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.

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

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

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

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 determine GJB2 allelic mutant and estimate probability of hereditary hearing loss in newborn with GJB2 heterozygous mutation in Beijing. METHOD: We performed genetic testing for sequencing of GJB2 gene for searching GJB2 allelic mutant in 915 newborn who received newborn deafness gene screening (GJB2 c. 235delC, GJB2 c. 299_300delAT, GJB2 c. 176191del16, GJB2 c. 35delG) in Beijing Tongren hospital, and the mutation were classified to pathogenic mutation,undefined variant and polymorphism. RESULT: Four hundred (43.72%, 400/915) newborn were detected to carry at least one mutation allele in GJB2. 3 (0.33%, 3/915) newborn had pathogenic mutations (c. 94C>T, c. 380G>T, c. 344T>G); 62 (6.76%, 62/915) newborn carried 14 undefined variant, 36 newborn had c. 109G>A (58.06%, 36/62),13 newborn had c. 368C>A (20.97%,13/62), six (c. 268C>G, c. 282C>T, c. 294G>C, 456C>T, c. 501G>A, c. 587T>C) are novel; 335 (36.61%, 335/915) newborn were polymorphism. CONCLUSION The probability of hereditary hearing loss is 7.09% in newborn with GJB2 heterozygous mutation in Beijing. It is noteworthy that c. 109G>A, c. 368C>A occupy a high proportion.
Alleles ; Beijing ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; Deafness ; genetics ; Genetic Testing ; Heterozygote ; Humans ; Infant, Newborn ; Mutation ; Neonatal Screening ; Polymorphism, Genetic

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.

Objective To study the chemical constituents of Abacopteris penangiana.Methods The compounds were separated and purified by various chromatographic techniques and their structures were elucidated on the basis of physiochemical properties and spectroscopic methods.Results Seven compounds were purified and their structures were identified as:(7'Z)-3-O-(3,4-dihydroxy phenylethenyl)-caffeic acid(1),caffeiein B(2),matteucinol(3),protocatechuic acid(4),p-methoxybenzoic acid(5),β-sitosterol(6),and daucosterol(7).Conclusion Compound 1 is a new phenolic acid compound named abacopteric acid,and the other compounds are isolated from the plant for the first time.