Objective To test the level of follicular helper T cells (Tfh) and interleukin (IL)-21,CXCL13 in the peripheral blood of patients with ankylosing spondylitis (AS),and to analyze the relationship between Tfh and clinic features and explore the possible immunological pathogenesis of AS.Methods The Tfh cells were obtained from patients and normal controls and detected by flow cytometry.While the levels of IL-21,CXCL13 in patients and normal controls were measured by enzyme-linked immunosorbent assay (ELISA) tests.Data analysis were performed by Student's t-test,Rank-sum test,Spearman's correlation test.Results The expression of CD4+CXCR5 qCOS + cells (Tfh) (mean rank 33.71) and IL-21 [(299±27) ng/L],CXCL13 [(5.8±1.0) μg/L] in the peripheral blood of AS was significantly higher than normal controls [mean rank 23.54,(176±26) ng/L,(4.2±0.8) μg/L] (Z=-2.258,t=17.221,t=6.464,all P＜0.05).It was similar in AS with peripheral joint involvement compared with AS of non-peripheral joint involvement,and there was no difference between AS patients with positive HLA-B27 and those without HLA-B27.Mean -while,no correlation was found between the expression of Tfh,IL-21,CXCL13 and level of ESR,CRP,BASDAI.And there was no significant correlation between the expression of Tfh and IL-21,CXCL13 (P＞0.05).Conclusion The expression of Tfh and the levels of IL-21,CXCL13 are increased significantly,but are not closely relatedto disease activity.These results indicate that the abnormality of Tfh may play an important role in the pathogenesis of AS.

Background Phlyctenular ophthalmia is a delayed hypersensitivity reaction to some microprotein and affected mainly by adolescent in high incidence.Objective This study was to investigate the microscopy findings of phlyctenular ophthalmia and evaluate the histological changes by laser scanning confocal microscope.Methods Twenty-nine eyes suffered from phlyctenular ophthalmia and twenty normal eyes were examined using laser scanning confocal microscope.The pictures were taken by a CCD camera.All the cases had initially chest X-ray,tuberculin test,bacterial and mycobacteria culture.Results Dendritic and inflammatory cells were increased and concentrated in conjunctiva,and epithelial cells were deformed and squamatizated.The capillaries engorged and the goblet cells were injured.The corneoscleral Vogt meshing of the phlyctenular keratitis was obscured and dendritic cells were intruded into the corneas.The corneal epithelium of phlyctenular keratitis was absent and the subepithelial nerve plexus were bended and fractured,and the dendritic and inflammatory cells were intruded.Scarring of corneal stroma was seen under the laser scanning confocal microscope.Conclusions Laser scanning confocal microscopy is valuable for basic research and clinical diagnosis of phlyctenular ophthalmia.

Objective To investigate whether allocardiac graft acceptance in the specific NF-κB impaired mice is due to regulatory T cell(Tr) and Th17 cells.Methods Mice abdominal heterotopic cardiac transplantation was performed and then divided in to control group(BALB/c→C57BL/6) and experimental group(BALB/c→IκBα/△N-Tg).Pretransplant and at day 7,30,100 posttransplant,spleens were harvested from the IκBα△ N-Tg mice,and then the Tr were detected by the fluorescence activated cell sorter.At day 5 posttransplant,the CD4 + Th17 cells from the spleens of the two groups were examined by the FACS.Additionally,at day 3 and 5 posttransplant,IL-17 expressed in the cardiac allograft was detected by Western blot.Results In the IκBα/ N-Tg mice group,the cardiac allografts were survived more than day 100,and without obviously lymphocytes infiltration.At the day 7 and 30 posttransplant,the Tr was obviously increased(21.23 ± 3.95,23.17 ± 4.11 vs 11.64 ± 1.96,P ＜ 0.05); however,the Tr decreased at the day 100 posttransplant,and had no difference with before transplant(10.79 ±2.48 vs 11.64 ± 1.96,P ＞0.05).Compared with the control group,at day 5 posttransplant,CD4+ Th17 cells in the IκBα/N-Tg mice and IL-17 expression of the cardiac allograft were both decreased.Conclusion In the early stage after transplantation,specific T cell NF-κB impaired could abrogate the balance of the Tr and Th17 cells,and induce the T cells differentiated into Tr and inhibit the Th17 cells differentiation,and then induce tolerance.

Calpain ; genetics ; Diabetes, Gestational ; genetics ; Female ; Genotype ; Haploidy ; Humans ; Polymorphism, Single Nucleotide ; Pregnancy

AIM To establish an HPLC method for the simultaneous content determination of six constituents in Xuanmai Ganju Granules (Scrophulariae Radix,Ophiopogonis Radix,Glycyrrhizae Radix et Rhizoma,Platycodonis Radix).METHODS The analysis of 80％ methanol extract of this drug was performed on a 35 ℃ thermostatic ZORBAX SB-C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile0.1％ phosphoric acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelengths were set at 210,250,278 nm.RESULTS Harpagide,liquiritin apioside,liquiritin,harpagoside,cinnamic acid and glycyrrhizic acid showed good linear relationships within the ranges of 2.177-43.539 μg/mL(r =0.999 6),1.713-34.261 μg/mL (r =0.999 5),1.946-38.916 μg/mL(r =0.999 6),2.070-41.395 μg/mL(r =0.999 7),2.06-41.2 pg/mL (r =0.999 6) and 3.623-72.454 μg/mL (r =0.999 6),whose average recoveries (RS-Ds) were96.08％ (2.1％),95.55％ (2.5％),95.04％ (2.6％),94.86％ (2.7％),95.70％ (1.9％) and 95.47％ (1.9％),respectively.CONCLUSION This simple and accurate method can be used for the quality control of Xuanmai Ganju Granules.

Objective:To compare the two-dimensional electrophoresis(2-DE) maps of rat spinal cord protein extracted by two different solution systems.Methods: Adult rat spinal cord protein was precipitated with 10% trichloracetic acid in acetone and resuspended in 8 mol/L urea plus 4%CHAPS (A solution) or, 5 mol/L urea, 2 mol/L thiourea, 2%CHAPS plus 2%SB3-10 (B solution). One hundred and fifty micrograms of protein was loaded on 18 cm IPG strip holder and run isoelectric focusing electrophoresis as the first dimension, then horizontal SDS-PAGE as the second dimension. Protein spots were visualized by silver stain.Results:There were 1 059 and 1 023 protein spots in each map, of which 790 spots were matched in two maps. There were 269 and 233 spots exclusively extracted by A and B solutions, respectively. Taken together, 1292 different spots were totally obtained by A and B solutions.Conclusion: Integrating protein spots extracted by different solution systems is beneficial for achieving intact 2-DE map of tissues.

OBJECTIVETo study the effect of heme oxygenase-1(HO-1) on proteins related to apoptosis in INS-1 cells with exposure to intermittent high glucose.

METHODSINS-1 cells cultured in vitro were divided into control group, persistent high glucose group (PHG), intermittent high glucose group (IHG), CoPP + intermittent high glucose group (CoPP+IHG), and ZnPP+ intermittent high glucose group (ZnPP+IHG). After 72 h of treatment with the corresponding protocols, the cells were examined for expressions of HO-1 protein by Western blotting and for expressions of Bax and Bcl-2 by immunocytochemistry.

RESULTSIn comparison with the control group, the cells in both PHG group and IHG group showed significantly increased expressions of HO-1 (P<0.01) and decreased Bcl-2/Bax ratios (P<0.05). The cells in CoPP+ IHG group exhibited a greater HO-1 protein expression but a lower Bcl-2/Bax ratio than those in IHG group (P<0.05) The ZnPP+IHG group demonstrated opposite changes in terms of HO-1, Bax and Bcl-2 expressions compared with the CoPP+IHG group.

CONCLUSIONIntermittent high glucose can lower Bcl-2/Bax ratio in INS-1 cells, and HO-1 may protect INS-1 cells against apoptosis possibly by up-regulating the Bcl-2/Bax ratio.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line ; Glucose ; administration & dosage ; adverse effects ; Heme Oxygenase (Decyclizing) ; metabolism ; Islets of Langerhans ; cytology ; drug effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.
Apoptosis ; Blotting, Western ; Centrifugation, Density Gradient ; Ficoll ; Flow Cytometry ; Glucose* ; Humans ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Protein Kinases ; Signal Transduction ; Stem Cells*

BACKGROUND/OBJECTIVES: The objectives of this study were to investigate the effects of lycopene on the migration, adhesion, tube formation capacity, and p38 mitogen-activated protein kinase (p38 MAPK) activity of endothelial progenitor cells (EPCs) cultivated with high glucose (HG) and as well as explore the mechanism behind the protective effects of lycopene on peripheral blood EPCs. MATERIALS/METHODS: Mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. EPCs were identified after induction of cellular differentiation. Third generation EPCs were incubated with HG (33 mmol/L) or 10, 30, and 50 microg/mL of lycopene plus HG. MTT assay and flow cytometry were performed to assess proliferation and apoptosis of EPCs. EPC migration was assessed by MTT assay with a modified boyden chamber. Adhesion assay was performed by replating EPCs on fibronectin-coated dishes, after which adherent cells were counted. In vitro vasculogenesis activity was assayed by Madrigal network formation assay. Western blotting was performed to analyze protein expression of both phosphorylated and non-phosphorylated p38 MAPK. RESULTS: The proliferation, migration, adhesion, and in vitro vasculogenesis capacity of EPCs treated with 10, 30, and 50 microg/mL of lycopene plus HG were all significantly higher comapred to the HG group (P < 0.05). Rates of apoptosis were also significantly lower than that of the HG group. Moreover, lycopene blocked phosphorylation of p38 MAPK in EPCs (P < 0.05). To confirm the causal relationship between MAPK inhibition and the protective effects of lycopene against HG-induced cellular injury, we treated cells with SB203580, a phosphorylation inhibitor. The inhibitor significantly inhibited HG-induced EPC injury. CONCLUSIONS: Lycopene promotes proliferation, migration, adhesion, and in vitro vasculogenesis capacity as well as reduces apoptosis of EPCs. Further, the underlying molecular mechanism of the protective effects of lycopene against HG-induced EPC injury may involve the p38 MAPK signal transduction pathway. Specifically, lycopene was shown to inhibit HG-induced EPC injury by inhibiting p38 MAPKs.
Apoptosis ; Blotting, Western ; Centrifugation, Density Gradient ; Ficoll ; Flow Cytometry ; Glucose* ; Humans ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Protein Kinases ; Signal Transduction ; Stem Cells*