Objective To establish a novel method to detect autoantibodies against platelatespecific receptors by flow cytometric bead assay and study its clinical application. Methods The beads were coated with monoclonal antibodies SZ2, SZ22, SZ21 and 7E3 against platelet GP Ⅰ b, GP Ⅱ b, GP Ⅲa and GP Ⅱ b/Ⅲ a, respectively. Captured platelet glycoprotein and beads complex was detected by FITC labeled polyclonal goat antihuman immunoglobulin using flow cytometer. The platelet samples that reacted with antibodies (SZ2, SZ22, SZ21 and 7E3) negatively and positively were tested, respectively. Each sample was repeated 20 times to generate intra-day CV for the MFI and once a day for 8 days to generate inter-day CV values. The 85 ITP patients, 17 NITP patients and 50 controls from the First Affiliated Hospital of Soochow University during March 2006 to December 2008 were included in the studies. The sensitivity and specificity of these four platelet antibodies to diagnose ITP were analyzed using ROC curve. The results were compared with MAIPA. Results The CV of the intra-day-assay for samples negative to antibody SZ2, SZ22,SZ21 and 7E3 were 3.26%, 2. 86%, 1.65% and 4. 94%, respectively; While the CV of the intra-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 6. 16%, 4. 88%, 5.20% and 5. 85%,respectively. The CV of the inter-day-assay for samples negative to antibody SZ2, SZ22, SZ21 and 7E3 were 5. 86%, 4. 74%, 5.69% and 7.56%, respectively; While the CV of the inter-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 7.53%, 5.49%, 7.11% and 6.25%,respectively. The MFI for SZ2 in ITP group, NITP group and healthy control group were 1.49(0. 88-16. 24),1.12(1.00-1.33), 1.01 (0. 83-1.37), respectively, which showed significant differences (H = 36.89,P＜0.01). The MFI for SZ22 in the three groups were 1.55 (0.84-11.30), 1.13(1.03-1.29), 0.98(0. 85-1.24), respectively (H=28.41, P ＜0.01). The MFI of SZ21 were 1.50 (0.87-11.04), 1.13(0.97-1.32), 1.05 (0.85-1.48), respectively (H=54.42, P＜0. 01). The MFI for7E3 were 1.51(0. 84-9.81), 1.05(0.86-1.13), 1.03 (0.74-1.28), respectively (H =31.97, P ＜0.01). Based on ROC analysis, with cut-off values of 1.37, 1. 24, 1.48 and 1.28 for SZ2, SZ22, SZ21 and 7E3,respectively, the AUC were 0. 86, 0.90, 0. 87 and 0. 84, respectively. The sensitivities of the assays were 58. 82% (50/85), 52. 94% (45/85), 52.94% (45/85) and 51.76% (44/85), respectively. When all four antibodies were used, the sensitivity was increased to 74. 12% (63/85), which was higher than that of MAIPA [ 50. 59% (43/85) ,χ2 = 6. 78, P ＜ 0. 05) ]. Conclusion Flow cytometric bead assay can be used to detect four platelet-specific autoantibodies simultaneously, and may be a useful method to aid in the diagnosis of ITP.

Objective To screen and identify the predominant T-and B-cell ( T-B) combined an-tigenic epitopes on the outer membrane protein Loa22 of pathogenic Leptospira interrogans ( L.interrogans) stains and to further analyze their immunogenicity.Methods PCR analysis was used to detect loa22 gene in L.interrogans strains belonging to eight different serogroups or serovars prevalent in China.The PCR prod-ucts were sequenced after T-A cloning.A prokaryotic expression system for loa22 gene of L.interrogans sero-group Icterohaemorrhagiae serovar Lai strain Lai was constructed.The expressed recombinant protein rLoa22 was extracted by Ni-NTA affinity chromatography.The rabbit anti-rLoa22 serum samples and IgG were pre-pared.The T-B combined antigenic epitopes on Loa22 protein were predicted by using professional bioinfor-matic softwares.Phage display in combination with Western blot assay and ELISA were performed to identify the immunogenicity of the recombinant phage PⅢ protein-displayed and artificially-synthesized T-B com-bined antigenic epitopes, respectively.MTS assay and ELISA were performed to detect the activation of T cells and the expression of IL-2, IL-4 and IFN-γinduced by T-B combined antigenic epitopes, respectively. Results All of the tested pathogenic Leptospira strains were positive for loa22 gene, sharing 85.5%-99.8%homologies in nucleotide sequences and 93.9%-99.5%homologies in amino acid sequences.The construc-ted prokaryotic expression system for loa22 gene efficiently expressed the rLoa22 protein.Among four T-B combined antigenic epitopes (Loa22-77, Loa22-90, Loa22-125 and Loa22-157), only Loa22-90 epitope presented a strong positive band in Western blot analysis.The proliferation of CD4+T cells and the expres-sion of IL-2 ( Th1 ) and IL-4 ( Th2 ) were significantly enhanced by the stimulation with Loa22-90 epitope peptide (P<0.05).Conclusion Loa22 protein is a sequence-conserved genus-specific outer membrane protein of L.interrogans.The Loa22-90 epitope is the predominant T-B combined antigenic epitope of Loa22 protein, which might be used as a candidate antigenic epitope in the development of multiple antigenic pep-tide ( MAP) vaccines against Leptospira infection.

AIM: To investigate the clinical significance in determination of the P- selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P- selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently - labeled SZ - 51 by direct FCM comparing with indirect FCM and enzyme- linked immunosorbent assay (ELISA). RESULTS: The level of P- selectin on platelet membrane is higher in DM (23.92 % + 15.83 % ), in hyperlipidemia ( 18.34 % + 9.46 % ) and in cerebral infarction ( 19.32 % + 10.38 % ) than normal subjects (3.38 % + 1.11% ) ( P < 0.01 ). In addition, similar results on P - selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. CONCLUSION: FITC - labeled SZ - 51 - IgG can be used in FCM, and it would be a new and sensitive method in detecting platelet activation.

Adult ; Datura metel ; poisoning ; Foodborne Diseases ; therapy ; Humans ; Male ; Middle Aged ; Young Adult

AIM: To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). METHODS: The P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, were analyzed using fluorescently-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). RESULTS: The level of P-selectin on platelet membrane is higher in DM (23.92%?15.83%), in hyperlipidemia (18.34%?9.46%) and in cerebral infarction (19.32%?10.38%) than normal subjects (3.38%?1.11%) (P

Angelica sinensis ; chemistry ; Animals ; Drugs, Chinese Herbal ; therapeutic use ; Lung Diseases, Interstitial ; drug therapy ; Male ; Phytotherapy ; Pulmonary Fibrosis ; drug therapy ; Rats ; Rats, Wistar

OBJECTIVE To investigate the clinical feature of nosocomial infections in general hospital,based on which effective methods are taken.METHODS An analysis was made on the basis of the survey,the data came from 73 690 hospitalized cases from 2000 to 2004.Bacterium appraise adopted French VITEK-32 fully automatic Expression.RESULTS Their prevalence rate was 5.34%,the main infection site was lower respiratory tract,and followed by upper respiratory tract and urinary tract.Among bacteria isolated from clinical cases,49.24% were G~-,(25.30%) were G~+,and 25.46% were fungi.The proportion of conditional bacteria showed an increasing trend from 2000 to 2004.CONCLUSIONS The conditional bacteria show an increasing trend in the nosocomial(infections.) Strengthening the nosocomial infection management,treating underlying disease actively,lessing(invasive) process,supervising towards bacteria,and using antibacterial drug rationally are effective methods to(reducing) the rate of nosocomial infections.

Objective To investigate the safety of the occipital condylar screw with vertical position and evaluate the selection strategy of the posterior approach of the posterior occipital condylar screw in Chinese people.Methods The clinical imaging data of 60 outpatients from September 2013 to September 2015 were retrospectively analyzed,36 male and 24 female,the average age was 41.6±9.2 (range from 25-58),Excluded occipitocervical injury,tumor and deformity patients.We built a three-dimensional digital model and simulated placing screw by utilizing CT data on Mimics software,after that we took the occipital condyle posterior medial and lateral midpoint as the entry point,then made 2 points equidistantly to the midpoint in vertical direction.We put 3.5 mm diameter virtual screws in 4 different conditions:largest cranial angle,smallest cranial angle,longest screw path and shortest screw path.Then we assessed the anatomical relationship between the screw and the hypoglossal canal or the atlanto-occipital joint by a three-dimensional window and measured the cranial angle,medial angle and length of screw path,then calculated the safety angle of the cranial angle,the successful rate of setting screw,and compared the safety of different screw points by 3-Matic software.Results 120 occipital condyles were obtained from the CT data of 60 patients by Mimics software.There was no significant difference in the data of the cranial angle,medial angle,safety range and length between both left and right sides.The obtained safe cranial angle of each point respectively was 20.9°±6.0° (lowest point),17.0°±6.2° (middle point),and 11.6°±7.1°(top point),obviously the largest angle was in the lower point and the smallest was in the top point.The difference was statistically significant.We then acquired the successful rates of different cranial angle of each point,the highest successful rate was 99.17％,96.67％,74.17％ in lowest,middle and top point when cranial angle were 3°or 4°,3°and 0°respectively.The successful rates of lower point and niddle point were significantly higher than the top point,and the difference was statistically significant.The medial angle parameters obtained were 34.41°±2.59°on left and 34.06°±2.44°on right,and there was no significant difference.The length parameters of the longest screw path acquired were 23.09± 1.47 mm,22.84± 1.40 mm and 23.15± 1.45 mm at top,middle and lowest entry point.The average value of shortest screw path of each point was 21 mm,and there was no significant difference among every entry point.Conclusion Among the occipital condyle posterior screw entering points,selecting the lower point can improve the success rate and safety;the change of nail enter point in the vertical direction has little effect on the length of the nail.We can increase the safety and reduce the risk of occipital condylar screw placement as far as possible through the three-dimensional digital technology.

OBJECTIVETo explore the molecular mechanism of microRNA-21 in myocardial damage of rats in the early stage of severe scald injury by observing the expression of microRNA-21 and programmed cell death 4 (PDCD4) in myocardial tissue of rat and to validate the relationship between them in cell model.

METHODS(1) Forty SD rats were divided into sham injury group (n =8, sham injured) and scald injury group (n =32, inflicted with 30% TBSA full-thickness scald on the back) according to the random number table. The left ventricular tissue was collected from rats in sham injury group at post injury hour 1 without any fluid infusion. Rats in scald injury group were given an intraperitoneal injection of lactic acid Ringer's solution and 8 rats were respectively sacrificed at post injury hour 3, 6, 12, 24 to harvest left ventricular tissue. The expression of microRNA-21 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. The protein expression of PDCD4 in myocardial tissue was assessed by Western blotting. (2) Rat myocardial cell line H9C2 was divided into microRNA-21 inhibitor group (cells were transfected with microRNA-21 inhibitor) and negative transfection control group (cells were transfected with negative control of microRNA inhibitor) according to the random number table. At post transfection hour 48, real-time fluorescent quantitative RT-PCR and Western blotting were performed respectively to determine the mRNA and protein expression levels of PDCD4 in cells. Data were processed with one-way analysis of variance, LSD-t and two independent samples t test. The relationship between microRNA-21 expression and PDCD4 protein level in myocardial tissue of rats was assessed by linear correlation analysis.

RESULTS(1) The expression levels of microRNA-21 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0. 96 ± 0. 13, 0. 44 ± 0. 08, 0. 42 ± 0. 10, 0.33 +0.07, and 0.61 0.10 (F = 27.331, P <0.001). Compared with that in myocardial tissue of rats in sham injury group at post injury hour 1, expression level of microRNA-21 was significantly decreased in scald injury group at post injury hour 3, 6, 12, 24 (with t values from 4. 558 to 9.410, P values below 0.01). The protein expression levels of PDCD4 in myocardial tissue of rats in sham injury group at post injury hour 1 and in scald injury group at post injury hour 3, 6, 12, 24 were respectively 0.44 ± 0.05, 0.60 ± 0.09, 0.92 ± 0. 15, 0. 86 ± 0.11, and 0.57 ± 0. 10 (F =8.622, P =0.003). Compared with that in sham injury group at post injury hour 1, protein expression level of PDCD4 was significantly increased in scald injury group at post injury hour 6 and 12 (with t values respectively 4. 968 and 4. 122, P values below 0.01). A significant negative correlation between the expression of microRNA-21 and PDCD4 protein in myocardial tissue of rats of scald injury group was observed at each time point (r = -0. 572, P = 0. 026). (2) The mRNA and protein expression levels of PDCD4 of myocardial cells in microRNA-21 inhibitor group were respectively 1.73 ± 0. 29 and 0. 38 ± 0. 08, which were significantly higher than those in negative transfection control group (0.95 ± 0.14 and 0.23 ± 0.03, with t values respectively 4. 857 and 3.356, P <0.05 or P <0.01).

CONCLUSIONSExpression of microRNA-21 was decreased, while expression of PDCD4 was increased, in myocardial tissue of rats in the early stage of severe scald injury. MicroRNA-21 might participate in myocardial damage in the early stage of scald injury by negatively regulating expression of PDCD4.

Animals ; Apoptosis Regulatory Proteins ; metabolism ; Blotting, Western ; Burns ; metabolism ; pathology ; MicroRNAs ; genetics ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Soft Tissue Injuries

Objective To evaluate the clinical effect of postural reduction combined with miniincision screw-rod system and percutaneous kyphoplasty (PKP) in treating osteoporotic thoracolumbar burst fractures.Methods A retrospective case series study was performed for data of 35 patients with osteoporotic thoracolumbar burst fractures without neurological deficits undergone mini-incision screw-rod system fixation and PKP between January 2012 and January 2014.There were 14 males and 21 females,with a mean age of 63.2 years (range,50-72 years).Operation time,intraoperative blood loss,complications,visual analogue score (VAS),height of fractured vertebrae and kyphosis Cobb angle were recorded.Results Operation time was (49.6 ± 6.8) min,and intraoperative blood loss was (45.6 ±7.8)ml.All patients were followed up for 9-18 months (mean,13.5 months).No intraoperative or postoperative serious complications occurred,including intracanal cement leakage,breakage or loosening of the screws.VAS of back pain was decreased from (8.4 ± 1.1)points preoperatively to (3.5 ± 0.6)points postoperatively (P ＜ 0.05).Height of the fractured vertebrae was improved from (49.62％ ± 5.68)％ preoperatively to (86.64 ± 6.63) ％ postoperatively (P ＜ 0.05).Kyphosis Cobb angle was improved from (28.12 ± 1.06) °preoperatively to (5.15 ± 1.08) °postoperatively (P ＜0.05).At the final follow-up,VAS was further decrease and vertebral height and Cobb’ s showed a slight loss of correction.Conclusion Postural reduction combined with mini-incision screw-rod system and PKP can relieve back pain,restore the height of injured vertebrae,correct kyphotic deformity and reduce operation time and blood loss,indicating a minimally invasive,safe and effective procedure for treatment of osteoporotic thoracolumbar burst fractures.