OBJECTIVETo study the association between Alzheimer' s disease (AD) and apolipoprotein E (apoE) polymorphism and apoE epsilon4 allele; and to investigate the role of apoE in senile plaque formation.

METHODSDuring the period from 1982 to 2003, 27 portmortem cases of AD from the archival files of Department of Pathology of Beijing Hospital, diagnosed according to the consortium to establish a registry for Alzheimer's disease (CERAD) criteria, were enrolled into this study. Among the 27 cases studied, there were 23 cases of definite AD and 4 cases of probable AD. Postmortem brain tissues from 67 neurologically unremarkable deceased were used as age-matched controls. Immunohistochemical study for beta-amyloid (Abeta) and Tau protein, as well as immunohistochemical study for Abeta/apoE, were performed in all AD cases using streptavidin-peroxidase (SP) and double immunostaining ( SP/ABC) methods, respectively. Senile plaques and neurofibrillary tangles in the 23 cases of definite AD were further quantified. The apoE genotypes in all cases were analyzed by polymerase chain reaction and restriction fragment length polymorphism technologies.

RESULTSImmunohistochemical study for Abeta distinguished 4 different types of senile plaques: diffuse non-neuritic plaques, diffuse neuritic plaques, dense-core neuritic plaques and dense-core non-neuritic plaques. Double immunohistochemistry for Abeta/apoE showed that some senile plaques were positive for both Abeta and apoE. The expression rates for Abeta and apoE in these 4 different types of senile plaques were 4. 28%, 84. 71%, 8.50% and 2.51%, respectively. The positivity rate for Abeta/apoE in diffuse neuritic plaques were significantly higher than those in other 3 types (P < 0.01). The frequency of occurrence of apoE epsilon4 allele in AD was significantly higher than that in the control group (P < 0.01). The numbers of senile plaques and neurofibrillary tangles in AD cases with apoE epsilon4 allele were also significantly higher than those in AD cases without apoE epsilon4 allele (P < 0.01).

CONCLUSIONSApoE polymorphism is associated with AD. The presence of apoE epsilon4 allele carries a higher risk for the development of AD. ApoE may also play an important role in the transformation of diffuse non-neuritic plaques to diffuse neuritic plaques.

Aged ; Aged, 80 and over ; Alleles ; Alzheimer Disease ; metabolism ; pathology ; Amyloid beta-Peptides ; metabolism ; Apolipoproteins E ; genetics ; metabolism ; Brain ; metabolism ; pathology ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Neurofibrillary Tangles ; pathology ; Plaque, Amyloid ; pathology ; tau Proteins ; metabolism

ObjectiveTo find out the risk factors causing iatrogenic spinal cord injury (ISCI) so as to provide theoretical support for reducing the spinal cord injury during spinal operation. Methods A retrospective study was done on 120 patients undergone cervical or thoracic spinal( C1-T12 ) surgery at Xiangya Hospital of Central South University from January 2002 to January 2009. The patients were randomly divided into injury group (n = 34) and control group (n = 86) and the univariate analysis was used to analyze 30 factors including clinical factors, iconography factors, operation and pathology factors as well as possible protective factors. Then, the factors with statistical difference were analyzed by using the multi-factor unconditioned Logistic analysis.Results The univariate comparison between the two groups showed statistical difference ( P ＜ 0. 05 ) in nine factors including combined hypertension, combined diabetes mellitus, preoperative ASIA grade, spinal canal stenosis rate, ratio of spinal cord area/efficient area of vertebral canal, spinal cord MRI T2WI high signal, bleeding amount during operation, intraspinal prominence adhesion to dura mate of spinal cord as well as intraoperative use of methylprednisolone. The multi-factor Logistic regression analysis revealed that ASIA grade, value of spinal cord area/efficient area of vertebral canal, spinal cord MRI T2W1 high signal and bleeding amount in operation had positive correlation with ISCI. Use of methylprednisolone during operation had negative correlation with ISCI. ConclusionsCombined diabetes mellitus, ASIA grade, spinal cord MRI T2W1 high signal, ratio of spinal cord/vertebral canal area and bleeding amount in operation are the risk factors for ISCI. Use of large dose methylprednisolone exerts preventive effect on ISCI.

OBJECTIVETo study the significance of cytokine IL-1α and S100β expression in formation and evolution of different types of plaques in Alzheimer's disease.

METHODSThirty-four autopsy cases of Alzheimer's disease encountered during the period from 1982 to 2008 were retrieved from the archival files of Department of Pathology, Beijing Hospital. Tissue blocks were taken from hippocampus for dual immunostaining for IL-1α/Aβ and S100β/Aβ.

RESULTSImmunohistochemical studied for IL-1α/Aβ and S100β/Aβ delineated four different types of senile plaques: diffuse non-neuritic plaques, diffuse neuritic plaques, dense-core neuritic plaques and dense-core non-neuritic plaques. The numbers of IL-1α-positive microglias and S100β-positive astrocytes associated with diffuse neuritic plaques were (7.29 ± 3.04) per mm(2) and (6.49 ± 2.20) per mm(2), respectively. In contrast, the numbers of IL-1α-positive microglias and S100β-positive astrocytes associated with diffuse non-neuritic plaques, dense-core neuritic plaques and dense-core non-neuritic plaques were (3.24 ± 1.53) per mm(2) and (4.14 ± 1.77) per mm(2), (2.09 ± 1.37) per mm(2) and (2.25 ± 0.83) per mm(2), and (1.38 ± 0.90) per mm(2) and (0.58 ± 0.36) per mm(2), respectively. The numbers of IL-1α-positive microglias and S100β-positive astrocytes associated with diffuse neuritic plaques were significantly higher than those of the other three types of plaques (P < 0.05).

CONCLUSIONThe IL-1α-positive microglias and S100β-positive astrocytes may be of certain significance in transformation of diffuse non-neuritic plaques to diffuse neuritic plaques in Alzheimer's disease.

Aged ; Aged, 80 and over ; Alzheimer Disease ; metabolism ; pathology ; Astrocytes ; metabolism ; Female ; Hippocampus ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Interleukin-1alpha ; metabolism ; Male ; Microglia ; metabolism ; Middle Aged ; Nerve Growth Factors ; metabolism ; Plaque, Amyloid ; classification ; metabolism ; pathology ; S100 Calcium Binding Protein beta Subunit ; S100 Proteins ; metabolism

Objective Through experiments in vitro,we explored the role of Chinese medicine monomer-Astragaloside Ⅳ with DC vaccine in the body immunologic function.Methods Peripheral blood mononuclear cells (PBMCs)from healthy volunteers were isolated,cultured and generated in vitro,pulsed with tumor antigen from SGC7901 gastric carcinoma cell lysates,produced DC vaccine.Observe T-cell proliferation responses stimulated by DC vaccine with AⅣ group,DC vaccine group and AⅣ group respectively,and the anti-tumor effects on SGC7901 cells in vitro.Results ①The T-cell proliferation rate of DC vaccine with AⅣ group and DC vaccine group were significantly higher than AⅣ group and T-cell group(negative control group)(P=0.000).The stimulating efficacy on T-cell proliferation of DC vaccine with AⅣ group was higher than that of DC vaccine group(S/R 1∶5,1∶10,1∶50,1∶100,P=0.013,0.014,0.017,0.019).Compared with T-cell group,the T-cell proliferation rate of AⅣ group had no statistically significance(P=0.185).②The killing rate of effector cells actived by DC vaccine with AⅣ group and DC vaccine group against SGC7901 gastric carcinoma cells were higher than that of AⅣ group and T-cell group(P=0.000).The Killing power of DC vaccine with AⅣ group was stronger than that of DC vaccine(E/T 5∶1,10∶1,20∶1,50∶1,P=0.023,0.012,0.016,0.011);while the group of AⅣ group and T-cell group cannot killing tumor cells.Both had no statistically significance(P=0.267).Conclusion AⅣ can stimulate T-cell proliferation and enhance the activity of killing tumor cells by DC,which induced specific antitumor response against stomach carcinoma cells effectively.

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

OBJECTIVETo explore the oxidative modification effect and its mechanism of low density lipoprotein (LDL) in hyperlipidemia (HL) rats after treating with tetrahydrobiopterin (BH4).

METHODSFifty four 8-week-old male Wistar rats were used, these 54 rats were randomly divided into control group, high fat diet group (HL group), high fat diet and injected BH4 group (HL + BH4 group), and 18 in each group. The BH4 levels of blood fats and blood serum and its metabolites, the aortic reactive oxygen species, the end product malondialdehyde (MDA) and the LDL oxidation level were all determined by killing 6 experimental rats in each group at the first 8, 16, and 24 weeks of age respectively.

RESULTSTreating with BH4 after 8 and 16 weeks, there was no significant difference in serum lipids among three groups (P > 0. 05); but ROS and MDA decreased significantly (P < 0.01); compared with control and HL groups, the BH4 level of HL + BH4 group increased a lot (P < 0.01); compared with control group, the BH4 content reduced obviously in aortic homogenate of HL group (P < 0.01), but the total petrin levels (TB = BH4 + BH2 + B) had no significant difference (P > 0.05); the serum TBARS formation increased gradually with the increase of week-ages, but compared with HL group, the serum TBARS formation of HL + BH4 group reduced significantly (P < 0. 01).

CONCLUSIONTreating with BH4 can reduced the LDL oxidation, the mechanism may be related to the correct of NOS uncoupling, the reduce of ROS generation and the decrease of LDL lipid peroxidation.

Animals ; Biopterin ; analogs & derivatives ; therapeutic use ; Hyperlipidemias ; blood ; drug therapy ; Lipid Peroxidation ; Lipids ; blood ; Lipoproteins, LDL ; metabolism ; Male ; Malondialdehyde ; metabolism ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

This study was aimed to explore the difference of NK cell receptor NKG2D and NKG2A expression on NK cells and CD3(+) T cells and their ligand MHC-I A/B (major histocompatibility complex class I-related chains A/B) and HLA-E expression in leukemia cells, as well as its immunological significance. Flow cytometry was used to detect the killing rate of NK92 cells to 8 leukemia cell lines, and the expression of NKG2D and NKG2A on NK cells and CD3(+) T cells as well as their ligand MHC-I A/B and HLA-E expression on leukemia cells. The results indicated that the NK92 showed different killing activity to different leukemia cell lines. The positive expression rate of NKG2D and NKG2A on NK cells and CD3(+) T cells in ALL patients was no significantly different from that in AML patients (p > 0.05), but positive expression rate of MHC-I A/B and HLA-E in ALL patients was obviously higher than that in AML patients (p < 0.05). It is concluded that there is difference of immune cell function between ALL and AML patients, this difference may be associated with the expression difference of NKG2D and NKG2A ligands on leukemia cells while does not associated with the killing and inhibiting receptors expressed on NK cells and CD3(+) T cells.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Infant ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; NK Cell Lectin-Like Receptor Subfamily C ; genetics ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; genetics ; metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Young Adult

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology