A two-step method for the purification of blocking-type anti-human CD80 monoclonal antibody 4E5 from mouse ascites was developed using anion exchange and gel filtration in combination. The ascites was first purified by anion exchange after centrifugation and filtration. The experimental parameters of sample loading and elution were optimized. The optimized loading condition was pH 8.0,50 mmol/L Tris-HCl and satisfactory results were obtained using a 0~0.5mol/L NaCl step elution. The fraction containing the protein of interest was directly loaded on gel filtration column and eluted using a 20 mmol/L phosphate buffer at pH 7.2. The purity of the obtained monoclonal antibody was up to 95% with a recovery of 61%. The purity of mAb could efficiently inhibit the growth of Daudi cells. The amplification of the method was also studied using a Bio-Scale Q5 column and the result was satisfied.

AIMTo study the effect of prothymosin alpha (Pro T alpha) as a fusion protein on secretion of IFN-gamma, IFN-alpha and TNF-alpha in vitro.

METHODSThe in vitro study was carried out on the culture of splenocytes, splenic and peritoneal macrophages isolated from Balb/c mice. Splenocytes were incubated with various concentrations of Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) with or without Con A (5 micrograms.mL-1) for 72 h. Splenic and peritoneal macrophages were respectively treated with Pro T alpha (1 x 10(-7)-1 x 10(-10) mol.L-1) in the presence of LPS (10 micrograms.mL-1) for 24 h. Then IFN-gamma, IFN-alpha and TNF-alpha levels in the supernatant were detected by ELISA.

RESULTSPro T alpha (1 x 10(-7) mol.L-1) was found to obviously increase IFN-gamma level (P < 0.05) in the supernatant of splenocytes compared with the control group. Moreover, Pro T alpha (1 x 10(-7) mol.L-1) significantly induced the secretion of IFN-alpha (P < 0.01) and TNF-alpha (P < 0.01) in splenic and peritoneal macrophages.

CONCLUSIONIn vitro, Pro T alpha could increase the secretion of IFN-gamma, IFN-alpha and TNF-alpha.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cell Separation ; Female ; Glutathione Transferase ; pharmacology ; Interferon-alpha ; secretion ; Interferon-gamma ; secretion ; Lymphocytes ; secretion ; Macrophages ; drug effects ; secretion ; Macrophages, Peritoneal ; secretion ; Mice ; Mice, Inbred BALB C ; Protein Precursors ; pharmacology ; Recombinant Fusion Proteins ; pharmacology ; Spleen ; cytology ; Thymosin ; analogs & derivatives ; pharmacology ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate pulmonary function impairment and the spinal factors that may determine pulmonary function in patients with scoliosis.

METHODSSeventy-eight patients with idiopathic scoliosis or congenital scoliosis and 78 age- and gender-matched healthy subjects were enrolled in this study. The radiographic parameters of spinal deformity were obtained from patients with scoliosis. Both two groups received pulmonary function tests.

RESULTSPatients with scoliosis demonstrated a restrictive pattern of pulmonary function impairment with a proportional decrease in both forced expiratory volume in one second and forced vital capacity. Total lung capacity and functional residual capacity were reduced. Carbon monoxide diffusion capacity was decreased, while diffusion coefficient remained normal or slightly higher. Airway resistance and conductance were not affected. In addition, airway resistance and residual volume were found abnormal in patients with congenital scoliosis. Multiple linear regression analysis showed that three spinal factors including involved thoracic vertebrae, vertical height from C7 to S1, and Cobb angle were independently responsible for 40%-51% of total variances of forced vital capacity, forced expiratory volume in one second, total lung capacity, and functional residual capacity.

CONCLUSIONSPatients with scoliosis have restrictive ventilation defects. More thoracic vertebrae involvement, lower vertical height, and larger Cobb angle are associated with severer impairment of lung volume.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Humans ; Lung ; physiopathology ; Male ; Radiography ; Scoliosis ; pathology ; physiopathology ; Thoracic Vertebrae ; diagnostic imaging ; pathology ; Young Adult

OBJECTIVETo investigate the impact of energy metabolism at the cellular level on the expression of the water channel protein aquaporin 1 (AQP1).

METHODSBalb/c mouse fibroblasts were incubated with iodoacetamide (IA) in vitro, and the changes in AQP1 expression were detected by immunoblotting and immunohistochemistry at 0, 4, and 6 h.

RESULTSIA induced the expression of AQP1 at 4 and 6 h accompanied with cell death. Reverse transcription PCR showed an increased expression of AQP1 mRNA in the cells. AQP1 expression was also upregulated by the inhibitor of microtubule and cytochrome C oxidase.

CONCLUSIONA pretranslational regulation occurs in IA-induced AQP1 expression in mouse fibroblasts, and the up-regulated AQP1 accumulation is characterized by mitochondria-related energy dependence.

Animals ; Aquaporin 1 ; genetics ; metabolism ; Cells, Cultured ; Energy Metabolism ; Fibroblasts ; cytology ; metabolism ; Iodoacetamide ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mitochondria ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects

Objective:To investigate the immunological features of 135 patients with corona virus disease 2019 (COVID-19), and to provide reference for the pathogenesis of the disease.Methods:The clinical and laboratory data of 135 confirmed COVID-19 patients in Guangzhou Eighth People′s Hospital from January 23 to February 29, 2020 were collected. The features of lymphocytes (CD4 + and CD8 + T lymphocytes, B lymphocytes, natural killer cells and natural killer T cells), and cytokines (interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ) of patients at a median of 19 (14, 27) days of admission were analyzed. Kruskal-Wallis test, Mann-Whitney U test, chi-square test and Spearman rank correlation were used for statistical analysis. Results:Patients were divided into three groups according to the relevant diagnostic criteria, including mild group (14 cases), ordinary group (92 cases) and severe group (29 cases). Decreased CD4 + T lymphocytes were found in 44.4% (60/135) patients, while decreased CD8 + T lymphocytes were found in 42.2%(57/135) patients. Compared to mild group and ordinary group, level of CD4 + T lymphocytes in severe group was significant lower ( Z=4.379 and 3.799, respectively, both P<0.01), and level of CD8 + T lymphocytes was also significant lower ( Z=2.684 and 3.306, respectively, P=0.022 and 0.003, respectively). Decreased B lymphocytes were found in 25.3% (24/95) patients and significant different among the three groups, with the lowest levels ((88(56, 189)/μL; Z=6.199, P=0.045) and most frequency of decreased levels ((52.2%(12/23); χ2=11.723, P=0.003) in the severe group. Compared to the mild group and the ordinary group, IL-6 level in severe group was significant higher ( Z=-4.022 and -4.108, respectively, both P<0.01) and IL-10 level was also significant higher ( Z=-3.261 and -4.006, respectively, both P<0.01). Similar levels of IL-2, IL-4, TNF-α and IFN-γ were found among three groups (all P>0.05). The IL-6 level was positively correlated with the persistence of viral shedding ( r=0.301, P=0.007). Conclusion:The immune-mediated inflammation may be the important cause of disease deterioration of COVID-19, which might be the key target of the treatment of severe cases.

Objective To analyse the relationship between Fourier transform infrared （FTIR） spectrum ofrat's spleen tissue and postmortem interval （PMI） for PMI estimation using FTIR spectroscopy combinedwith data mining method. Methods Rats were sacrificed by cervical dislocation, and the cadavers were placed at 20 ℃. The FTIR spectrum data of rats' spleen tissues were taken and measured at different time points. After pretreatment, the data was analysed by data mining method. Results The absorption peak intensity of rat's spleen tissue spectrum changed with the PMI, while the absorption peak position was unchanged. The results of principal component analysis （PCA） showed that the cumulative contribution rate of the first three principal components was 96%. There was an obvious clustering tendency for the spectrum sample at each time point. The methods of partial least squares discriminant analysis （PLS- DA） and support vector machine classification （SVMC） effectively divided the spectrum samples with different PMI into four categories （0-24 h, 48-72 h, 96-120 h and 144-168 h）. The determination coefficient （R2） of the PMI estimation model established by PLS regression analysis was 0.96, and the root mean square error of calibration （RMSEC） and root mean square error of cross validation （RMSECV） were 9.90 h and 11.39 h respectively. In prediction set, the R2 was 0.97, and the root mean square error of prediction （RMSEP） was 10.49 h. Conclusion The FTIR spectrum of the rat's spleen tissue can be effectively analyzed qualitatively and quantitatively by the combination of FTIR spectroscopy and data mining method, and the classification and PLS regression models can be established for PMI estimation.

Objective To investigate the interaction between vessel and vena cava filter (VCF) with different numbers of support poles and the influence on blood flow during its implantation by using finite element method and computational fluid dynamics method, so as to provide more scientific guidance for intervention treatment and design & development of the filter. Methods Three kinds of VCF models with 4, 6, 8 support poles were established by using Solidworks, respectively. The stress distribution and radial support stiffness of the vessel and filter under working condition were then simulated and analyzed by using ABAQUS. The distributions of blood flow velocity, pressure and shear stress after VCF implantation were simulated by using Fluent. Results All the mechanical and fluid properties of the three kinds of VCFs were within the scope of security. The comprehensive mechanical performance of the 6-pole filter was better, while for the 8 pole-filter, it showed larger stress under working condition, larger support intensity, higher exit velocity, larger shear stress on the filter mesh, and the trail of the flow was changed from laminar to transition flow, which might cause some damage to the vessel wall. The 4 pole-filter under working condition had a larger stress on the vessel, while its exit velocity was lower, which could be likely to cause local damage on the vessel wall, and disadvantageous to accurately positioning the filter in diseased region of the vessel. Conclusions The 6 pole-filter has a superior hemodynamic effect and comprehensive mechanical properties, which reduces the potential possibility of damage to the vessel wall caused by implantation of the filter. The simulation analysis on VCF provides a good reference for design of the filter and its intervention treatment in clinic.

Animals ; Coronary Vessels ; surgery ; Drug-Eluting Stents ; adverse effects ; Lactic Acid ; chemistry ; Polyesters ; Polymers ; chemistry ; Sirolimus ; chemistry ; therapeutic use ; Swine

AIM To establish an LC-MS/MS method for the simultaneous content determination of forsythin,forsythoside A,chlorogenic acid,neochlorogenic acid,amygdalin,emodin,rhein and salidroside in Lianhua Qingwen Capsules.METHODS The analysis was performed on a 35℃thermostatic ACQUITY UPlC-HSS T3 column(100 mm×2.1 mm,1.8 μm),with the mobile phase comprising of 0.1%formic acid-acetonitrile flowing at 0.3 mL/min in a gradient elution manner,and electron spray ionization source was adopted in negative ion scanning with multiple reaction monitoring mode.RESULTS Eight constituents showed good linear relationships within their own ranges(r≥0.999 5),whose average recoveries were 99.20%-100.96%with the RSDs of 0.62%-1.23%.CONCLUSION This simple,sensitive and reliable method can be used for the quality control of Lianhua Qingwen capsules.

OBJECTIVETo introduce a novel Cobb protractor and assess its reliability and rapidity for measuring Cobb angle in scoliosis patients.

METHODSThe novel Cobb protractor had two endplate markers. A measurement was performed just to align the two markers to each endplate of the curve. The Cobb angle on the posteroanterior radiographs of 24 patients clinically diagnosed with adolescent idiopathic scoliosis was measured by three orthopedic surgeons with both standard Cobb method and the new technique, and the time of measurement was recorded. Intraclass correlation coefficients (ICCs) were calculated to assess the reliability of the new method.

RESULTSThe time for a measurement with the new tool was approximately 10 seconds less than the time that used to finish a measurement with the standard method (P<0.05). The overall mean Cobb angle for the major curve of the 24 patients was 47.8°. The mean overall intraobserver and interobserver ICC was 0.971 and 0.971 for the Cobb method group, while the overall intraobserver ICC and the interobserver was 0.985 and 0.979 for the new tool group.

CONCLUSIONSThe novel Cobb protractor could perform quick measurement and measure almost all forms of radiographs. The Cobb protractor might be an ideal instrument to measure the Cobb angle.

Adolescent ; Child ; Equipment and Supplies ; Humans ; Radiography ; Reproducibility of Results ; Scoliosis ; diagnostic imaging ; Spine ; diagnostic imaging