Objective To establish the expression of membrane-bound HLA-G1-G4 isoforms in choriocarcinoma cell line JAR and to investigate its roles in NK cytotoxicity in vitro. Methods Stable expression of HLA-G1, -G2, -G3 and -G4 in JAR cells was established by gene cloning and transfection.HLA-Gtranscripts and protein expression in the transfected JAR cells was tested by RT-PCR, flow cytometry, Western blot and immunocytochemistry, respectively. High-affinity peptide KIPAQFYIL pulsing was performed to evaluate its effects on HLA-G expression. Effects of HLA-G1-G4 isoforms on NK cytotoxicity was performed with lactic dehydrogenase (LDH) releasing method. Results RT-PCR, Western blot and immunocytochemistry results showed that exogenous HLA-G1-G4 gene were successfully transfected and proteins were stably expressed in the HLA-G negative JAR cells; Flow cytometry data showed that only HLAG1, but not HLA-G2-G4 isoform was detectable in those transfected JAR cells and the peptide pulsing did not affect their expression status. However, all HLA-G1-G4 isoform expressed JAR cells could significantly decreased the NK cell cytotoxicity (P＜0.05). Conclusion HLA-G1-G4 isoform expression could dramatically inhibit NK-92 cell lysis, indicating that membrane-bound HLA-G isoforms are importantly immunotolerant and may play immune regulation roles in various physio-pathological situations.

Objective To investigate the mechanism of acquisition of HLA-G1 antigen by NK cells.Methods K562 cells stably expressing HLA-G1 antigen (K562-G1) were constructed.K562-G1 cells, K562 cells and shed HLA-G1 were respectively co-cultured with NK-92MI cells to observe the acquisi-tion of HLA-G by NK cells.To further investigate the mechanism , NK-92MI cells with blockage HLA-G re-ceptors were further co-cultured with K562-G1 cells and HLA-G1 proteins expressing on K 562-G1 cells were blocked and then co-cultured with NK-92MI cells. Acquisition of HLA-G 1 by NK-92MI cells was analyzed by flow cytometry and fluorescence microscopy .The effects of HLA-G1 expression on the cytotoxicity of NK-92MI cell were evaluated by flow cytometry analysis based on CD 107a labeling.R esults NK-92MI cells could quickly acquire HLA-G1 from K562-G1 cells in co-culture experiments .Blockade of HLA-G1 or its re-ceptors KIR2DL4 and ILT2 with specific mAbs did not affect the acquisition of HLA-G1 by NK-92MI cells. Moreover, HLA-G1 could significantly inhibit the cytotoxicity of NK cell ( P<0.01).Conclu sion NK-92MI cells acquire HLA-G1 from K562-G1 cells via trogocytosis , which is not associated with affinity be-tween receptor and ligand , extracellular domain of HLA-G1 or passive adhesion .

Objective To investigate the expression of heat shock protein 70(HSP70) and c-fos,the presence of apoptosis and the neuroprotective effects of Cudrania tricuspidata root extrate(Ecr) after focal cerebral ischemic reperfusion in rats.Methods Models of middle cerebral artery occlusion(MCAO) were used in this study.The rats in Ecr pretreated ischemia reperfusion(IR) group were fed with Ecr(2 ml tid) for 5 days before MCAO.The brain specimen were took at different reperfusion times: 1 h,6 h,12 h,24 h,3 d and 7 d after recirculation.Then immunohistochemistry,insitu hybridization,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) were used to detect the expression of HSP70,c-fos gene and cell apoptosis in the brains.Positive results were semiquantitatively analyzed.Results IR could induce the expression of HSP70 and c-fos.HSP70 was detected at 6 h following reperfusion and peaked at 24 h both in cortical and basal ganglia regions.c-fos was markedly expressed at 1 h and the level peaked at 6 h in the ischemic hemispheres,and then reduced gradually.The TUNEL positive cells were markedly observed at 6 h.After treatment with Ecr,the positive reaction cells both of HSP70 and c-fos were significantly increased(all(P

Objective To investigate the mechanism of different HLA-G isoform mRNA patterns in different cells alters its cell membrane expression.Methods RT-PCR was used to analyze HLA-G isoform mRNA(HLA-Gl-6)of ovarian cancer cell lines HO-8910,HO-8910PM and OVCAR-3,leukemia cell lines Jurkat,K562,HIJ60,MUTIZ-1,and the chorioeareinoma cell lines JEG-3,JAR.HLA-G between cellular membrane and intracellular expression were analyzed by flow cytometry.Results All HLA-G mRNA isoforms were observed in the positive control cell line JEG-3,but none in the negative control cell line JAR.HLA-G1 isoform mRNA was expressed in HO-8910,HO-8910PM,OVCAR-3,MUTZ-1 and Jurkat cells.HLA-G2 mRNA was not detected in any cell line but JEG-3.HLA-G3 mRNA was found in HO-8910,HO-8910PM,K562,HIJ60,MUTZ-1,OVCAR-3 and Jurkat cells.HO-8910,HO-8910PM,HIJ60,Jurkat cells expre8sed HLA-G4 mRNA.Only the Jurkat cells expressed HLA-G5 mRNA.FACS results showed that JEG3 and HO-8910PM cells membrane expressed HLA-G,however,the intraeellular HLA-G expression was detected in all tested cells except the negative control cell JAR.Conclusion Only the HLA-G1 isoform could be exDressed on cell membrane in particular cell lines. Other isoforms including HLA-C2,-G3,-G4,-G5 and HLA-G6 could not reach cell snrface.

Human leukocyte antigen G ( HLA-G) is a member of the non-classical HLA classⅠb family. It is considered to play a crucial role in immune tolerance. A unique feature of HLA-G is the struc-tural diversity as surface expressed and as secreted molecules, which is mainly attributed to alternative spli-cing of the primary transcript. HLA-G can promote the invasion and metastasis of tumor cells through various ways. In addition, HLA-G has been described included in exosomes. Exosomes released by most cell types are nano-sized vesicular bodies that contain lipid bilayer and rich contents. As a new marker for diseases, exosomes are extensively involved in the occurrence and development of diseases. Recent studies have found that exosomes can express soluble HLA-G, which reveal a new way by which HLA-G regulates tumor micro-environment. In this review, we focus on the expression of HLA-G on exosomes to provide new thoughts for the early detection and treatment of tumors.

Objective To investigate the clinical significance of noninvasive detection of end-tidal carbon dioxide partial pressure (PetCO2) in the management of children with acute asthma, and to evaluate the association between PetCO2 and artery blood gas carbon dioxide partial pressure ( PaCO2 ). Methods This was a prospective,double blinded study of children aged 5 ～ 14 years old treated for acute asthma in a pediatric emergency department. PetCO2 and PaCO2 measurements were taken before therapy and after each nebulization treatment ( maximum of three). Various clinical parametesr were recorded. Patients with PaCO2 and PetCO2 measurements within 8 minutes of each other were eligible for inclusion. Patients with cardiac disease,chronic pulmonary disease, poor tissue perfusion, or metabolic abnormalities were excluded. Results Sixty five children were enrolled. The initial PetCO2 value was (34. 8 ±8. 6) mm Hg (95% confidence interval =34. 0 to 36. 1). The PetCO2 value of post-treatment was (33.2 ±8.2) mm Hg (95% confidence interval =32. 5 to 34. 4) ,which was decreased significantly than that before treatment(P ＜ 0. 01 ). Fifty seven PetCO2-PaCO2 paired values were available from 57 patients. The values of PetCO2 and PaCO2 were ( 34. 8 ±7. 6) mm Hg and (40. 6 ± 8. 3 ) mm Hg, respectively. PetCO2 and PaCO2 values were highly positively correlated ( r = 0. 92,P ＜ 0. 000 1 ). Conclusion Noninvasive bedside measurement of PetCO2 in children with acute asthma in emergency department is feasible. Continuous PetCO2 monitoring can provide a reliable assessment of pulmonary status. PetCO2 can serve as an important adjunct index in the clinical management of pediatric patients with acute asthma.

Objective To evaluate the relationships of the serum levels of matrix metalloproteinase-9 (MMP-9), intercellular adhesion molecule-1 (ICAM-1) and adiponectin with the mild cognitive impairment (MCI) in senile metabolic syndrome (MS)patients. Methods The 74 cases with MS and 30 health controls (control group) were enrolled. Mini-mental state examination (MMSE), montreal cognitive assessment (MoCA), digit-symbol test (DST), auditory verbal memory test (AVMT), trail making test(TMT), sunderland clock drawing test (CDT) and verbal fluency test (VFT) were applied to evaluate cognitive function. Based on the cognitive assessment, MS patients were divided into two groups: 39 cases with MCI (MS+MCI group) and 35 cases without cognitive impairment (MS group). The levels of MMP-9, ICAM-1 and adiponectin were measured by ELISA. Biochemical variables were measured by routine methods in all subjects. Results (1)MS+MCI group showed the higher levels of BMI, SBP, FBG and MMP-9 (all P＜0.05) and lower level of adiponectin (P＜0.05) than did the MS group. And MS group had higher levels of MMP-9 and ICAM-1 (P＜0.01) and lower adiponectin level (P＜0.01) than did the control group. (2)Spearman's correlation analysis showed that the serum levels of MMP-9 (r=-0.794, P＜0.001) and ICAM-l (r=-0.501, P＜0.001) were negatively correlated with adiponectin. However, MMP-9 was positively correlated with ICAM-1 (r=0.481, P=0.006). (3)Multiple linear regression analysis indicated that there was linear relationship of MoCA with MMP-9 (β=-3.438, P=0.0019), adiponectin (β=1.337, P=0.006), SBP (β=-0.058, P=0.003) and FBG (β=-0.227, P=0.049). (4)Stepwise logistic analysis showed that both high MMP-9 (OR=1.007) and low adiponectin (OR=0.359) were risk factors for the decline of cognitive function. Conclusions Elderly patients with MS may show deterioration in memory, calculation and visuospatial perception. Elevated inflammatory factors might contribute, in combination with abnormal metabolism, to MCI. MMP-9 might contribute to neuronal degeneration. However, adiponectin could strongly counteract the risk factors for cognitive impairment.

Objective To explore issues of the human tissue biobank, ranging from its establishment, collection and preservation of samples, quality control, management and application. Methods Development of standardized operational procedures, collection of such samples as fresh frozen tissues from the surgery, paraffin-embedded tissues, whole blood, serum, plasma, and cerebrospinal fluid. Specimens were classified and made aliquots according to different requirements, and then stored at room temperature, -80℃ refrigerator or in liquid nitrogen. Microsoft Access database system was used in the management of these specimens. Results From September 2004 to September 2008, a total of 11 872 samples from patients with benign and malignant diseases were collected and preserved. Among them, 4360 tubes of fresh frozen tissue samples from 2500 cases were provided within and beyond the province. These samples were also applied to DNA, RNA, protein extraction and tissue microarray, and immunohistochemistry-related research. Conclusion Human tissue biobank is highly useful in sharing human tissue resources effectively, as it can provide high quality specimens from benign and malignant diseases and normal control. In addition, it plays a very important role in exploring pathogenesis, developing new technologies for early detection of disease and new therapeutic strategies.

Toxoplasma gondii tachyzoites were isolated from an ocular patient in the Republic of Korea and maintained in the laboratory (designated KI-1). In the present study, its genotype was determined by analyzing dense granule antigen 6 (GRA6) gene and surface antigen 2 (SAG2) gene as typing markers. Digestion of the amplification products of GRA6 and of the 5' and 3' ends of SAG2, respectively, with Mse I, Sau3A I, and Hha I, revealed that KI-1 is included in the genotype I, which includes the worldwide virulent RH strain. In addition, when the whole sequences of the coding regions of SAG1, rhoptry antigen 1 (ROP1), and GRA8 genes of KI-1 were compared with those of RH, minor nucleotide polymorphisms and amino acid substitutions were identified. These results show that KI-1 is a new geographical strain of T. gondii that can be included in the genotype I.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/*genetics ; Base Sequence ; Genes, Protozoan ; Genotype ; Polymorphism, Genetic ; RNA, Protozoan ; Research Support, Non-U.S. Gov't ; Toxoplasma/*genetics

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.
Animals ; Antigens, Protozoan/analysis ; Female ; Humans ; Korea ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Middle Aged ; Parasitemia/parasitology ; Sarcoma 180 ; Serial Passage ; Specific Pathogen-Free Organisms ; *Toxoplasma/classification/growth & development/isolation & purification/pathogenicity ; Toxoplasmosis, Ocular/*diagnosis/parasitology ; Tumor Cells, Cultured ; Virulence