Interleukin-18 ; immunology ; metabolism ; Male ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; metabolism ; Receptors, Interleukin-18 ; immunology ; metabolism ; T-Box Domain Proteins ; metabolism ; Th1 Cells ; immunology ; metabolism ; Young Adult